Apparatus and method using centripetal acceleration to drive fluid flow in microfluidic systems by i

专利摘要:
The present invention relates to apparatus and methods for performing microanalytical and microsynthetic analyzes and procedures. The present invention provides a microplatform and micromanipulation device for manipulating the platform to utilize the centripetal force generated from the rotation of the platform to move fluid through the microchannel. The microsystem platform of the present invention includes an information system coded on the surface of the disk opposite the surface containing the fluid component and an information system of data acquisition, analysis and storage and retrieval. Certain methods of the present invention are provided for carrying out a wide variety of microanalytical or microsynthetic methods.
公开号:KR19990072013A
申请号:KR1019980704305
申请日:1996-12-05
公开日:1999-09-27
发明作者:알렉 미안；스티븐 지. 키이퍼-히긴스；조지 디. 코레이
申请人:피.리차드 이글링；가메라 바이오사이언스 코포레이션；
IPC主号:

专利说明:

Apparatus and method using centripetal acceleration to drive fluid flow in microfluidic systems by informatics
In the field of medical, biological, and chemical analysis, mechanical automated fluid handling systems and instruments that are manufactured to operate on a macroscopic scale (ie, in millimeters and milliliters) are well known in the art.
U.S. Patent No. 4,279,862, filed July 21, 1981 to Applicant "Bertaudiere", discloses a "centrifugal photometric analyzer."
U.S. Patent No. 4,381,291, filed April 26, 1983, to Applicant "Ekins," discloses "analytic measurement of free ligand."
U.S. Patent No. 4,515,889, filed May 7, 1985, to Applicant "Klose" et al., Discloses "automated mixing and incubating reagents to perform analytical determination." Started.
U.S. Patent No. 4,676,952, filed June 30, 1987 to Applicant "Edelmann," discloses a "photometric analysis apparatus."
U.S. Patent No. 4,745,072, filed May 17, 1988 to Applicant "Ekins," discloses "immunoassay in biological fluids."
U.S. Patent No. 5,160,702, filed November 3, 1992 to Applicant "Kopf-Sill," discloses "centrifuge rotor for analyzing solids in a liquid." Started.
U.S. Patent No. 5,171,695, filed December 15, 1992 to Applicant "Ekins," discloses "determination of analyte concentration using two labeling markers."
U.S. Patent No. 5,173,262, filed December 22, 1996 to applicant "Burtis", discloses "centrifuge rotor for processing liquids."
U.S. Patent No. 5,242,803, filed September 7, 1993, to Applicant "Burtis", discloses "rotor assembly for carrying out an assay."
U.S. Patent No. 5,409,665, issued April 25, 1995, to Applicant "Burd," discloses "cuvette filling in a centrifuge rotor."
U.S. Patent No. 5,413,732, issued May 9, 1995 to Applicant Buhl et al., Entitled "Preparation of lyophilized reagent spheres for use in automated centrifugal blood analyzers." automated centrifugal blood analyzers ”.
U.S. Patent No. 5,432,009, filed July 11, 1995, by Applicant "Ekins," discloses "method for analyzing analytes in a liquid."
U.S. Patent No. 5,472,603, issued December 5, 1995, to Applicant "Schembri," discloses "analytical rotor for performing fluid separations."
"Multiple cuvette rotor for cell fractionation" is disclosed in Anderson's 1968 page 545-562 of "Anal. Biochem."
"Minidisc module for a centrifugal analyzer" is disclosed on pages 955-960 of "Clin. Chem." No. 20 of "Renoe" et al.
"Clin. Chem.", Page 20, pages 932-941 of "Burtis" et al. "Method for dynamic introduction of liquids into a centrifugal." analyzer "is disclosed.
On pages 240-246 of Clin. Biochem. No. 8 of 1975 by "Fritsche" et al., "Enzymatic analysis of blood sugal levels using a centrifugal analyzer "is disclosed.
Page 1225-1233 of Clin. Chem., Et al., "Burtis," describes a "multipurpose optical system for use with a centrifugal analyzer." Has been disclosed.
On pages 802-805 of Clin. Chem. No. 22, 1976, Hadjiioannou et al., "Automated Enzyme Ethanol Determination in Biochemical Fluids Using a Miniature Centrifugal Analyzer. enzymatic ethanol determination in biological fluids using a miniature centrifugal analyzer.
"Automated blood fractionation system" has been disclosed in pages 1361-1365 of "Clin. Chem." No. 24 in 1978 by Lee et al.
In 1982, "Clin. Chem." No. 28, pp. 1965-1961, a "multichannel electrochemical centrifugal analyzer" was disclosed.
"Clin. Chimica Acta" No. 119, pages 275-284 of "Bertrand" et al., "Automated Measurement of Serum 5'-Nucleotidase Using a Centrifugal Analyzer ( automated determination of serum 5'-nucleotidase using a centrifugal analyzer. "
A "portable whole blood analyzer" is disclosed in pages 1665-1670 of Clin. Chem., 1992, "Schembri" et al.
In the third edition of "Basic Mecical Laboratory Technologies" published by "Delmar", Boston, USA, 1995, "A variety of automated medical research and analysis techniques (a variety) of automated medical laboratory analytic techniques. "
Recently, microanalytical devices for performing select reaction pathways have been disclosed.
US Patent No. 5,006,749, issued April 9, 1991, filed by Applicant "White," describes a method and apparatus for using ultrasonic energy to move microminiature elements. Started.
U.S. Patent No. 5,252,294, filed on October 12, 1993 to Applicant "Kroy" et al., Discloses a "micromechanical structure for performing certain chemical microanalyses."
U.S. Patent No. 5,304,487, filed April 19, 1994 to Applicant "Wilding," discloses "fluid handling on microscale analytical devices."
U.S. Patent No. 5,368,704, filed November 29, 1994, to Applicant "Madou" et al., Discloses a "microelectrochemical valve."
Applicant International Publication No. WO93 / 22053, published November 11, 1993, of the University of Pennsylvania, discloses "microfabricated detection structures."
Applicant International Publication No. WO93 / 22058, published November 11, 1993, of the University of Pennsylvania, describes "microfabricated structures for performing polynucleotide amplification." Started.
"Fluid management of biological fluids" is disclosed in Columbus et al., Pages 1531-1537 of "Clin. Chem." No. 33, 1987.
"Multianalytical microspot immunoassay" is disclosed in pages 337-353 of "Ann. Biol. Clin." No. 50, 1992, by "Ekins" et al.
On pages 43-47 of 1994 "Clin. Chem.", "Wilding," et al. "Manipulation of fluids on straight channels micromachined into silicon ".
The prior art discloses synthetic microchips for performing microanalytical and microsynthetic methods. Microanalytical methods and apparatus of the prior art have had problems designing reservoirs in the range of 10-100 μm and systems for moving fluids on microchips through multiple channels. In addition, the devices disclosed in the prior art require separate data analysis and storage media to be incorporated into the instruments that perform microanalysis, thereby not increasing the flexibility or usefulness of the instruments designed to use the microchip, Only increase complexity unnecessarily.
Thus, simple, flexible, reliable, fast and economical microanalytical and microsynthetic chemicals that perform biological, biochemical and chemical analysis and synthesis to flow fluids within the components of a microsystem platform. There is a need to provide a phosphorus reaction platform. Such a platform must be able to flow nanoliter to microliter fluids, including reagents and reactants, at high speed to effect proper mixing of the reaction components, removal of the reaction products, and separation of the desired products and intermediates. There is also a need for a mechanism for manipulating the microsystem platform to provide fluid flow, thermal control, reagent mixing, reactant detection, data acquisition, data analysis, and data and systems connected to the user. Such devices may be complex in use (professional, eg in hospitals), easy to use (consumers, eg in home monitoring, etc.), or portable (outdoors, e.g. For example, for use in environmental experiments).
This application is directed to US patent application Ser. No. 60 / 008,215, filed Dec. 5, 1995, US patent application 60 / 008,267, filed Dec. 6, 1995, US patent application No. 60, filed Dec. 18, 1995. / 008,819, and US patent application Ser. No. 60 / 023,756, filed August 12, 1996.
The present invention relates to methods and apparatus for performing microanalytic and microsynthetic assays and procedures. In particular, the present invention relates to the miniaturization of genetic, biochemical, and chemical processes involved in analysis, synthesis, and purification. More specifically, the present invention relates to a microsystem platform and a micromanipulation device that rotates the platform, and through microchannels inserted into a microplatform. It uses centrifugal force as the platform rotates to drive the fluid flow. In addition, the microsystem platform according to the present invention acquires, analyzes, stores, and retrieves system informatics, and data in an encoded state on the surface of the disc opposite the surface containing the rheological component. It can be provided to informatics. Also provided are methods for performing a wide range of microanalytical or microsynthetic chemical processes using the microsystem apparatus according to the invention.
1A (top view) and FIG. 1B show the reservoirs 12, 14, 18, 20, valves 13, 15, 17, 19, 21, 23, 25, respectively, in the discs constituting the microplatform of the present invention, respectively. Figures show the arrangement of reaction chambers 16, 22, 24, holes 11.32, and air outlets 29, 33, 34, 35, and FIG. 1C shows the arrangement of multiple microsystems on a disc. One drawing.
2A and 2B are graphs and schematics showing the arrangement of the channels on the disc of the present invention, respectively, described in relation to equation (5).
3A and 3B are graphs and schematics showing the arrangement of the channels on the disc of the present invention described in connection with equations 12 and 13, respectively.
4A and 4B are graphs and schematics showing the arrangement of the channels on the disc of the present invention, respectively, described in relation to equation (14).
5A, 5B, and 5C are graphs showing the arrangement of the channels on the disc of the present invention, respectively, described in relation to equation 15 and FIG. 5D is a schematic diagram thereof.
6 is a schematic diagram of a piezoelectric stack microvalve.
7 is a schematic representation of a pneumatically actuated microvalve.
8 is a schematic diagram of a device for flowing air pressure to a rotating disk.
9 is a schematic diagram of a bimetallic microvalve.
10 is a schematic diagram of a pressure balance type microvalve.
11 is a schematic of a polymer relaxed microvalve.
12A and 12B show two different embodiments of the fluorescence detector according to the present invention, respectively.
13A, 13B, and 13C are schematic diagrams of a multiple loading apparatus for a disc, respectively.
14A to 14F are diagrams showing the performance of the laser light source-operated CD-ROM of the disc according to the present invention, respectively.
Fig. 15 is a flowchart showing the processor control structure of the player and / or reader device according to the present invention.
16 is a schematic diagram of the transverse spectroscopic detection chamber.
17A-17E are schematic diagrams illustrating different structural and functional layers of a disc according to the invention, each shaped into a DNA array.
17F is a schematic diagram showing the basic area and design of the analyte disc.
17G is a schematic diagram of a disk formed as a home test diagnostic disk.
17H is a schematic of the disk formed for a simple immunoassay.
17I is a schematic representation of a disk formed as a gas and particle disk.
17J is a schematic diagram of a hybrid disc including separately assembled chips.
17K is a schematic diagram of a sample recognition disk.
17L is a schematic view of a disk formed for use in the pathology field.
17M is a schematic diagram of a disc with removable analysis layers.
17N is a schematic representation of a disk for aerosol analysis.
17O is a schematic representation of a disk for flow cytology.
17P is a schematic representation of a disk used in the field of microscopy.
17Q is a schematic representation of a disc used in the field of immunology.
17R is a schematic representation of a thin layer chromatography disk.
18 is a schematic diagram of a disk formed for cold blood measurement.
19 is a schematic diagram of a disk formed for SPLITT cleavage of blood components.
20 is a schematic diagram of a disk formed for a DNA meltometer.
21 is a schematic diagram of a disk formed for DNA amplification.
22 is a schematic of a disk formed for automated restriction enzyme action of DNA.
Figure 23 is a partial schematic view of a disk formed for DNA synthesis.
23B is a schematic of a disk formed for the synthesis of multiple DNA deficient nucleotides.
24 is a schematic of a disk formed for DNA disruption.
25 is a schematic of a disk formed for iron analysis.
26 is a schematic view of a disk formed for a solid phase reaction.
27 is a schematic diagram of a disk formed for sample extraction.
28 is a schematic representation of a disk formed for capillary electrophoresis.
29 is a schematic diagram of the transverse optical path in the microplatform.
30 is a block diagram of a process flow for controlling information according to the present invention.
31 is a more detailed schematic diagram of a processing flow for controlling information according to the present invention.
32 is a more detailed schematic diagram of a processing flow for controlling information according to the present invention.
Example
The present invention provides microplatform and micromanipulators for performing microanalytical and microsynthetic chemical analysis of samples for biological, chemical, environmental, and industrial use. For the purposes of the present invention, the term "sample" is meant to include any chemical or particulate state species that have been separated or detected as a rather complex mixture or synthesized from previous stage species. The present invention provides a fluid flow on a platform that results from rotatable analytical and / or synthetic chemical microvolume analysis platforms (hereinafter collectively referred to as "disks") and centrifugal forces exerted on the platform by rotation. It provides a combination of micromanipulators for manipulating the platform to provide. The platform of the present invention is preferably provided as a circular disk, but any platform that can be rotated to provide centrifugal force for the fluid on the platform is possible within the scope of the present invention.
The microplatform of the present invention (preferably, hereafter collectively referred to as "disk", for the purposes of the present invention, the terms "microplatform", "microsystem platform", and "disk" are considered to be compatible with each other. Are provided to include one or more microsynthetic chemistry or microanalysis systems. Such microsynthetic chemistry or microanalysis systems also include a combination of components that are optionally interconnected to provide fluid flow between the various components upon rotation of the disk, as described in detail below. These components may be manufactured integrally with the disk, or attached to or installed on the disk, or inserted into or in contact with the disk, as described in more detail below. The present invention also includes a micromanipulator for manipulating the disc of the present invention, wherein the disc is rotated in such a micromanipulator to provide centrifugal force to enable fluid flow on the disc. Thus, such micromanipulators provide a means for stopping or starting the rotation of the disc, and preferably for rotating the disc at a controlled rotational speed to change the direction of rotation of the disc. As will be described in more detail below, both electrochemical and control means are provided as components of the apparatus of the invention. In addition, user connection means (eg, a keyboard and a display) are also provided.
The present invention provides methods and apparatus for manipulating samples consisting of fluids, cells and / or particles (collectively referred to as "samples") containing the analytes of interest. The platform of the present invention includes a sample inlet, a microchannel for fluid flow, a reagent reservoir, a mixing chamber, a reaction chamber, an optical reading chamber, a valve for fluid flow between various components, a temperature control member, a separation channel, an electrophoretic channel, Electrodes, air outlets, sample outlets, product outlets, mixing means including magnetic and acoustic mixers and mechanical mixers, built-in power sources such as batteries or electromagnetic generators, liquid and gaseous reagents, and It includes many other components known to those skilled in the art. Movement of the sample is facilitated by appropriately providing air holes or air movement channels that allow the flow of air and at the same time prevent loss of fluid and / or particles during acceleration. Preferably, the disk comprises mechanical, optical, and rheological control elements microfabricated on a platform made of, for example, silica, quartz, metal, or ceramic. In the description of the present invention, the term "microfabricated" means the process by which these structures are manufactured on a scale of millimeters or less. These processes include photolithography, etching, stamping, and other means well known to those skilled in the art, of course, but these processes are by no means limited to these means.
The flow of fluid (including reagents, samples, and other fluid components) is dependent on the centrifugal acceleration due to the rotation of the platform and the selective operation of the various valves that control the connections between the components of the platform's microsystem. Controlled by each. The magnitude of the centrifugal acceleration required for a fluid to flow at a constant velocity under appropriate pressure for a particular microsystem is determined by the effective radius of the platform, the angular position of the structure on the platform relative to the direction of rotation of the platform, and the rotational speed of the platform. Of course, the magnitude of acceleration is by no means limited to these factors.
Chemical and biochemical reactions are performed in the reaction chamber by selective opening of the microvalve to control adjacent reagent reservoirs. As will be described in more detail below, microvalvees include not only mechanical, electrical, and thermal valve mechanisms, but also capillary microvalves whose fluid flow is controlled by the relationship between capillary attraction and centrifugal forces acting on the fluid. The contents of the reagent reservoir connected to the reaction chamber through the microchannels controlled by these microvalve are introduced into the reaction chamber by rotation of the microplatform and opening of the appropriate microvalve. The amount of reagent entering the reaction chamber is controlled by the speed of rotation and the time the valve is open to the reagent reservoir. Reaction products performed in the reaction chamber are likewise removed from this reaction chamber to the assay device, second chamber, or product outlet by controlled opening of the microvalve in the reaction chamber.
The analytical device constituting the microplatform of the present invention includes a detection system for detecting, adjusting, quantifying or analyzing a reaction process, a product or a secondary product. The detection system is easy to assemble and the use of the micro platform of the present invention is used in fluorescence, chemiluminescence, color, electrochemical and radioactive means, but not limited thereto. Optionally, the upper extremity detection means includes a platform and includes an apparatus for adjusting the platform.
Thus, the microplatform and microadjustment apparatus according to the present invention generates the analytical and synthetic data to be processed. The processing of data is performed by a processor and memory modules on disk, a microprocessor and memory device, and a border computer connected to the adjusting device. Removable media for withdrawal and storage are provided by a device using a computer diskette, tape or optical media, or by the disk itself. In contrast, data is recorded on the microplatform using CD-reading / writing techniques and conventional optical data storage systems. In such an embodiment, data is recorded on the microplatform on the underside of the platform opposite the wet side holding the plurality of microsystem components described above.
The physical parameters of the microplatforms of the present invention can vary widely. When provided as a disk, the disk radius is 1 to 25 cm and the disk thickness is 0.1 to 10 cm, preferably 0.1 to 100 mm. The most advantageous preferred embodiment for making and operating the disc of the present invention has dimensions belonging to one of four forms. That is, (1) a 3 inch compact disc (CD) having a radius of about 3.8 cm and a thickness of about 1 mm, (2) a 5 inch CD having a radius of about 6 cm and a thickness of 1 mm, (3) about 10 8 inch CDV with a radius of 2 cm and a thickness of 2 mm (commonly referred to as a "leisurevision" disk), (4) 12 inch CDV with a radius of 15 cm and a thickness of 2 mm.
The size of the microchannels and reagent chambers can be optionally determined by the reagents and reagent distributions required for the particular application and for each particular example of the microanalysis and microsynthesis methods of the present invention. In the application of microanalysis, for example 5 or 6 cm × 6 mm with reagent chambers that can accommodate up to 5 ml (which is the amount that can actually be converted by the disc) is suitable. The size of the microchannels ranges from 0.1 m to values close to 1 mm in thickness of the disk. The microchannels and reagent chambers may be trapezoidal, circular or otherwise shaped as desired. The microchannels are realized with microsystem platforms having a thickness of about 0.1 to 100 mm, where the cross-sectional dimension of the microchannels across the thickness dimension of the platform is 500 μm or less and 1 to 90% of the cross-sectional dimension of the platform. The reagent chamber, reaction chamber, detection chamber and sample inlet and outlet are formed in a micro system platform having a thickness of about 0.1 to 100 mm, the cross sectional dimension of the micro channel across the thickness dimension of the platform being the cross sectional dimension of the platform. 1 to 75%.
Input and output ports (inlets and outlets) are components of the microplatform of the present invention used to remove various fluid components. The inlet allows samples and reagents to be placed or introduced onto the disc, and this type of port is generally located towards the center of the disc. The outlet allows air to flow inside the on-disk "muffler" or "baffle" system to allow free fluid motion on the disc. In addition, an air conversion channel is provided in the air handling system on the disc such that the fluid motion converts air through a channel connected to the fluid receiving microchannel in a direction opposite to the direction of motion of the fluid, Provide positive pressure to further promote exercise. The outlet also allows product to be removed from the disk. The shape and design of the port will vary depending on the particular application. For example, the sample input port enables capillary motion that allows the sample to be sucked effectively into the disc. In addition, the ports can be designed to be shaped to automatically inject samples / reagents or remove products. The inlet and outlet can be provided in the most advantageous arrangement in which multiple samples can be provided on a disc using a specially designed dosing tool. Similar tools can be usefully designed so that the product can be effectively removed from the microplatform. Representative arrangements for sample ports, vents, reagent chambers, reaction chambers, and microvalvees are shown in FIGS. 1A-1C.
Operable Arrangement of Multiple Disk Components and Elements Optimal positioning depends on the dynamics of the fluid motion relative to the centripetal force. Centripetal force is a function of platform radius, disk velocity and fluid concentration. Certain variables associated with the platform microsystem of the present invention are understood by the following equation. These represent system performance limitations that are presumed to be incomplete and complete (turbulent) losses for well-developed fluid flows.
The driving force for the fluid motion or the force generating the fluid pressure is the force resulting from the centripetal acceleration. The device rotates at each frequency and at an angular ratio of pin rotation.
ω = 2πｆ (1)
The centripetal acceleration (or acceleration directed along the radius of curvature at radial distance from the center of the regularly rotating disk) is
α _c = ω ² R (2)
The weight m in the uniform circular motion is subjected to a centripetal force directed inward along the radius of curvature with respect to the center of rotation.
F _c = ma _C = mω ² R (3)
If the mass is kept uniform in the radius, the rotating device supplies the force, which is the origin of the liquid static pressure described below. If the mass is placed on top of a trap door over a radially oriented tube, the trapdoor is open, and the mass inertia is the source that accelerates the tube downwards to drive the fluid in the outer radial direction of the rotating disk. do.
Rotation causes static pressure in the non-flowing fluid. It is assumed that the liquid column extends from the inner radius R. The tube may go along the radius and incline along an angle at the radius. The pressure in the position (R _O), for example, is defined as the atmospheric pressure P _O. The excess pressure due to the rotation of the liquid at position R such that R _O <P _O is obtained by integrating the centripetal force per unit area for the liquid with a density p from position R _O to.
_{P - P O = ∫ρa C =} ρω 2/2 × (R 2 -R O 2) (4)
If the tube is filled and extends from the center, the pressure
P-P _O = (2.834 × 10 ^-4 ) pf ² R ² (5)
R in pounds per square inch (psi) is the radial position in units of cm, ρ is the density in units of gm / cm 3, and f is the frequency in units of revolutions per second. Thus, the pressure (or fluid centripetal force value) changes linearly to the density of the fluid, the square of the radial position from the center of rotation, and the square of the rotational frequency.
To determine the velocity of the liquid in motion in the channel on the rotating disk, the equation of motion for the liquid must be solved. The length dR term filling the fluid radius and the circular channel has an accelerating mass dm.
dm = πρa ² dR (6)
The equation of motion for a fluid element is force = (mass) x (acceleration).
The force is a centripetal force, capillary force due to the difference in interfacial energy between the fluid and vapor and the liquid and solid surface, and the dispersing force due to the velocity and uneven flow of the liquid. Capillary forces are ignored, and it can be understood that centripetal force and / or external pressure are necessary to introduce liquid into the wet channel. Over-estimating the dispersing force includes the force for fully developed laminar flow of Newtonian fluid F _L and the force F _D due to uneven flow.
F = ma
F _C + F _L + F _D = dma _R (7)
F _C + F _L + F _D = (πρa ² dR) a _R
Where a _R is the acceleration of the fluid mass element along the radius.
F _C = (πρa ² dR) ω ² R
F _L =-(8μπa ² dR) u (8)
F _D =-(2πρa ² dR) u ²
Where μ is the velocity and u is the fluid radial velocity. The last two formulas are standard mechanical equations for fully developed and not fully developed laminar flow at the channel inlet / flow droplet end. Also, for a tube or channel that is inclined at an angle θ with respect to the radius F _C , it must be replaced with (F _C ) × co θ. The final equation is
(πρa ² dR) ω ² R- (8μπa ² dR) u-(2πρa ² u ² dR) = (πρa ² dR) (du / dt) (9)
Where the radial acceleration of the fluid is defined as a ^2- (du / dt). This is the differential equation for the fluid flow rate.
The equation is applied to a specific example, ie, a fluid droplet of length L moving in a radial channel having a length greater than that of the droplet.
Since the fluid droplets all move at the same speed, dR can be replaced by L and R, the average positions of the droplets, <R> = (R + L / 2).
Driving common factor:
(ω ² (R + L / 2) / 2)-(8μ / ρa ² ) u-2 (u ² / L) = (du / dt) (10)
The equation must be solved numerically. An approximation can be obtained by comparing the numerical values. The approximation value is obtained as follows. Negative terms on the left can be offset with positive terms. The right hand term is set to zero and the solution is solved by the final equation for the "terminal speed" at position R, u ₀ .
(ω ² (R + L / 2) / 2)-(8μ / ρa ² ) u _o -2 (u _o ² / L) = 0 (11)
The equation has the following solution.
u =-(B + √B ² + 4AC) / 2A (12)
here,
A = L / 2, B = 8μ / ρa ² , C = (ω ² (R + L / 2) 2) (13)
In conventional units, they are A = 2 / L, B = 320μ / ρD ² , and C = (19.74) f ² (2R + L), fluid velocity with u _o = unit cm / sec, droplet length with L = unit cm , μ = velocity of unit poise, ρ = fluid density of unit gm / cm 3, D = 2a = tube diameter of unit cm, R is the radial position of fluid droplets of unit cm. As mentioned above, the equation gives the approximate velocity of the fluid droplets in the tubular channel, with the volume of the droplets being shorter than the channel length. This can be assumed to be viscous and non-viscous loss. The velocity of fluid droplets increases with increasing droplet volume (length) and density, and decreases with increasing viscosity. The speed increases with increasing channel diameter, rotation speed, and radial position.
The fluid flow velocity in the filled channel connecting the filling channel at position R _O with the receiving chamber at position R ₁ is equal to equation (11) when the channel velocity is L = R ₁ -R _O. Calculated by defining L in the equation Equation (13) and then equation (14) are used to calculate the flow velocity in the filling channel as a function of radius.
The fluid flow ratio is the product of velocity and channel area.
^{_{Q = u O πa 2 = u}} O πD 2/4 (14)
Where Q is the flow in units mL / sec, u _o is the speed in units cm / sec (calculated from equations 12 and 13), and D is the tube diameter in units cm.
The time required to deliver volume (V) from the reagent chamber to the receptacle through a length (L) tube or channel may be such that the tube fills the volume by volume (V) or the droplet length of volume (V) in the tube Longer than the tube itself). In the former case, the time is the value of the volume V of the fluid divided by the flow ratio Q, and in the latter case it is roughly calculated as the ratio of the tube length times the resulting droplet length.
If Dt = V / Q, L ≤ (4V / πD ² ), (15)
When Dt = (V / Q) × (4πD ² L / 4V), L> (4V / πD ² )
Where Dt is the same time as unit seconds for a fluid of volume V that is unit mL flowing at a rate Q of unit mL / sec to flow from the filled reagent chamber to the receptacle through a tube of length L and diameter D (units) to be. The flow ratio Q is calculated by the variables defined in equations (12) and (13) and equation (13). The time Dt increases with increasing volume delivered and decreases with increasing flow ratio.
Flow characteristics, such as pressure and velocity, are related to the physical variables of the disk, such as the disk radius and rotational speed, as described above. These relationships are shown in FIGS. 2-5, which are derived from the above equation for water with ρ = 1gm / cm 3 and μ = 0.001 poise at room temperature. From these figures one can derive the closest variables for fluid motion on the rotating disk.
FIG. 2A shows the static pressure relationship in a fluid filled tube 30 cm in length as a function of radius distance R and rotation ratio f calculated from equation (5). The arrangement of the tubes on the rotating disk is shown in Figure 2b. It can be seen that pressure in the range of 0 to 10,000 psi can be generated in the tube at 0 to 10,000 rpm rotation speed. Pressures of this magnitude can be commonly used, for example, to drive high pressure liquid chromatography (HPLC).
FIG. 3A shows the radial velocity of water droplets with moving tubes of 1, 10, and 100 μL, tubes 1 mm long and 30 cm in diameter, in the hollow. The radial velocity is calculated from equations (12) and (13). The lubes are arranged to extend from the center along the radius of the disk, the disk rotating at a speed of 100, 1000 or 10,000 rpm. The arrangement of the tubes on the rotating disk is shown in Figure 3b. These velocities are used to calculate the delivery time for fluid droplets. For example, 1 μL of water droplets flow at about 20 cm / sec when located 2 cm away from the center of the disk rotating at 1,000 rpm. Thus, the time to pass through the 1 cm tube is calculated to be about 0.05 seconds (the velocity drops to 30% for tubes oriented in the non-radial direction at 45 ° to the direction of rotation).
4A shows the flow ratio within a 5 cm fluid filled tube of different diameters. The tubes are each placed on a rotating disk and connected to two radially directed reagent chambers, as shown in FIG. 4A. According to equation (14), the flow ratio is the radial position of the tube (which varies in the range of 2 to 30 cm in this embodiment), the tube diameter (10, 100, or 1,000 μm), and the rotation frequency (100, 1,000 or 10,000 rpm). ) Function. (As mentioned above, for a 45 ° non-radial oriented tube, the velocity drops to 30%.) The droplet rate shown in FIG. 3A is calculated by Equation 3 and the flow ratio is given by Equation (4). Is determined by
5A, 5B, and 5C show the time required to deliver 1, 10, and 100 μL droplets through a 5 cm tube, respectively. The tube connects two radially directed reagent chambers as shown in FIG. 5D. The transfer time is a function of the radial position of the tube (0-30 cm), the tube diameter (10, 100, or 1,000 μm), and the rotational frequency (100, 1,000, or 10,000 rpm). The curves shown in FIGS. 5A-5C are calculated using equation (15).
Taken together, the equations and graphs show the interrelationship of fluid properties such as disk radius and rotation speed, channel length and diameter, and speed and density that determine the fluid velocity and flow ratio on the disk. Behind this deviation is the loss of viscosity due to fuzziness (non-swirl) flow and the additional loss due to uneven flow of droplets at the tube inlet and outlet. The equations and graphs provide the lower limits of velocity and flow ratio. Flow rates range from less than 1 cm / sec to 1000 cm / sec and fluid flow rates range from 1 pL / sec to 10 mL / sec when having a rotation ratio in the range of 1 to 30,000 rpm. By combining the channel diameter and location on the disk, fluid transfer can be performed in a time range of millions of seconds to several hours for various processes.
Disc coating and its composition
Microplatforms, such as disks, and components comprising such platforms may have surface coatings of various compositions to be particularly suited to the various applications described above. The composition of the disc is a function of structural requirements, fabrication process, and reagent compatibility / chemical resistance properties. In particular, the discs are for example inorganic materials such as silica, quartz, metal or organic materials such as amorphous or plastics, for example poly (methyl methacrylate) (PMMA), acetonitrile-butadidine-styrene (ABS) , Polycarbonate, polyethylene, polystyrene, polyolefin, polypropylene and metallocene. These can be used with altered or unchanged surfaces as described below.
One of the important structural considerations in the manufacture of the microsystem discs of the present invention is mechanical failure due to stress during use. Fracture mechanisms for high-speed rotating discs may occur as a result of tensile loads or cracking and crazing as described in Hertzberg (1989, Deformation and Fracture Mechanics of Engineering Materials, 3rd Edition, New York, Willy and Suns). have. The failure occurs when the stress due to the load of the disc (defined as load per unit area) exceeds the critical characteristic value of the material used to make the disc. The "load" that may occur at any point of the disc is the tensile force due to rotation (ie the total load at a given radius on the disc is the centripetal force required to hold the material at a larger radius moving in a circle) and the load / area Or the stress is the force divided by the total area of the disk (2πr × the thickness of the disk). The threshold of stress at which the material breaks is called the yield stress, which depends on the intrinsic energy that bonds the materials together and the presence of defects in the material. The defect-free material ruptures, while the material with small defects propagates the cracks through cracks or "crazing" (ie, plastic deformation and fracture of the glass plastics described above). For example, the yield strength of commercialized silicon can stretch a 30 cm disc at about 10,000 rpm without mechanical breakdown when the diameter of the inner channel and chamber is about 80% or less of the disc's total thickness. In discs made of plastic, the stress on the disc is generally reduced due to the low density of the plastic (which reduces the load / unit area). However, the yield strength is about twice less than the yield strength in silicon (as described in more detail in New York, Marcel Decker, Computing Modeling of Polymers (Bretz et al.), Lewis and Janis (1992)). One way to solve this problem is to solve the problem by rotating a 30 cm disc of plastic at low speed (1,000 rpm) or by reducing the radius of the disc (such as when using a 4 cm plastic disc when 10,000 rpm speed is required). Can be. Therefore, the choice of material for a particular application is sufficient to accommodate the disc's compositional requirements regarding the disc's functional properties and characteristics.
The disc material in contact with the fluid should not degrade the reagent solution under rotational stress during heating and cooling, and will respond when illuminated with high-intensity ultraviolet or visible light (which may occur with the use of any detection means as described below). Must have In addition, surfaces (such as microchannels, reagent chambers, and reaction chambers) in which reagents and reaction mixtures are present should have desirable surface properties for each application. Silicon, silica, and quartz are particularly robust materials as substrates for microplatform fabrication. Silicon and silicon oxides (particularly silica) are a few peroxides (such as mixtures of hydrogen peroxide and sulfuric acid), hydroxides (such as KOH), hydrofluoric acid (HF), alkali-based nitrates or mixtures thereof, and many (such as SF ₆ ). Chemically corroded by perfluorinated solvents (see London, Inspect, Silicon Properties 10th Edition, 1988; Silica Chemistry, published by Willy and Suns, New York, 1979, et al.). Silicon-based substrates are chemically inert to aliphatic and aromatic carbon hydroxides (such as tetrahydroprene, toluene, etc.) and are almost inert when exposed to water and neutral hydroxide solutions.
Many polymer-based (plastic) substrates are suitable for making the microsystem platform of the present invention. The most chemically resistant polymer, poly (tetrafluoroethylene; PTFE), is not melt processable but is easily machined. PTFE is chemically inert and can be used in most applications using strong acids, strong salts, strong alkalis, strong halogenated solvents, or other reinforcing reagents. Other fluoropolymers (such as FEP, PFA) can be processed much easier than PTFE and retain the chemical resistance of most PTFE. The material to be more easily processed can be altered depending on the choice of resistance. For example, although polyimide has high resistance to alcohols, alkalis, aliphatic carbon hydroxides, and bases (eg, NAOH), resistance to most halogenated solvents (eg, dichlorobenzene) is poor. Do. Poly (vinyl chloride) has a strong resistance to the oxidation of acids and aliphatics, but the oxidation resistance to aromatic compounds is poor. In addition, many materials that do not have high resistance to any chemical application provide sufficient resistance to the dilute solution and a single use device (e.g., polyimide and polyimide may be used with any acidic diluent such as acetyl acid and hydrochloric acid). Provide sufficient resistance). Most polymer materials are water resistant.
Specific chemical polymer mixtures include formamide, lutidine, and acetonitrile with nonaromatic, nonpolar polymers (polypropylene, polyethylene); Dichloromethane with polycarbonate and aromatic polymer (polystyrene); Ethanolamine and dimethyl sulfide with aliphatic and non-aromatic polymers (poly (methyl methacrylate), polyimide, polyamide). The fluoropolymer is resistant to all of the above chemicals. Other solvents and reagents, including pyridine, tetrazole, trichloracetic acid, iodine, acetic anhydride, N-methylphyllolidine, N, N-diethylpropylethylamine and piperidine, are available from PVC (1989). Suitable for use with fluoropolymers and some solvent resistant polymers, such as John Willy and Suns, New York, Encyclopedia of Polymer Science and Technology, 2nd edition, 3, pp. 421-430.
The materials provide sufficient flexibility for any application.
The surface properties of the materials may vary depending on the particular application. For example, appropriate surface modifications can be made to facilitate or inhibit cellular and / or protein uptake. Surface modification can be accomplished by silanization, ion implantation, and chemical treatment by an inert gas plasma (gas through which current passes through). A strong correlation is established between water contact angle and cell uptake when there is a hydrophilic surface that exhibits significantly less cellular uptake than the hydrophobic surface (see 1994 1994 Biomaterials 15, 725). Silicon, silica, and quartz are present and have inherently high energy hydrophilic surfaces. Modification of the surface properties is carried out by hydroxylation or silanization (achieved by NaOH treatment at high temperature). Silanes and siloxanes are particularly suitable for increasing hydrophilicity on hydrophobic surfaces. These compounds consist of one or several reactive head groups, for example the core region of the alkane (-CH ₂ O-), which bind (via chemical or hydrogen bonding) to the substrate. These compounds provide a solution to more complex alterations of surface properties (such as departure from functional groups to obtain problematic surface properties). Many such functional compounds are introduced to surfaces containing vinyl, phenyl, methylene and methoxy groups as well as surfaces containing mixing functions. These functional groups not only change the overall properties, such as the liquid contact angle, as a result of the continued binding of certain binding molecules such as peptides, antibodies, etc., but also provide a place for preferential absorption of molecules. Silation is often performed by immersion in an aqueous solution at slightly elevated temperatures. Chemical resistance to silane and siloxane coatings is determined by the binding properties in chemically absorbed molecules (Ackl, Chemtech 7, 125, 1977). Properties such as hydrophobicity are maintained for a considerable period of time when the organic silane is in contact with highly corrosive acids, which means that single or limited use is possible under these circumstances.
Plastic-based disks can also be easily processed to obtain the required surface properties. Inert gas or reactive gas plasmas are typically used to alter surface energy by forming surface complexes such as, for example, hydroxyl enriched surfaces with increased hydrophilicity or perfluorinated surfaces for increased hydrophilicity. Surface graft polymerization is a technique used to graf polymers with intended surface properties to substrate polymers selected for bulk processability and manufacturing properties such as plastics. Common methods of initiating graft polymerization include audit radiation, laser radiation, thermal or mechanical processes, photochemical processes, plasma, and wet chemical processes (Polymer Science and Technology Dictionary, published in 1989 by Wilcy & Sons., New York). Further discussed in pp675-689 of the second edition). Chemical modifications of the polymer (and suitable polymer) surface include oxidation (polyethylene), reduction (fluorinated polymers), sulfonation, polymerization (vinylidene fluoride) dehalogenated hydrogenation (hydrofluorination) and hydrolysis. The chemical properties of the surface are altered by chemical modification, but the mechanical properties, durability and chemical resistance are mainly the action of the substrate plastics. Surface grafting of polymers on polyethylene (ethylene glycol) (PEG) yields a surface that is hydrophilic (different from polyethylene) and resistant to water (PEG itself is water soluble, but polyethylene is not). Finally, the silencing of the organic polymer surface can be performed, providing a broad surface of the surface of the surface energy and chemical combination.
Embodiments involving thin film disks can be carried out, including the use of microsystems that include layers of microsystems stacked on a solid support, which are useful for continuous analysis by switching disks and which include consumable disks. And low price. Such a disk is shown in FIG. 17L. Such discs may have unique markings, for example, bar codes directly on the disc.
Specific examples applied for various applications are provided in the examples below.
Disk Related Devices and Elements
The microsystem platform (microplatform) of the present invention is provided with a number of mounting components and disposed on the disc either as a direct manufactured or prefabricated module. In addition to the integral parts of the disc, certain devices and elements are arranged on the outer surface of the disc and are optimally located on or in contact with the disc of the invention.
1. Temperature control element
Temperature control elements, in particular heat elements, include heat lamps, direct laser heaters, Peltier heat pumps, resistive heaters, ultrasonic heaters and microwave excitation heaters. Cooling elements include Peltier devices and heat sinks, radiant heat fins, and other components that promote radiant heat loss. The thermal device may be applied entirely on the disk or may be applied to a specific area on the disk. The thermal element can be manufactured directly on the disc or can be made integrally with the disc. The temperature of any particular area on the disk is monitored by a resistive temperature device (RID), thermistor, liquid crystal bidirectional refraction sensor or by an infrared signal using an IR searcher. The temperature in any particular region of this disk can be controlled by the feedback control device. The micro thermal control device may be manufactured directly on the disc, and may be manufactured on a microchip and integrated on the disc or controlled by a device located on the outer surface of the disc.
2. Filter
It is a sieve structure that selectively retains or improves the passage of particulates, such as cells, cell aggregates, protein aggregates, or other particulate matter applied to the fluids applied on the microanalysis or microsynthetic discs of the present invention. Filters, and other structures are provided. Such filtering means may include microsieve structures, which may be manufactured directly on the fluid control structure on a disc (see, eg, Automat Analyt, published in 1994, US Patent Application 5,304,487, Publication No. WO93 / 22053, Wilding et al. Tech 40. 43-47) are manufactured separately and assembled in a fluid control structure. The sieve structure is provided with an orifice in a negligible range of sizes and optionally to classify the sample based on the size of the sample's components.
Another type of filter includes a material that selectively removes a sample component based on the electrostatic force between the filter material and the sample component. The electrostatic composition of the sieve material is either intrinsic to the material or given by the nature of the charge transferred to the material by the electronic circuit. The material held by the filter material may be irreversibly bonded or further processed to selectively separate by adjusting the composition and ionic strength of the buffer or by controlling the electrical state of the material, in the case of an electrically regulating material.
In another embodiment of a filter of another microsystem platform of the present invention, a particular component of the sample may be retained within the surface of the disc component or by interacting with a particular protein, peptide, or antibody of the disc of the present invention. It can be held in reservoirs, microchannels, or in certain areas. The material held by the specific binding can be separated from the surface of the disc and transferred to a collection reservoir by treatment with a buffer of appropriately selected ionic strength, using conventional methods that have been improved for immunological or chromatographic techniques.
The present invention also provides a compartment formed by a microchannel region or chamber or reservoir where the inlet and outlet ports of the chamber are limited by the filter device. In certain embodiments, the chamber thus formed comprises a reagent, such as a bead, in particular with a compound, such as an antibody, having an affinity for contaminants, unused reactants, reaction by-products, or other components that are undesirable in the final product. Coated beads. In using a disk comprising such a filter forming chamber, fluid containing necessary and unnecessary compounds is moved through the filter chamber by the centripetal force of the rotating disk. Accordingly, the unnecessary compound is bound by the affinity material and the required compound flushes the free chamber by fluid flow that is driven by centripetal force. Optionally, the necessary compound may be retained in the filter forming chamber so that the unnecessary compound is flushed. In these embodiments, for example, the outlet of the chamber is provided by the opening of the valve.
3. Mixer
Various mixing components are preferably provided as an embodiment of the micro system disk of the present invention, which is added from the reaction reservoir and requires mixing of the components in the reaction chamber. The stationary mixer is coupled to the fluid control structure of the disc by providing a textured surface to the chamber or microchannel containing the mixer. The two or more channels are mixed with each other by the hydrodynamic activity exerted by the mixing surface or the fabric surface of the chamber or by the action of the centripetal force exerted by the rotating disk. Mixing is achieved by quickly changing the direction of rotation or by physically stirring the disk by a system on the outer surface of the disk.
In another embodiment, a flexible flat plate (FPW) device (see White, US Pat. No. 5,006,749) is used to mix the fluid on the disk of the present invention. The FPW device uses aluminum and piezoelectric zinc oxide transducers at one end of a very thin film. The transducer sends and searches acoustic plane waves that propagate along the membrane. Stiffness and mass per unit area of the membrane determine the velocity of the plane wave. When coupled with an amplifier, the waveform forms a delay line oscillation proportional to the acoustic wave speed. Structures based on FPW phenomena are used to mix liquids as well as to detect pressure, acceleration, organic chemical vapors, protein adsorption, liquid density and viscosity. The FPW device may be integrally formed on the disk or may be located adjacent to the disk to mix the fluid components, especially in the reaction chamber on the disk.
4. Valve device
Control of fluid motion and delivery on the disk typically uses a valve device (micro valve) to encourage or prevent fluid motion between the components. An example of such a microvalve is a piezoelectric functional group comprising a sandwiched sheathed plate between two silicon wafers, as described by Nakagawa et al. (Napa Vallcy. CA proc.IEEE Workshop on Microelectromechanical Devices, page 89). This valve is schematically illustrated in FIG. 6. In this embodiment, the lower wafer and the glass plate have one or two inlet channels and may have outlet channels between them. The upper wafer may have a circular center platform and a concentric platform surrounding it. The piezoelectric stack base can be arranged on a central platform whose upper end is connected on the concentric platform by circular bridge means. The center of the SiO ₂ / SiN ₄ arcuate structure is connected to the piezoelectric element. The valve seat is made of nickel or other sealing material. In a three passage embodiment, the fluid is moved from the central fluid port to the outlet without providing voltage. When voltage is applied, the piezoelectric element compresses downward from the arcuate center to lift the end and blocks the central inlet to allow fluid to flow from the outer inlet. Alternatively, in the embodiment of the two passage embodiment, the fluid is flowed without applying a voltage and is limited when a voltage is applied. In another embodiment of the two way valve, the fluid is restricted when there is an applied voltage and is allowed to flow when a voltage is applied. In any of these embodiments, the piezoelectric stack may be perpendicular in the plane of rotation, may be inclined to the plane of rotation, or may remain in the plane of rotation.
In another embodiment, the fluid control is operated using a pneumatically actuated microvalve and the fluid channel is etched in one layer of material with the valve seat raised at the control point (a schematic diagram of this type of valve is shown in FIG. 7). For other layers, the corresponding holes are drilled by the laser to provide holes of sufficiently small diameter to provide a pneumatic access. In a second structure, the layer of silicone rubber or other flexible material is spun disposed. These structures are then joined together. Fluid movement is stopped by applying an air pressure that compresses by pushing the flexible membrane down on the raised valve seat. Valves of this type have been described by Vcider et al. (Sweden Stockholm, Aero. Sensor ⅸ pages 284-286). The measuring device manufactured by Vcider et al. Shows that the pseudo valve is completely shut off by applying 30 KPa above the fluid inlet pressure. This valve corresponds to 207 psig and can be adjusted by changing the diameter of the compressive orifice and the thickness of the membrane layer. Compressed pneumatic pressure is applied to the disk to actuate the valve as shown schematically in FIG. 8. Compressed pneumatic pressure is applied to the disc to actuate the valve as shown schematically in FIG. 8.
In addition, the pneumatic action is carried out by a microfabricated gas valve which uses a bimetallic functional device as shown in FIG. 9. This functional group may include an integrated resistance element that heats when applying a voltage, thereby deforming the diaphragm. This variant causes the functional structure to impinge on the valve on the valve opening to control the flow of fluid through the opening. These valves allow for proportional control based on voltage inputs, typically 0-15V DC. These types of valves are commercially available (Milpitas, Redwood Microsystems, Menlo Park., ICsensors).
An example of a pneumatically actuated membrane valve may be to integrate the components on a single disk or the compressed air outlet on one disk may be aligned with the second disk so that the compressed air acting orifice of the other disk. It can include two disks arranged to collide. In each embodiment, the pneumatic pressure source may be delivered to the disc by a concentric ring of material such as Teflon. In this embodiment, the upright core and the rotating element are adjacent to the disk. Pneumatic pressure is transmitted through the inner surface of the upright core and directed by the channel to the outer edge of the upright core. Properly arranged channels are machined into rotating elements and impinge on the channels in the upright cores and direct compressed air pressure to the gas valve.
Another embodiment of the invention is a pressure balanced microvalve as shown in FIG. This type of valve consists of three layers of material comprising two layers of silicon separated by a thin layer of electrically insulated oxide (eg silicon dioxide). The glass layer is coupled to the top of the valve and preferably comprises inlet and outlet ports. The central plunger in the form of a central silicon layer is deflected into a gap contained on the lower silicon layer by applying a voltage between the silicon layers. Optionally, the plunger is deflected by providing a pneumatic pressure drop into the gap in the bottom layer. Irreversible trimming of the micromachined portion may be prevented by applying Cr / Pt of the thin film layer to the glass structure. In electrostatically driven devices, this type of valve has many advantages, such as being able to be wired directly when manufacturing the disc. In this embodiment, the functional group is a fine adjustment device and it requires minimal input energy to open the valve even at relatively high pressures. These types of valves are described by Huff et al. (Seventh International Conference on Solid State Sensors and Actuators 1994. pages 98-101).
Other types of single use valves involving polymer relief valves comparable to disks are discussed below with reference to FIG. This valve acts on the basis of the relaxation of the non-equilibrium polymeric structure. This phenomenon is observed when the polymers are stretched at temperatures below their glass transition temperature (T) resulting in an unbalanced structure. When heated above the temperature T, relaxation and shrinkage of the polymer chains is observed as the structure is in equilibrium. A typical example of this phenomenon is the shrinkage of polyolefins (used in heat shrink tubing or sheaths), whose polyolefin structures are stable at room temperature. However, when heated to 135 ° C., the structure shrinks. Examples of PR valve polymers include, but are not limited to, polyolefins, polystyrenes, polyurethanes, poly (vinyl chloride) and certain fluoropolymers.
One method of making a PR valve is to place a polymer seat or to laminate over a channel (shown in FIG. 11) that requires the valve. The cylindrical valve is then cold stamped in such a way as to block the microchannel. This valve is acted upon by storing local heat, for example by contact with a laser or heating element. The valve is then retracted and fluid flow is possible.
Another type of microvalve useful for the discs of the present invention is a single use valve illustrated herein as a capillary microvalve (described in US Patent Application No. 60 /-filed Aug. 1996, which is incorporated herein by reference). . This type of microvalve is based on the use of rotating induction fluid pressure to overcome capillary forces. Fluids that completely or partially wet the material of the microchannels (or reservoirs, reaction chambers, detection chambers, etc.) comprising them are resistant to flow as they move from the narrow cross-section microchannel to the large cross-section channel, and these materials Fluid that has not wetted is resisted from flowing from the large cross-section microchannel (or reservoir, reaction chamber, detection chamber, etc.) to the microchannel with a small cross section. The capillary pressure is inversely proportional to the size of the two microchannels (reservoir, reaction chamber, detection chamber, etc. or combinations thereof), the surface tension of the fluid and the fluid contact angle on the material of the microchannel (or reservoir, reaction chamber, detection means, etc.). Change. In general, the detailed cross-sectional shape is not critical, but the dependence on the cross-sectional size results in significant capillary pressure for microchannels of size 500 micrometers or less. By varying the cross sectional shape, material and cross-sectional area of the components of the microsystem platform of the present invention, the "valve" is shaped to require the application of a specific pressure on the fluid to induce fluid flow. This pressure is applied on the disc of the present invention by rotating the disc (the disc is adapted to vary according to the square of the rotational frequency, the radial position and the range of the radial fluid as described above). By varying the position and range along the radial direction of the fluid of the fluid adjustment component of the microplatform of the present invention, as well as the capillary valve cross-sectional size, the capillary valve is operated in a rotationally dependent manner using a rotational speed of 100 rpm to thousands of rpm. It is formed to free. The device increases the speed of rotation in a predetermined monotonous manner to enable complex multistage fluid processes.
Control of the microvalve provided by the present invention is accomplished using a disc control element, a specific device controller, or a combination thereof.
6. Control device
Integrated electrical processing devices (generally “controllers” herein), including microprocessors and I / O devices, may be manufactured directly on the disc, or may be manufactured separately and assembled on or within the disc, or Preferably it can be arranged completely away from the disk as part of the fine adjustment device. The controller can be used to control rotary drive motors (speed, durability and direction), device temperature, optics, data acquisition, analysis and storage and to monitor the status of the system integrated with the disk. An example of a rotation controller is to use a rotation sensor adjacent to the motor itself to determine the rotational speed, and to use a motor controller chip (eg Motorola MC33035) for the drive direction and speed of this motor. Such sensors and chips typically use sensor data to control the rotation of the disk at rotation set points. Similarly, rotational data from these sensors can be converted from a digital train of pulses to an analog voltage using a frequency-to-voltage switching chip (eg, Texan Instruments Model LM2917). In this case, the analog signal provides feedback to control the analog voltage set point corresponding to the required rotational speed. The controller may also use data encoded on the data storage surface of the disc in a manner similar to that used in commercially available compact disc (CD) players. In these embodiments, this digital material read into the laser is used to control the rotational speed by means of a phase locked loop. Rotational speed information inherent in the data bit read frequency may convert the analog voltage as described above.
The controller can also include communication components, which can access an external database and use a modem for remote data transfer. In particular, the controller is integrally formed in the optical reading apparatus to recover the information stored on the disk and records the information generated by the analyzing apparatus on the disk in the optical data storage area integral with the disk. In these embodiments read and write operations are performed on the surface of the disc that is symmetrical to the surface comprising the microsystem components described herein.
(Instructions and data in the term " informatics ") The information allows the control of any particular microanalysis device on the disc and most preferably to a computer connected to the memory or device of the microprocessor or the disc device of the present invention. It is most desirable to be controlled by. This information is used by the controller to control the opening and closing states and timing of the microvalve on the disc, determine the optimum disc rotation speed, control the heating and cooling elements on the disc, monitor the detection system and generate by the disc. Integrate the collected data and implement a logical structure based on the collected data.
7. Power supply
The electrical necessity of the system contained on the disk can be transmitted to the disk by a brush that impinges on a connection integral to the disk. Optionally, the electrical connection is made by the point of contact between the hub, where the micro platform connects the rotating spindle or disk to the rotational inducing means. The battery is integral to the disc to provide a mounted electrical supply. The battery can also be used to power a device used to adjust a disk. Batteries used according to the invention are rechargeable, such as cadmium or lithium ion cells, or conventional lead acid or alkaline cells.
The power delivered to the disk can be either AC or DC. The need for electricity is determined by the particular analytical device mounted on the disk, but the voltage can be megavolts at microvolts, more preferably kilovolts at millivolts. Electricity can be supplied for the manipulation of the components or electricity is used to control and regulate the electrons on the disc.
Optionally, an induced current can be generated on the disc by the nature of the rotation and the current is provided by an induction loop or an electric brush. This current can be used to supply current to the device on the disk.
8. Detectors and sensors
Detection systems used in the microplatforms of the present invention include spectrometers, electrochemical, physical, light scattering, radioactivity and mass spectrometer detectors. Spectroscopic methods using detectors were improved by electrical spectroscopy (ultraviolet and visible light absorption, luminescence, and refractive index), vacuum spectrometers (IR and Raman) and x-ray spectroscopy (screw jet propulsion laboratory in Pasadena, California). X-ray fluorescence using microfabricated field emitters as described and conventional x-ray analysis methods.
The general case of each detection and representative example used with the microsystem device of the present invention is described below. This case is based on the center type (light basics and electrochemistry). In addition, the apparatus for performing detection using the detector of the present invention may be located outside of the platform either integrally or adjacent to the disk platform.
a. Spectroscopic method
1. Fluorescence Detection
The fluorescence detection system improved for use with the naked eye has been used in the prior art and is structured for use with the microsystem platform of the present invention. 12A and 12B illustrate two representative fluorescent devices. In Fig. 12A, an excitation circle such as a laser is focused on the light transparent region of the disc. Light from the analytical useful portion of the electromagnetic spectrum can be coupled to the disk material, which is particularly transparent to light of a particular wavelength and is emitted by the light, to a reagent or product occupying a reservoir in which the light spectral characteristics are indicated. To be determined by Optionally, the light at a particular wavelength is paired with a material having a refractive index feature of geometric shape that makes up the entire internal reflection of the emitted light. This is multiple refraction by the sample searching for the material on the disk surface by infinite light propagation or significantly increasing the path length.
A suitable arrangement for an infinite light wavelength device is shown in FIG. 12A (see Glass et al., 1987, Appl. Optic 26, 2181-2187). The fluorescence detector is connected to the waveguide of the disk to increase the detection efficiency.
In this embodiment, the optical component ahead of the detector includes a dispersing element to have spectral resolution. When the noise is not proportional to the passage length in the same way as the signal, the fluorescent excitation can be increased through multiple reflectors from the surface in the device.
Another form of fluorescence detection arrangement is illustrated in FIG. 12B where light of the fluorescence excitation wavelength and the emitted light wavelength is guided through one side of the device. 90 ° is used to separate the excitation and collection optical heat. It is also possible to use other angles, including 0 °, where the excitation and emitted light travel in the same line. Once the light source is classified from the fluorescence signal, any optical shape is used. Optical windows suitable for measurement by the spectrometer and transparent to the wavelengths used are included in appropriate locations on the disc ("reading" reservoir of the detection chamber). The use of this type of fluorescence in systems visible to the naked eye is described by Haab.
2. absorbance detection
Absorbance detection can be used to detect any analyte that changes the transmitted light by changing the specific absorbed energy (direct absorbance) or the absorbance (indirect absorbance) of another component in the system. The optical path is designed so that the absorbance detector is focused on the light path that receives the maximum amount of tube delivered from the illuminated sample. The light source and detector may be located outside of the disk. It is adjacent to the disk and moved simultaneously with the disk or integrated with the disk. The sample chamber on the disk consists of a cuvette, which is illuminated and transmitted light detected in a single passage or in multiple passages. In particular, it is used as an optical signal of the strobe to illuminate the detection chamber, which is the same frequency as the rotation frequency or plural rotation frequencies. In addition, the sample chamber may be a planar wavelength, the analytes interacting in terms of wavelength and the light absorbance is a result of reduced overall internal reflection (ie, the analyte may contain compounds having the analyte inserted or attached to the chamber surface). By specific binding, retreating from the surface of the sample chamber reduces the intensity of light).
Indirect absorbance can be used with the same optical design. For indirect absorbance measurements, the analyte does not absorb the light source. Instead, the drop in absorbance of the auxiliary material is measured as when the analytes are located in the same chamber. The increased transmission corresponds to the analyte concentration.
3. Vibration Spectroscopy
Vibration spectroscopic detection means are provided for generating data from the detection chamber or “read” section of the microplatform of the present invention. Infrared (IF) optical design is similar to the design parameters described above with respect to the visible range of the optimized component and electromagnetic spectrum instead of absorbance spectroscopy and infrared frequency in uv. To take full advantage of this, all materials in the optical path must transmit IR light. The placement of the optical components to provide Raman light to disperse the information is similar to that shown in FIGS. 12A and 12B for fluorescence measurements. However, due to the illumination time required to generate the signal, the rotational speed of the disc must be slowed down. In some cases, the IR or Roman scattering analysis, which depends on the use, is most preferably performed offline in a separate IR or Raman instrument suitable for analysis of the disc of the present invention.
4. Light scattering
Turbidity can be measured on a disc. The optics are arranged as in absorbance measurement. In this analysis, the intensity of the transmitted light is related to the concentration of light particles dispersed in the sample. An example of this type of detection method is particle aggregation analysis. Larger particle precipitates in the rotating disk move faster than smaller particles, and the turbidity of the solution in the sample chamber before or after rotating the disk is related to the size of the particles in the chamber. Turbidity measurements can be used specifically to detect the presence of analyte in the sample chamber if small particles are to be aggregated only in the presence of the analyte. For example, the small particles may be coated with the antibody from one or more particles to the analyte resulting in the aggregation of the particles in the presence of the analyte such as the antibody binds to the analyte. After the disk rotates and interaction occurs, the sample chamber contains analytes less confused than the sample chamber containing no analytes. The system can calibrate the standard amount of analyte to provide analyte concentrations associated with turbidity of the sample under standardized conditions.
Another form of light divergence detection method is provided for using the microsystem platform and apparatus of the present invention. Light source Preferably, the monochromatic light from the laser light source is directed across the cross section of the flow channel on the disk. Light scattered by particles in a sample, such as a cell, collects at some angle over the illuminated portion of the channel. Data reduction is designed directly into the device based on standard values, such as beads of appropriate size, to relate the signal to interpretable results. Using the sized beads, finer identification between particles of different sizes is possible. Still other applications of the system include flow cytometry, cell counting, cell sorting, and cell biological assays and tests, including chemotherapy sensitivity and toxicology.
b. Electrochemical Detection Method
Electrochemical detection requires contact between the sensor element and the sample or between the sensor element and a material such as a gas in equilibrium with the sample. In the case of direct contact between the sample and the detector, the electrode system is built directly on the rotating disk and moves in contact with the disk after rotation is complete or after the rotation is complete. Detectors constructed using gas vapor in code information about a sample can be manufactured with a detector external to the disk provided in the gas vapor disposed in contact with the sample chamber and the detector. The electrochemical detector may include any electrochemical transducer that is compatible with the potentiometer, voltmeter, and ammeter device and compatible with the materials used to form the microsystem.
1. Electrical potential measurement
One form of microsystem platform and useful electrochemical detection means of the present invention is an electrical potential measurement system. The system provides a means of characterizing the interfacial properties of a solution passing over an activated flow channel in the device. Depending on the temperature control characteristics of the microplatforms of the invention, the flow potential can be measured in the device. To generate the flow potential, the voltage potential difference between two platinum leads in contact with the solution inside and outside the disc is measured in comparison with the reference electrode. As the fluid flows under controlled centripetal movement through the channel, the flow potential is in fluid interaction with the disk surface in the field of movement.
Optionally, platinum electrodes are used to generate electroluminescent ions. At this time, chemiluminescence is detected using one of the above-described optical detectors depending on the chemiluminescence signal. Volatile current components are also useful on the microsynthetic platform of the present invention to generate reaction media or products.
2. Electronic chemical sensor
The electrochemical sensor is preferably included inside the disk. In one embodiment, an electrochemical detector is provided for using a reducing circulation reaction. This embodiment utilizes microalignment electrodes that are interlocked within a micromechanized chamber containing a species of interest. The potential of one electrode is set at the oxidation potential of the material of interest and the potential of the other electrode is set at the reduction potential. The volume of fluid containing the material of interest is directed to the chamber. The electrochemical reversible material is oxidized or reduced by circulating the electrode. In this embodiment, the molecule is detected by an apparent increase in the reduction current. Since the irreversible material is not converted to the signal after the first cycle, the overall distribution of the final signal is suppressed. Data analysis software is used to suppress signals due to irreversible materials.
In another embodiment, the multichannel electrochemical detector is provided with 16 lines of electrodes fabricated in a chamber having a dimension between 50 μm and 100 μm between lines (see Aoki's Analytical Chemistry 62 2206, 1992). In an example, the fluid volume containing the material of interest is directed to the chamber. Within the chamber, each electrode is set at a different potential so that electrochemical measurements of 16 respective channels are made. In addition, each electrode potential is removed step by step by the action generator. This document leads to information pertaining to the reduction current and the reduction potential of the substance. This form of analysis identifies the molecules via the voltammogram.
c. Physical method
Physical detection methods are also provided for using the disc of the present invention. For example, the disc is used as a viscometer. The microchannel containing the fluid to be tested preferably contains beads inserted on the disc. Bead movement through the fluid is analyzed and converted into viscous data based on standard values stored and stored in microprocessor memory.
Another embodiment is a capacitive pressure sensor (see, Micro Electromechanical System 11 43 by Esashi, 1992). In this embodiment, the silicon and glass substrates are anodized and bonded with a sealed reference cavity. The pressure can be detected by a capacitive change between the silicon barrier and the aluminum electrode formed in the glass. The capacitor-to-frequency converter output of the CMOS circuit can be integrated on the silicon substrate or included to control the electrodes of the disk.
With the preferred arrangement of the pressure sensor, the pressure due to the centrifugal force can be determined at any position on the disc. Regarding the microchannel diameter information and the direction pattern of the channel on the disk, the pressure data can be used by the microprocessor to adjust the disk rotation speed to control fluid movement on the disk.
Surface acoustic wave (SAW) devices are also provided as part of the micro system platform of the present invention. The device may be located on a disk or included in a fluid channel above the device to detect headspace gas. When placed in a fluid system, SAW is used to detect density changes in solution as an indicator for changing the buffer or reaction composition (see, Analytical Chemistry 61 by Ballantine, 1989).
Blocked in the head space surrounding the disk or volatile gas on the disk can be measured in various ways. For example, a Clark electrode placed in contact with a gas solution on a disk is used to detect oxygen concentrations (see Analytical Chemistry 62 by Collision, 1990).
d. Radiation detection parts
The microsystem platform of the present invention may comprise a radiation detector. Radioactive decay of analytes or synthetic products on disks of the present invention can be detected using CCD chips or similar channels capable of integrating signals over time. Alternatively, radioactivity can be determined directly by placing a solid state detector in contact with the radioactive analyte (see nucleic acid 22 2121-2125 by Lambture, 1994).
Modular structures
The analyte system provided as part of the platform of the present invention consists of a controller, detector, buffer and reactor, chamber, microchannel, microvalve, heater, filter, mixer, sensor, and other components. The components that make up the analyte system on the disk are one or more fully integrated systems completely fabricated on the disk, fully integrated systems fabricated on the disk or made of parts, components fabricated directly on the disk and manufactured on the disk or assembled on the disk. A secondary set of parts connected by an auxiliary set of parts, consisting of parts connected to the disc outwards through a simultaneously rotating disk, and parts connected to a rotating disk from a fixed position in relation to the disk (eg a rotating spindle) do.
Method and use
The present invention provides numerous possible applications and examples due to their adaptability. Any characteristic is common to most embodiments, but the characteristics are the same collection, sample application to disk, sufficient testing at sample application, various specific analyzes performed on disk, data collection, processing and analysis, data transfer and This includes storage, memory at a remote station or communication section using communication software, data output to the user (including printing and screen display), and sample disk processing (including disk disinfection if necessary).
Samples or analytes are collected using the appropriate means for the particular sample. For example, blood is collected into vacuum tubes using lancets in the hospital or laboratory for home or consumer use. The urine is collected in a disinfection container and applied to the disc using conventional liquid delivery techniques. Saliva is applied to the disc by diluting with a small amount of distilled water solution, mild detergent and sugar water. The solution is used for gargles to detect antigens, biological secretions and microorganisms. Optionally, small bags made of fishnet polymer material containing detergents and chewing resins can be chewed to the user to promote the secretion of saliva, which is removed from the mouth and the saliva is regenerated and applied. Amniotic fluid and sap can be collected with medical knowledge by outstanding talents as needed.
Environmental and industrial samples are collected from groundwater or factory wastewater into containers made to prevent leach contaminants in the samples. Soil samples are collected and mixed with a solvent designed to dissolve the analyte of interest. Industrial applications such as pyrogen screening are carried out using specially designed sample vessels.
The sample or analyte is loaded on disk by the user. The sample is selectively loaded onto the disc at a location close to the center of rotation, with the result that a large amount of centripetal force is applied to the sample to maximize the number, length, or arrangement of fluid handling components available to interact with the sample. Provide the widest path across the surface.
The plurality of samples can be applied to the disc using the plurality of loading apparatuses shown in FIGS. 13A to 13C. In embodiments of a plurality of loading apparatuses, the various pipette cylinders are equally spaced and arranged radially. The pipette is spaced apart to provide the tip portion of the pipette to fit snugly into the inlet port on the surface of the disc. This tip is a simple pin shape that uses a combination of surface and fluid properties to maintain a specific volume of sample. Optionally, the tip is a cavity tube, such as a capillary or plastic tip, and a manually controlled fluid that responds to positive or negative pressure, such as a manual or automatic pipette device. The loader can be operated automatically or manually.
The cylinder can be arranged in a flexible assembly, and the tips can be arranged linearly in one arrangement and in a radial arrangement. In each embodiment, the loader consists of an alignment device to identify the reoccurrence location of the loading tip on the disk of the present invention.
The loader is specially designed for the material to be studied. Examples include medical use (samples include amniotic fluid, sap, pleura, pericardium, peritoneum, body fluids including semen and synovial fluid, blood, sweat, saliva, urine and tears, tissue samples, and excretion). The loader device is preferably compatible with standard blood manipulation devices such as vacuum tubes that fit into the septum. The septum loading device is also compatible with sheet collection devices such as lancets by obtaining small amounts of blood samples. The disc has an integral lancet and rubber seal to sample the blood directly.
Static and dynamic loading of the disc is envisioned within the scope of the present invention.
Micro-manipulation devices for treating the platform to form a combination of the microsystem platform as described above and to exert a centripetal force on the fluid on the platform are selected and located on a disk or device. Mechanical, electronic, opto-electronic, magnetic, magneto-optical, and other devices are included within a disk or disk surface. Some devices between the disks have been described above in detail, and additionally, the disks include electronic circuitry, including microprocessors for the disk function co-operation, and devices in communication with the disk operating devices and other devices. The disc may optionally include a detector, sensor, or component of the device and an energy source (such as a power source for electrochemical systems, an electromagnetic radiation source for spectrometer systems), or valves, channels, and other fluid compartments. Detectors, sensors, actuators, communication and data processing devices, including electromechanical (laser, infrared, radio frequency (RF), microwave) electrical or other means, including mechanical, electrical, and electromagnetic devices for controlling fluid movement on the surface; Optionally transparent materials that facilitate data generation and operation using circuits designed to control procedures and processes on disk, including intermediary communication between player / reader devices, system diagnostics, analytical protocols, and analysis of analytical data. Contains materials. It is available in the form of ASICs or ROMs designed only at the manufacturing location, FPGA's, EPROMs, flash memory (UV-erasable EPROMs), or designable IC arrays, or similar devices that can be designed by the user through platform controls or other devices. do. Also included in the components of the present invention are assembly RAMs that can be designed in high-level languages or assembly language that can be designed through CPU and microprocessor units and components that communicate with other devices in communication with disk communications, remote displays or data analysis systems. .
Off-disk devices include microplatform operating devices and other devices that can read information in proximity to the information and initiate processes on the disk. Figure 15 is a division of the apparatus and auxiliary devices which are part of the micromanipulator and the interactive parts "interaction" means the exchange of "data" between the disk and the device or the parts of the device itself. The relationship will be described by the detailed embodiment of the parts.
It includes mechanical drive and circuit design for rotational monitors and controls, comprehensive system control, data read / write devices, external detectors and actuators for use with discs, dedicated data and analysis processors to withdraw data display and data analysis, A central processor unit, a user interface, means for communicating with the disk, a user, and other devices. Mechanical drive and associated circuitry includes devices for precisely controlling and measuring the rotational speed and angular position of the discs, and devices for selecting or mounting cassettes, turntables, or other plurality of disc storage devices. The system control unit provides a comprehensive device control, predesigned or accessible device to the user interface. Disc data reading / writing apparatus is provided for reading displayed information from a disc or other medium. Optionally, the record-disc output includes analysis data resulting from the analysis in which the disc section was performed on the disc. This option is undesirable in the use of the disc, which is contaminated with a biological or another hazard, such as absence of means (such as sterilization), to neutralize the risk. The device is an optical, magneto-optical, magnetic and electrical component for actuating components of detectors and sensors and microvalve and initial processes on the disk that operate in conjunction with external detectors and sensors or other components on the disk, including analysis and diagnostic devices. It also includes. Such aspects of the disk micromanipulator are illustrated in FIGS. 14A and 14F.
The disc data processor is preferably embodied in the apparatus of the present invention that enables processing and manipulation of recorded disc data. The part includes software used by a micromanipulator CPU, program circuits (such as FPGAs, PLAs, etc.) and dedicated chipsets (such as ASICs). An analysis processor is provided for processing data arising from events and analysis performed on a disk, detected by an external detector, and communicating from on-disk components. The apparatus also includes a central processing unit or computer in which disk data and analysis result data analysis is processed (via the preliminary program), and conventional computer output (word processor, graphics production, etc.) may be provided.
User interfaces including keypads, light pens, monitors, indicators, flat panel displays, interfaces with communication options to host devices and peripherals, printers, plotters, and graphics devices are provided as part of the microplatform of the microcontroller of the present invention. . Communications and telecommunications include standard hard-wired interfaces (such as RS-232 and IEE-488M SCSI buses), infrastructure-red and optical communications, short or long distance telecommunications ("cell" remote radio frequencies), and manual or automatic Provided via internal or external modem for telephony.
The disc information includes software recorded on the disc to facilitate the microsystem test operation structured thereon and to analyze data generated during use of the microsystem by the user. Disc information includes information recorded on the disc (such as optically coded data) and disc-specific information (such as the current state of the valve, accessible by magnetic pickup or by the reflectivity of the coating material at the valve-position). Include. Data recorded on the disc may include, but is not limited to, audio / video / test and machine format information (eg, binary, binehex, assembler language). Such data may include information about the disc configuration, the identity of the disc, the license, the analysis protocol and programming, the protocol description, the diagnostic and test results, the point of use of the information, the analysis result data, and the background information of the control program that analyzes or rotates the disc. Contains system control data used for initiation. The acquired data information may be stored analog or digital, and may be raw data, processed data, or a combination thereof.
System control data includes data relating to the physical variables of the disk and tuning data that allows micromanipulation at precise angular velocity and acceleration. Disk configuration and compatible data includes data that takes into account the type of disk used to determine the adaptability of the desired test protocol (configuration of on-disk devices, valves and reagents, configuration of reaction and detection chambers), and such data includes the type of disk It provides the functional identity and capacity of the disk. It may also form part of an interactive feedback system for checking microsystem platform factors prior to commencement of disk testing. Disc identity and sequential numbers are coded and provided on each disc to enable exact identity of the disc by production date disc type and usage rights, and the data is encoded by the producer and user information, which information is provided by the user. Are written to disk. Further, the history of the process executed by the user with the disc is included in the disc data. Also included in the disk data is the history of the execution process with the disk, which is typically recorded for information recorded by the user and machine recognition (how many and which tests were or were not used).
30 to 32 show the operation of the software encoded on the disk used for controlling the apparatus for driving the disk. 30 illustrates a process flow. The control program, which is encoded as data on the disk, is recorded via conventional means, for example by means of an optical storage medium (such as a small disk or a "laser vision" disk) and stored in a random access memory (RAM) of a micromanipulator. In the usual manner. This program is then executed. In some applications, running the complete program will run automatically without any real interaction by the user. In other applications, the user will have various options (typically a menu) to run the program. For example, the user's choices such as whether to perform full or limited diagnostics, test processing, analysis, or other disk functions, or to determine how the test results are output and the degree of detail are provided through the user interface.
31 and 32 show one particular set of programmed steps for performing a test using the capillary microvalve described above, with another arrangement of steps in the program signaling for example to activate other actuators and microvalves. To demonstrate one of the common techniques, it is easily integrated. The program consists of blocks, in which different rotation rates are set during the change time, allowing for capillary valve adjustment, mixing, and latencies, and adjusting the spindle motor through (eg) vibration acceleration and deceleration. Blocks are possible but not shown. These program blocks consist of output commands for changing electrical devices (motors, detectors, etc.) and reading data from the device to yield measurements of the device and process conditions. If the condition is "bad" (eg, the motor cannot reach the proper speed, the neighboring device is not close, and no power is detected in the light source for the spectrometer's measurement), the facility for stopping the program is placed in the program. It is equipped. This state may lead to a program stop (as shown) or send the program back to the user for another mechanism via the interface.
The program acquires data, analyzes data. And a data output block additionally. The particular acquisition process involves the use of the symbolic signal on the disc for the data acquisition means (eg an optical signal associated with the detection chamber via the detector). In this way, data is acquired for a certain time when the detection chamber is near the detector. It is also possible to use the features of this data to obtain data continuously and to determine which parts of the data are useful for analysis, for example, the shape of the non-signal can look like a square wave for the arrangement of windows on a transparent disk. It is like a function of time. Data analysis includes endpoint analysis (data at endpoint time for reactions) or internal return of data as a function of time, nonlinear rectangular fitting as well as other methods. The data output may be in the form of a user interface, a "yes / no" answer to numeric data, stored in an internal or external storage medium.
Not all elements of these programs need to be included on disk. For example, a program can reside in a computer and is designed to be written to a disc to obtain the rotational speed profile required to use the disc. All other aspects of the program, such as when and how to read and analyze the data, can be part of the program to be applied and read from other media.
Analysis / reading protocol data is a description of the analysis and testing that can be performed on disk. Such data may be simplified to the title given to the disc and may include a detailed description of the disc usage, data analysis and handling, including test protocols and data analysis protocols. Analysis / test protocol programming can be used as system-specific subroutines in more general software configurations, or provided that the device can be supplied with programmable logic to perform desired analysis. Analysis / protocol descriptions are provided as audio, video, text, or other descriptions of the analysis process performed on the disc, including background information, status for use, prevention, and other aspects.
Symbolization and validation data / programming is provided to ensure the safety of programming and data generated from the analysis performed by the disk. The signing and decoding routines are used to restrict access to data contained on disk. Such routines are also used in the field of medical diagnostics.
In addition, system self-diagnosis is provided. System diagnostics include diagnostic test results for other factors, thermal factors, valves, reagent chamber states, and detector functions, recorded to disk or stored in disk memory by the separator used in the diagnosis.
The point of use information is symbolized on the disc at the point of use (eg, sample loading) in the form of a textual image, video, or audio including position, time and composition. In addition, test result data recorded by the disc executor / reader or by its own disc at the time when this procedure is executed is included at the time of use information.
Certain data is native to the disk and accessible through the microcontroller. These include sample sufficient test data to record the presence of samples or reagents in other fluid handling areas of the disc and in appropriate reservoirs, and are accessible through external detectors and sensors. The valve status is also recorded including a record of changes in the valve status during the procedure performed in the disc. The valve state is determined using, for example, a magnetic pickup in the device applied to the magnetic valve mechanism, and the state is visible to the naked eye through an optical window on the disc. The presence of radioactive, chemical or physical contaminants on the outer surface of the disk may be recorded upon detection by a sensor comprising a device, which is optically concluded in a message conveyed to a user interface such as a display or output.
Disc data and information are stored using a variety of media, including recording media of disc material (ie, optical reading discs, in particular read / write CD-ROMs) by means of their own devices using electrical factors. Information is encoded using conventional or advanced techniques used for storing computer information. Video, audio, and text information is digitized using methods improved by digital video, audio, and computer equipment. Analog signals from test processes, such as those observed in photoelectric distribution tubes or photodetectors, can be converted by analog-to-digital conversion, or low or amplified through process jackets for off-disk or off-device May be supplied. Various embodiments of a disc operating device according to the present invention have the ability to use data that can only be read from any of these media forms or to read and write data from a disc. The sign and authentication code can be used for stability purposes. Disc data storage media include audio CD, CD-ROM, and optical media that utilize reflective / non-reflective planes and holes on the surface, using techniques adapted from “laser disc” technology and barcodes. In addition, magneto-optical and magnetic media are provided using the internal arrangement of electronic elements (FPGAs, PLAs, EPROM, ROM, ASICs, IC networks) for information processing. In addition, chemical recording means comprising simple chromatographic staining of the detector or chamber of the device provides a simple optical record of the test results. Such simple chemical recording means provide a home-diagnostic method without the need for expensive, more complex devices in capacity than simply needed to determine the arrangement depending on the presence of a chemical recorder.
Software and communication
Software that provides information and instructions for microsystem execution, property control, data acquisition, handling and processing, and communication is within the scope of the present invention. For the purposes of the present invention, such software is called "machine language command". Control and analysis software is provided in high-level languages such as C / C ++, Visual Basic, Fortran or Pascal. The driver is provided for an interface board (inside the device or inside the host computer that interfaces with the device) that translates commands on the host computer's bus into micromanipulator commands. In addition, drivers for experimental-control software, such as LabView, may be created corresponding to using conventional industry standard interface protocols. Such applications can run on a number of popular computer platforms including Unix / Linux, X-Windows, Macintosh, SGI, and the like. For example, test validity can be guaranteed (at least in part) through the use of a ROM-based test procedure where all programming is performed at the time of manufacture without the possibility of end-user modulation. In addition, discrete application software can be improved so that data from disk-executors can be analyzed on an uncontrolled platform using useful applications (such as Excel, SigmaFloat, Oracle, Sybase, etc.).
Because some areas of disc technology disclosed herein relate to important issues related to human health, disc diagnostic software analyzes the diagnosis of discs, factors (samples, reagents, devices), operators, and analysis software to ensure the validity of the results. You should be able to. The type of information used by such diagnostic software may be sufficient for sample sufficiency and leakage, verification of disk format and software / test procedure compatibility, on-off disk software testing, feasibility, on-disk and off-disk sensors and detectors. Location and functionality, operator communications and microprocessors, microprocessor / CPU diagnostics, power stability, and more.
Diagnosis of mechanical electronic factors is carried out in a manner familiar to those skilled in the art. Software self-diagnosis is accomplished by using a checklist / verification of software routines or subroutines to detect compatibility with system hardware (from micromanipulation devices or disks) or with other elements of system software.
Sample-related disk diagnostics include analysis of runoff, sample sufficiency, and reagent sufficiency, and the type and nature of the assay to be performed. Device-related disk diagnostics include checking of detector / sensor functions, electronic factor self test, valve control, and thermal control test. Software diagnostics provide self-tests, tamper protection, read-only and read-write tests of software components coded in a disc or device. In addition, the disk format is checked using disk diagnostics so that the disk format and analysis type are read exactly and in the same state as the protocol maintained in the device memory.
On-disk software includes useful read-only software, such as ROM, in particular CD-ROM, for diagnostic, analysis control and data analysis. Read-only software is designed for specific procedures and processes that cannot be altered and ensures the safe use of tamper-proofs and the useful use of discs. In addition, software may be embodied in a symbolic medium (such as optical, magnetic, etc.) or alternative media (such as a barcode). Reprogrammable software (such as FPGAs, PLAs, EPROMs, or IC analysis) may be reprogrammed by a device designed for this purpose or by a disk micromanipulator. Similar kind of software is optionally provided on the device. In other cases, a user interface is provided through the display, contact pad elements, and / or keyboard of the device.
The application is provided in a read-only or reprogrammable software format. The above components of the fluid microcontroller according to the present invention include software that can be read from standard computer storage media. Examples include medical or analytical diagnostics of forestry and online services such as newsletters and newsletters, including pattern recognition, static analysis software, etc., or integrated databases accessible from network workstations or contained within disk or device memory. Include the program.
The integration of control and application programs can be made either by adaptation of the existing OS or through the use of unique operating systems developed for micromanipulators and discs according to the invention. Optionally, the OS uses production software to combine text, graphics, video, and audio to facilitate the use of a "point and click" system. In addition, the OS provides a fax or goal-adaptive environment for custom programming (e.g., LabView-based systems) by sophisticated users, while providing independent software developers or disk reader / executor authors. By providing additional software improvements.
In addition, the OS may be selected to allow the design of a disk-based analyzer. Mechanical designs, including simulations of rotational hydrodynamics and stability and flow simulations, are included in the disc design software package.
The communication aspects of the present invention include hardware and software embodiments related to remote control and analysis or input and output data from a user. Features of hard-wired communication include local buses (e.g. VGA buses for video signals), conventional hard-wired interfaces (e.g. RS-232, IEEE-488, SCSI buses), Ethernet connections (Eithernet) connections, Appletalk, and high-speed data-video or image-transmission and communication over various local area networks (LANs). The telecommunications device includes a cellular transceiver for short range communications, a microwave transceiver for telecommunications, and an internal or external modem for manual or automatic telephony. In addition, video input / output ports, analog output lines for data transmission, input jackets for input of analog signals from other devices, and optical and infrastructure-red communication ports are provided for communication with peripheral devices.
Configuration of Micromanipulators for Specific Applications
Micromanipulating devices include various combinations of hardware and software as described above. 15 illustrates a general combination of communications, devices, detectors, and control devices within a device. Certain applications may not have features, for example a portable device may not have a visual user interface. Micromanipulating devices include, for example, devices comprising computers, printers, and image-processing facilities, or hosts for peripheral elements such as control pads, data entry / read-output devices (such as Newtonian devices, etc.), or integrated systems. It can be a peripheral or "independent" device for a large set of devices. The apparatus in all embodiments includes hardware for rotating the disk at a fixed or variable rate and a system for monitoring the rate of rotation. The apparatus also initiates sample and disk diagnostics, executes "external" tests and detections described above, initiates sample and disk diagnostics, executes "external" tests and detections described above, and implements specific actuators such as valves. May include a device for initiating on-disk diagnostics, reading symbolic or other data / information storage medium information and disc specific information in the disc, and in some devices, writing information to the disc.
Additional elements within the device, including system controls, data processors, arrays of analysis processors, external detectors, external actuators, analysis outputs and data output lines, communications, and software are specific devices and / or applications.
For example, in a "use point" portable or home application, sample storage is accompanied by the operator's program initiation. System control can be provided by an indicator or front panel controller that can access various programs stored on a disk or device. Such " hard-wire &quot; programs utilize controller circuitry to read or read / write to / from disk or memory and / or to run tests using external devices. The apparatus may be designed for the execution of a single procedure or may be a preliminary program that executes multiple embodiments or a set of procedures of the same procedure using a single disk. Device operation is visually obtained by pressing a single button. Such procedures and data processors of this kind of device include chipware and circuitry designed to process analysis data (analysis processor) and symbol data (data processor). Information from such a processor may be useful for output to the user on the front panel or video display, and may also be used internally to ensure correct operating conditions for analysis. This internal information processing is based on the results of system diagnostic tests to ensure disk identity and test type compatibility, the presence of reagents and samples determined by the adsorption of light through a detection port that inspects reagents and sample chambers, and prior to initiation of the test. The presence of contaminants detected and the results of self-diagnosis on external detectors and actuators. These results are used by the system controller to determine if the required tests can be run.
After loading and activation, the analysis results can be encoded on disk or stored internally in electronic memory. The results of this analysis and procedure are then sent to the front panel display (flat panel LCD, etc.) using an appropriate video driver. The processed analysis data can also be sent to one of a number of standard digital I / O systems, including RS-232, RS-232C, IEEE-4888, and other systems similar to digital I / O and interfaces. The low analog signal can also be connected to one or more outer jackets for off-device storage or processing.
An embodiment of the least technically sophisticated device is a portable device which is no larger than a portable audio CD executor consisting of a selector, controller, or disk drive for a programmable or pre-programmed angular acceleration / deceleration profile to limit the number of procedures. Such devices are beneficial for toxic chemical / pollution test aspects. The analysis for testing enters the disc, the disc is inserted into the executor, and the appropriate program is selected. The analysis results
It is immediately stored on disk that is displayed to the user and / or read later by the large launcher / reader. In addition, the results are stored as intrinsic state of the indicator (eg, positive / negative state of litmus paper in different cuvettes) without analysis or other data collection performed by the device. Such data may be accessed by a large launcher / reader or by other means outside of the workplace environment. Information about the location, time and other status of sample collection is entered via the user interface.
Another embodiment is an independent device with high performance communication capabilities and a high degree of functionality. Typical applications for such devices are home blood analyzers. This device is used by dropping a drop of blood onto the disc, inserting the disc, and starting the analyzer, preferably by pressing a single button. Next, one or more analytical procedures are performed. Analysis data is sent to software that performs the required analysis on disk or in a device. In addition, the device may be permanently or temporarily attached to a home telephone line and automatically transmits low or reduced data to a central computer used to analyze the transmitted data, thereby allowing existing and / or standard data obtained from the same patient to Compare, and maintain a permanent record as part of the patient's device to confirm receipt of data for data analysis, and suggest measures (such as communicating with a physician).
The desktop peripheral / host application station consists of a device as described above capable of receiving commands or responding from a host computer by one of a number of possible data protocols. The system can act as a host or transfer data to peripherals or other network devices and workstations. In addition, remote access to the preliminary program function, reprogram function, and real-time control capability are provided.
Another embodiment of this application is a centralized or peripheral execution / reading device associated with software located in a nursing home of a hospital. As the test is run on disk, the information is relayed to the physician by telephone, fax, or pager via a short range transceiver. The identity of the patient can be entered simultaneously with the collection of the sample by using a light pen and barcode attached to the device that clearly provides the patient / sample identification.
The apparatus also provides an interface by an integrated computer having the above-described performance and functions, and features such as high resolution graphics, image processing, and the like. The computer controls the device performing the above-described functions for the peripheral system while the physical integration greatly increases the data transfer rate.
Example
In addition, integrated systems are provided with large-scale analysis software, background databases, and information. Carousal disk-storage cassettes are also an advantageous feature of such systems. This type of integrated system is useful for large scale assay devices.
Standalone systems are useful in isolated environments. Examples of such systems include devices used in isolated or climatically adverse spaces such as air, water, and soil testing devices used in the Arctic for environmental protection purposes.
In accordance with the present invention, the microsystem platform also allows samples of other assays such as mass spectrometers, gas chromatographs, high pressure liquid chromatographs, liquid chromatographs, capillary electrophoresis, conductive plasma spectroscopy, and X-ray absorption microstructures. It is useful for manufacturing. In some applications, the finished product is removed from the disc to be analyzed.
Samples may be preaggregated and washed on the device by an aqueous anomalous separation system. This can be done by mixing two phases that are separated from each other by thermodynamic differences, such as polyethylene glycol (PEG) and dextran, for example, wherein the biopolymer is usefully separated by this method. Optionally, environmental tests such as color analysis can be enhanced by integrating cloud-point separation to focus and enhance the optical signal. In addition, small scale reverse current chromatography can be performed on the device (see Analytical Chemistry, 1991, p. 63). Centrifugal forces on the disk can be used to force forces of different densities to flow against each other, thus allowing the components to be separated by increasing or decreasing the density to develop a chromatogram.
Application and use
The microsystem platform and micromanipulators that make up the fluid engineering micromanipulator of the present invention are used in a variety of microsynthesis and microanalysis applications because of the flexibility of the design, where the fluid is synchronized on the platform by centrifugal forces that occur when the platform is rotated do. The short and typical samples of the following applied forms are within the scope of the instant invention, which does not limit all embodiments of the present invention.
The present invention is advantageously used for microanalysis in the field of biological research. Such microanalysis includes immunoassays including polymerase chain reaction, ligase chain reaction, and magnetic chain reaction. Molecular and microchemical analysis, including enzymatic digestion restriction and DNA fragment size separation / division of DNA, can be accomplished using the microsystem discs of the present invention. Microsynthesis control, such as DNA fragment ligation, replacement synthesis, radiolabeling, and fluorescent or antigenic labeling can be performed using the disks of the present invention. Nucleic acid sequencing using various synthetic protocols using enzymatic replacement synthesis of DNA can also be performed, in sequence using software modified from software that is very useful for macroscopic and automated DNA sequencing machines, where the digestion and analysis of DNA fragments of a single strand is very useful. The discs can be separated on the disc to be identifiable and arranged. Other applications include microbiological applications including pH measurement, filtration and ultrafiltration, chromatography including affinity chromatography and reverse phase chromatography, electrophoresis, microculture, pathogen identification, cytometry, immunoassay, and Conventional experimental methods performed at the macroscopic scale.
The example shown is an immunoassay. While there are a number of experimental methods for detecting antigen / antibody responses used in current research and clinical trials, the most robust immunoassay protocols include "sandwich" analysis. In this assay, non-flowable antibodies are given as samples to be tested in the antigenic assay. Second antibodies with different epitopes of the same antigen are immobilized to form a “sandwich” of the antigen between two immobilized antibodies in succession. In this assay, the second antibody is linked to a radiolabel or fluorescent label, or a removable moiety such as enzyme or catalytic functionality. For example, horseradish peroxidase or alkaline phosphatase is used to change the color of the substrate, the strength of which depends on the amount of the second antibody immobilized on the sandwich.
One example of a disk suitable for conducting such an immunoassay is shown in FIG. 17Q. In this embodiment, the second antibody is linked to alkaline phosphate (AP). The presence and amount of AP activity is measured by monitoring the conversion of one of the following substrates by the chromatic enzyme. B-naphthyl phosphate is converted to an insoluble azo dye in the presence of diazo salt, i.e. 5-bromo-4-chloro-3-indoli phosphate is 5.5-dibromo-4- in the presence of copper salts. It is converted to 4-dichloroindigo, or 4-methyllumbeliferilyl phosphate is converted to 4-methyllumbeliferione which emits light of 450 nm.
In one embodiment, the reaction chamber comprises an antibody against an antigen, which antibody does not flow by being absorbed into the reaction chamber. Contact with the reaction chamber is advantageously placed in a reagent vessel containing the second antibody, which is identical to an enzyme such as alkaline phosphate. Samples that may contain the antigen identified by the antibody are mounted at the inlet. The disc is rotated to introduce the sample into the reaction chamber containing the non-flowing antibody, and then after a sufficient time, the second antibody is introduced into the reaction chamber to saturate the non-flowing antibody with the amount of antigen present in the sample. Optionally, the sample may be in contact with the second antibody while reacting with each other, and then introduced into the chamber. Incubation of the sample with the antibody is done without spinning for about 1 minute. After each incubation, the wash buffer is rotated from the buffer vessel into the reaction chamber to remove unfixed antibody. In alkaline phosphatase assays, 2 mg / mL o-Dianisidine aqueous solution, 1 mg / mL B-naphthyl phosphate buffer in 50 mM boric acid / 50 mM KCl, pH 9.2, and 100 mM magnesium chloride are delivered to the reaction chamber in an appropriate amount. The amount of enzyme-secondary antibody binding is measured by detection of purple light precipitates using a photodiode or CCD camera.
An immunoassay disc is shown in FIG. 17r.
In an alternative embodiment of the immunological analysis of the present invention, the present invention is directed to determining and measuring the presence and amount of particular cell or cell fluids, most preferably biological fluids such as blood, urine, amniotic fluid, semen, and milk. Means are provided. In this embodiment of the present invention, the microsystem platform includes a solid surface on a chamber or disk to selectively couple specific cell or cellular fluids. After attaching the cell to the surface, nonspecific binding cells and other components are removed using fluid flow (cleaning) or centrifugal force (including inertial flow of fluid corresponding to the centrifugal acceleration of the disc). Cells still attached to the microplatform surface or chamber are detected and metered using means not limited to microscopic means, spectroscopic means, fluorescent means, chemiluminescent means, or light scattering means. The present invention also performs toxicity monitoring, such as metabolic monitoring, on cells that are attached to a particular surface to determine the efficacy of an energetic or other drug product. The ordered arrangement of these surfaces is provided in certain examples to fully measure purity, infertility, and cytomegalovirus containing biological samples.
The surface or chamber of the disk for joining a particular cell or cellular fluid is prepared to provide a specific binding site. Generally, an antibody, preferably a single cell antibody, is attached to a surface or chamber, which antibody is specific for cell surface antigens that appear on a cell or cellular fluid. Optionally, ligands for cell surface receptors that appear on specific cell or cellular fluids are used to provide specific attachment sites. An array of specially prepared surfaces or chambers is provided in the disc according to certain embodiments. Such surfaces and chambers are provided by contacting the surface with an aqueous solution of a suitable antibody. In this method of preparation, contact of the surface with the antigen is continued by contacting the surface with a non-specific blocking protein, such as bobbin cerium albumin. Antibodies and blocking proteins can be contacted to the surface or chamber using piezoelectric drive point heads (such as those applied to ink-jet printing). Optionally, injection of the antibody receptor onto the chamber or surface using screen printing or an airbrush can be applied. This method preferably prepares a surface or chamber of 0.1 to 10 mm. Optionally, microlithographic techniques and microstamping techniques can be used to prepare the surface or chamber.
In an embodiment of the present invention, a biological sample or other fluid sample containing a particular cell or cell fluid is applied to the prepared surface or chamber and contacted with the prepared surface or chamber for a sufficient time to allow the cell or cell fluid to bind to the surface. Since contact with the surface may be inhibited by cell setting properties in the volume of the fluid, the chamber and surface preferably have a minimum height across the microsystem platform.
Non-specific cell bonds are minimized or removed from the chamber or surface by cleaning the surface or chamber with a sufficient amount of fluid. Cleaning is accomplished by flowing fluid over the surface or chamber or by centrifugal force.
After cleaning, the cells still attached to the surface or chamber are detected and computed. In a preferred embodiment, detection and computation are accomplished by fluorescence microscopy. In an embodiment of the present invention, certain dyes may be used to impart the fluorescence signal of the cells remaining on the disk. Such salts can be added directly to the surface or chamber, for example using membrane osmotic dyes. Optionally, specific antibodies can be linked to such dyes. The dye may be added to the biological fluid containing the cell before it is introduced on the microsystem platform, or such dye may be contacted with the cell on the disk. The presence of the cell is detected using detectors such as light sources, source filters, chromatic filters or mirrors, fluorescence detectors including emission filters and photomultiplier tubes.
In another embodiment, thin layer chromatography is achieved on a microplatform disk comprising 100 pm square channels that radiate outward from the center of the disk. Each channel is filled with a separate substrate, which typically contains binder materials (0.1-10%) such as starch cast, polyacrylic acid salts, etc., to provide mechanical strength and stability (the use of such compounds in conventional TLC applications Disclosed in Analytical Chemistry 66.27A, proposed by Poole et al.). Absorbents are also included in the material comprising the separation channels, which include cellulose, polyethylene powder, aluminum oxide, diatomaceous earth, magnesium silicate, and silica gel. Such substrates can be modified with sealline molecules such as, for example, dimeltyl-ethyl-octa-3-aminopropy-silane. Preferably, the separating substrate comprises a glass fiber or PETE matrix containing the absorbent.
The sample is loaded through a port adjacent to the center of the disc. When the disk is rotated, the flowable phase is allowed to flow out through the separation substrate while moving the sample component around the disk at a certain rate. The flowable phase can be selected from a number of suitable solvent systems including hexane, methanol, dichloromethane. The choice of particular solvent depends on the nature of the diss material, the separation substrate, and the components of the sample to be separated. Similarly, visualization reagents used to detect separated sample components are characterized on a separated substrate. For example, ninhydrin is used to detect amino acids, alimony chloride and potassium permanganate are used to detect hydrocarbons, sulfuric acid and anisealdehyde are used to detect carbohydrates, and sulfuric acid and bromine are used to detect olefins. Used. The estimation of the separation channel after separation is achieved using a CCD camera. A disk configured such that this layer is applied in the field of chromatography is shown in FIG. 17R.
The medical field using the microsystem of the present invention varies. Various embodiments of the present invention are provided in a portable device for rapidly analyzing homes, beds, hospitals, blood components, blood gases, pharmaceutical concentrates, metabolites, and infectious reagents. In a home monitoring embodiment, the present invention provides a simple and readily available device for adding a blood, urine sample, or saliva sample to a particular application area on the disc, inserting the disc in the device, and operating the device by pressing a button. to provide. In hospital facilities, it applies to both bedside and clinical trial embodiments, which are advantageously electrically connected to a central processing unit located in the nurse's waiting room, where embodiments of the clinical laboratory can quickly and automatically diagnose a patient's sample. Medical resources. Medical applications of the present invention include blood tests (such as platelet measurements important for patients receiving chemotherapeutic drugs), immunoassays for metabolites, drugs, and other biological chemical types, vaccine efficacy measurements, myeloma or lupus. Measuring redness, measuring blood glucose and / or ketone levels in diabetic patients, testing cholesterol, measuring drug concentrations in blood, and measuring other medically relevant clerettes in patients with disease, sepsis / intramuscular Toxin measurement, allergy test, and thrombus measurement.
The invention also provides an analytical device for environmental testing, industry, and compliance control. A broader embodiment as well as a portable, preferably portable embodiment, installed as part of an industrial characterization scheme is provided. Application to this embodiment of the present invention includes analytical tests, in particular tests for industrial wastewater and industrial waste, in which compliance control and industrial characterization, and most advantageously human consumption, in particular pharmaceutical and endotoxin measurements, Include. Applications for testing, mixing, and evaluating perfumes and other complex mixtures are also possible within the scope of the present invention.
The present invention also provides chemical reaction and synthesis modeling, where reaction schemes or industrial production schemes can be tested and evaluated in miniaturized simulations. The present invention provides for low cost phototypes of potential research, medical, and industrial chemical reaction summaries that can be measured at macroscopic levels after analysis and maximization using the microsystem platform of the present invention.
The invention is also provided in many other fields, including in the field of microsynthesis methods and forensics.
The following examples illustrate certain preferred embodiments of the present invention without departing from the nature of the invention.
Example 1
Microplatform Disc Manufacturing for Chemical Analysis, Synthesis, and Applications
The microplatform discs of the present invention are made of thermoplastic materials such as teflon, polyethylene, polypropylene, methylmethacrylate, and polycarbonate, which are easy to mold, stamp, and mill. Optionally, the disk can be made of silica, glass, quartz or inert metal. Fluid control systems are prepared by successively applying one or more of the above materials that are cascaded onto a thermoplastic substrate. 17A-17E illustrate disks suitable for performing DNA sequencing. The disc of the present invention is made by injection molding, and the optically clear base layer has an optical pit like a conventional compact disc (CD). Such discs are round polycarbonate discs with a diameter of 120 mm and a thickness of 100 pm. The optical pit provides a device control program, user interface information, graphics, and a means and driver form for reading a particular sound. The driver type depends on whether the microcontroller is portable, benchtop or floor model, and also on external communication elements and other types of hardware. This layer is overlaid with reflective surfaces with suitable windows for external detectors, in particular optical detectors, which remain clean on the disk. Other polycarbonate layers of varying thickness are placed on the disk in the form of channels, vessels, reaction chambers, and other structures, which include provision for valves and other regulating elements. These layers can be prefabricated and cut into appropriate geometrical forms depending on the given field and shape to be assembled on the disc. Layers comprising materials other than polycarbonate may also be applied to the disc. The composition of the layer on the disc depends on the large part for the particular application and the needs of the chemical compatibility of the reagents to be used with the disc. The electrical layer can be applied to discs requiring electrical circuits such as electrophoretic applications and electrically adjustable valves. Regulators such as valves, integrated circuits, laser diodes, photodiodes, and resistive networks or flexible monolithic structures capable of forming optional heating zones can be suitably applied to wired resets by installing modular directly on the disk. . Reagents that can be stored in a dry state can be introduced into a suitable open chamber by spraying into the container using a means similar to an inkjet printing head, and then dried on a disc. Thereafter, an upper layer comprising an entry port and an air hole or shaft is then applied. Thereafter, the chemical reagent is injected into a suitable container, and then a protective cover layer including a thin plastic film is applied.
Various types of discs for use in particular applications are shown in FIGS. 17F-17P.
Example 2
Blood composition
Blood components may be analyzed via cold blood analysis using a prepared microplatform disk as described in Example 1 held in a device comprising a microchannel layer with multiple microchannels shown in FIG. 18. The microchannel layer has a thickness of 100 pm and is treated with heparin to prevent solidification during the analysis. The blood sample to be analyzed is introduced by capillary action into channels arranged perpendicular to the direction of rotation as shown in FIG. 18, and these multiple channels may be arranged radially on the disc. When all the samples to be tested enter the channel, the disk is rotated at a speed of 8000 to 10000 rpm to settle the red blood cells. If the centrifugal force is operated for a suitable time (3 to 5 minutes), the cold blood of each sample is determined simultaneously by stroboscopic questions of each channel using a conventional CD laser system in the above described apparatus. As the laser passes through the boundaries of the red blood cells, the change in the light scattering pattern detected by the photodiode detector is converted into a cold blood value based on standard light scattering cold blood information stored in the device's internal processor and memory. Optionally, pure information is converted into an analytical microprocessor via an infrared port or hard-wire interface. Such a central microprocessor may be placed on a site or in a central location such as a nurse waiting room in a medical center or, optionally, a hospital, which is connected to a cold blood analysis device by telephone or other connecting means. Cold blood applies lancet-collected blood droplets and automatically cold blood analysis and data are processed on site or sent to a central location. Embodiments of the present invention are provided for chronic patients with cold blood increased disease (such as leukemia, lymphoma, myoma, and anemias).
In addition, blood gas can be measured using the device in combination with a disk with integral electrodes implemented in a cold blood channel, or using a device with a separate channel to determine blood gas on the cold blood disk. Can be measured. Blood oxygen content (PO ₂ ) is measured by a Clark-type electrode composed of a thin Cr-Au cathode and an Ag-AgCl wire anode. The amount of carbon dioxide in the blood is determined by the seversing electrode using an ISFET (field-effect transistor) as the pH monitor. Blood pH is measured using a S ₃ N ₄ gate ISFET with a reference electrode consisting of a liquid junction and an Ag—AgCl wire electrode. Analytical methods for blood gases, electrolyte concentrations, and other information advantageously performed using cold-blooded disks or optionally altered disks are a modification of Shoji and Esashi's macroscopic method (Sensors and Operators, 1992, Volume 8, page 205). Is disclosed.
Blood analysis is also performed using the Spiel Flow Thin Cell Sorting Method (SPLITT), described by Bor Pew et al. A schematic form of a disk configured for SPLITT analysis is shown in FIG. 19. This process can produce proteins, lipoproteins, platelets, red blood cells, lymphocytes, monocytes, and neotrophils. Discontinuous circular channels are etched into the disc to connect the thin wall and the inlet stream separator at the end. Sample and carrier streams are introduced on opposite sides of one end and the chamber is rotated in that direction. In the rotating chamber, two separate separation planes are installed with an inlet separation stream (ISP) and an outlet separation stream (OSP) based on hydrodynamic forces. The ISP is adjustable by adjusting the ratio of samples in the carrier stream. Two separate separation modes, the equilibrium and delivery modes, are possible by the sample input method.
In equilibrium mode, the separation is based on the equilibrium of components with respect to the centripetal force to be applied. Separation is maximized by adjusting the outlet flow ratio. Suspended fragments may be collected from one side of the outlet stream separator. In the delivery mode, the component is introduced as thin lamina over the ISP. Due to the difference in the settling coefficients, components with high delivery ratios are selectively directed at the orifice to the opposite side of the outlet valve. Variable flow values are disclosed elsewhere in this document. In another embodiment, each SPLITT chamber may be introduced as a separate type of flow controlled by an ISP or OSP, and a fixed flow control orifice.
In order to completely classify the blood into the above-described fragments, a five step separation process is performed, where each stage classifies two fragments. One embodiment of the microsystem of the present invention used for this classification is shown in FIG. Five concentric SPLITT cells are labeled C1 (adjacent to the center of rotation) to C5 (toward the circumference) in the figure. The blood sample is introduced into C1 and subjected to delivery mode separation by rotating the disk at an appropriate speed. Platelets and proteins (fragment 1) are sorted towards the center of rotation and blood cells (fragment 2) are moved towards the perimeter. Fragment 1 proceeds to the inlet of C2 while fragment 2 moves to C3 by opening and closing the valve properly seated on the disk. The fragments are then subjected to delivery mode separation and equilibrium mode separation, respectively.
Fraction 1 (fraction 1) directs platelets toward the center of rotation and protein toward the periphery. Oil 2 yields oil 3 and oil 4 consisting of lymphocytes and mononuclear leukocytes towards the center of rotation, erythrocytes and neutrophils towards the center of rotation, and mononuclear leukocytes towards the periphery. Oil 4 produces neutrophils towards the center of rotation and red blood cells towards the periphery. Thus, fractionation of the blood into five independent components is achieved.
Activation of enzymes in protein fractions can be determined using immobilized enzymes (Heineman, 1993, App. Biochem 41. 87-97). For example, blood-specific enzymes (such as glucose oxides, alkaline phosphorus, and lactate oxides) are immobilized in polyvinyl alcohol (PVAL). Lactate oxide is passivated by sandwiching a thin layer of enzyme on the platinum-clad graphite electrode between two layers of PVAL. The sensor reacts to the lactate by electrochemical oxidation of hydrogen peroxide water, which is caused by enzyme-catalyzed oxidation of the lactate that diffuses into the network. The current generated is proportional to the concentration of peroxide, which in turn is proportional to the concentration of lactate. The sensor has been shown to be sensitive to the lactate concentration range of 1.7-26 μM.
During separation, the cach fraction is signaled by the sensing system to determine the relative components of the fraction. Alternatively, each fraction can be removed from the disc through the outlet port for further study outside the device. For example, each fraction can be measured simply by passing the cells through a lean vapor passing through two electrodes comprising a resistance regulator. As the cell passes through the electrode, the corresponding increase in resistance is adjusted and measured. These data are then integrated in relation to a standard set of particles distributed according to size to determine the relative number of each cell type in the original sample.
The oil can be well stained with fluorescent antibodies unique to each cell type. The cells are held in position by a micromechanical filter which is an essential component of the stained and washed channel (US Pat. No. 5,304,487) on the disc. The final labeled cells can then be quantified as a grading function of fluorescence staining associated with the cells.
Example 3
DNA size and mutation detection
The detection of unique mutations of DNA at the size and specific location of the DNA is performed by using double chain fusion analysis with disks prepared as shown in Example 1 and FIG. 20. The DNA fusion phase (as described in US patent application Ser. No. 08 / 218,030, filed March 24, 1994, shared and co-filed and incorporated herein by reference in its entirety) preferably comprises the structure of the disk of Example 1 Is merged in. DNA fusion technology takes advantage of the fact that the point of denaturation of the DNA duplex is affected by the length, the basic configuration, and the degree of complementation of the two chains in the duplex. The point of denaturation may be determined in relation to any physical state of the molecule (eg, temperature or concentration of denaturing drug such as urea or formamide), and the standard state to derive information that may be stored in the memory of the microprocessor and / or device. Set of can be used. To size any particular DNA duplex, one chain is immobilized on disk by attaching it to a bed encoded with streptavidin. The bed is held by a filter mechanism in the channel (see US Pat. No. 5,304,487). Alternatively, the bed may be a paramagnetic bed held in the channel by applying a magnetic field using a permanent magnet integrated into a disk located adjacent to the channel. Electromagnets may be used. The electromagnet can be integrated directly into the disk and can be activated by applying a 500 mA direct current 0.8 volt. The other chain is generally labeled using a fluorescent die or radioisotope. In contrast, the distinct optical characteristics (ie hyperchromaticity) of the DNA molecules themselves are detected using DNA molecules that are not labeled by absorbance at 260 nm. Although this aspect of the method requires more complex devices to generate and detect ultraviolet light, the user's DNA preparation is minimized and the DNA preparation cost per sample is reduced. In an embodiment of the present invention, an immobilized, labeled dual structure is placed on the disk and subjected to the fluid flow of the buffer solution contained on the disk. During the development of fluid flow, the increased denaturation agent that adds DNA to the DNA is more susceptible to the controlled denaturation gradients that occur in the fluid flow. With an effective radius of 3.5 "and a rotational speed of 600 rpm, 100 μL / min of fluid flow can be generated in a channel with a diameter of 100 μm. Four buffer solution chambers each containing 300 μL are provided for each quadrant of the disc (at 25 mm). 800 μm depth extending from a radial position of 50 mm), at 10 μm / min, this allows for 30 minutes of melting ramp, each dual structure dissociates at a specific denaturant concentration on the slope, Michael processor and / or Or by comparison with standard degrading agent profile information stored in the device's memory Degeneration is a suitable detection means (photooptical means for ultraviolet absorption or fluorescence detection, or radioactive for DNA chains labeled with radioisotopes). Isotope detectors (Geiger-Müller counters) are used to detect by flowing down the fusion chamber.
Experimental use of disks and devices of this aspect of the present invention is directed to chain reactions or magnetic chain reactions of polymerases (the latter being filed on Jan. 9, 1993, US application Ser. No. 08 / 074,345, and Dec. 8, 1994). DNA fragments generated by U.S. Application No. 08 / 375,226, filed Jan. 19, 1995, each of which is part of an ongoing application of US 08 / 353,573, each of which is incorporated by reference in its entirety. fragment identity) and size determination. Amplification is performed using one primer labeled with a detectable label, such as a fluorescent die or radioisotope, and the other primer is covalently attached to a molecule (e.g. biotin) that enables the immobilization of the primer. .
After amplification (more specifically off the disk or on the disk as described in Example 4 below), the labeled and covalently attached double-stranded DNA product fragment, for example, may be used to The streptavidin is encoded by moving it to a channel or compartment on the disc so that Dean coats the wall, or the amplification reaction mixture contains a binding matrix such as Dynal M-280 Dynabeads (polystyrene coated with paramagnetic particles of 2.8 μm diameter). Streptavidin is attached to the encoded solid support by moving to a compartment on the disk. Normalized size labels are included in the post-amplification compartments to provide a reference set of DNA fragments for comparison with amplification product fragments. In such an assay, multiple different double-structured DNA molecules from multiple amplification reactions or multiple separate amplification reactions can be sized simultaneously, so that each fragment or set of fragments is reaction-specific, detectable label or fragment-specific. Detectable labels are distinguished from others by use or by differences in some other physical characteristics of the fragment. In order for amplification to be performed outside the disk, a bed attached to the fragment is mounted in a channel on the disk that has the ability to hold the bed (as in "optical tweezers" or size exclusion by magnetic aspiration). In the latter embodiment, magnetic retension means (permanent magnets or electromagnets) are accommodated on the second disk which rotates simultaneously with the first disk and incorporated into the disk, or the DNA fragments are immobilized in the appropriate compartment. So that it is located on the device.
DNA size analysis is also performed essentially as described above, wherein the retained particles are susceptible to thermal degeneration gradients. For the thermal gradient used to modify the binding of the DNA fragments, a Peltier heat pump or resistive element directed to laser heating is used to increase the temperature of the binding compartment by increasing the heat energy through the denaturation range. As mentioned above, a flow rate of 10 μL / min can occur in a 100 μm diameter channel, allowing for 30 minutes of melting ramp. The compartments are also susceptible to fluid flow as described above for extracting denatured and labeled chains from the combined fusion chamber. The downstream flow from the binding / fusion chamber is an appropriate means of detecting denaturation of the DNA fragments, such as laser excitation at the resonance frequency of die label and photodiode detection. The intensity of the raw absorbance or other signal and the corresponding temperature are integrated by the microprocessor and the size of each DNA fragment is compared to internal DNA size label control and DNA fusion profiles and properties stored in the microprocessor and / or device memory. Is determined by.
DNA mutations are also detected by fusion analysis. The DNA fragments tested (amplification-induced fragments and restriction enzyme assimilation or cloned fragments) are prepared and crossed using gene binding standard replication (typically native) or gene fragments of interest. The mating can be performed on-device or using conventional DNA mating methods (as described in Hames & Higgins, Nucleic Acid Hybridization. A Practical Approach. Rickwood & Hames. Eds. IRL Press: Oxford, 1985). Can be. Extraction of hybridized fragments depends on the degree of complementarity between the two DNA chains (ie, native and mutant).
Hybridization assays are performed using naturally-typed DNA in which one chain therein is covalently attached to a molecule allowing passivation. The uncovalent chains are then extracted by washing at a temperature higher than the Tm of the dual structure (generally the DNA is heated above 90 ° C., or at a low temperature in the presence of denaturing agents such as formamide). Extraction is further adjusted to determine the concentration of single-chain binding products for use in crosses. Typically the amount of DNA extracted is adjusted, for example by ultraviolet absorbance, and the border DNA is considered to be completely single-chain when no further DNA can be extracted. Naturally typed DNA is prepared by covalent attachment of only one chain to make a double structure to require detectable labeling of only one chain (complementary) of the mutant DNA being tested. Alternatively, either chain may be covalently attached, allowing both mutant chains to be detectably labeled. The advantage of double-labeling a mutant fragment only when the native chain is covalently attached to the immobilized molecule is that denaturation and extraction of the non-complementary chain can be coordinated during the crossover and into the naturally-typed DNA chain of the mutant DNA chain. Non-specific binding / crossing of can be detected.
After the mating is completed, the degree of complementarity of the chains is determined by altering the thermal or chemical denaturing protocol as described above. Analysis of the final pattern of dual structure fusion can be performed using standard or predicted single base or multiple mismatches, mismatched DNA duplexes that are prepared concurrently with or prior to experimental analysis and stored in the device microprocessor and / or memory. This is done by comparison with the structural fusion pattern. This comparison forms the basis for the determination of rapid screening of individuals for the various polymorphisms that are characterized and related to disease.
DNA mutations are also detected by fusion assay. In this example, the test DNA is immobilized on the disk and subjected to cross / denaturation analysis using a pair of test probes first characterized. Using this method, preferably, DNA fragments are prepared using in vitro amplification so that one chain is immobilized because the binding molecule is covalently attached to one of the primers. Using this method, the tested DNA fragments are successively crossed and extracted by denaturation from a series of detectably labeled well-characterized DNA probes. Alternatively (depending on the nature of the DNA mismatch that is expected for each probe), the detection and labeling probes are provided with different detectable labels so that the identity of each probe can be identified. Denaturation is multiplexed. This method is useful for screening genes as described above.
Example 4
DNA Amplification and Analysis
DNA fragments are amplified in vitro by polymerase chain reaction (PCR) or magnetic chain reaction and analyzed by capillary electrophoresis. Reagent mixing, primer heating cooling, elongation and denaturation in an amplification cycle resulting in amplification of the 500 bp target fragment and its accompanying analysis is performed using a device and disc as described in Example 1 above. A schematic diagram of the disk structure is shown in FIG.
The disc comprises at least three sample inlet ports (A), (B) and (C). Port (A) allows injection of 30 attomole (about 100 pg) of linear bacteriophage lambda DNA. Ports (B) and (C) allow influx of 5 μL of a 20 μM solution of primers 1 and 2, each having the following sequence: The sequences of primers 1 and 2 are
Primer 1: 5'-GATGAGTTCGTGTCCGTACAACTGG-3 '(SEQ ID No. 1)
Primer 2: 5'-GGTTATCGAAATCAGCCACAGCGCC-3 '(SEQ ID No. 2).
The disk also includes three reagent chambers (D), (E), (F) in the figures, with 54 μL of distilled water; 10 μL of 100 mM Tris-HCI (pH 8.3), 500 mM KCI, 15 mM MgCl2, 0.1% gelatin and 1.25 μM solution of each dNTP, and 1 μL of Taq DNA polymerase at a concentration of 5 Units / μL, respectively. .
In addition, the disk includes a reaction chamber (G) configured to facilitate mixing these reagents using a flexible-plate-wave component (as described in patent 5,006,749). Cooling and heating means are also included through the Peltier component in the construction of the reaction chamber. These components may be integrated into the disk or may be located on the device to provide heating and cooling specific to the reaction chamber. The disc is also provided to contain a number of sets of reaction components A through G.
Amplification is initiated by introducing sample DNA and primers into each set of ports (A), (B) and (C). When all samples and primers have entered the port, the disk is rotated at a speed of 1 to 30,000 rpm to achieve mixing of the reagents into the reaction chamber. At the same time, the valves controlling the reservoirs (D), (E) and (F) are opened and the contents of these reservoirs are also sent to the reaction chamber (G). Mixing sample DNAs with primers and reagents is facilitated by the activity of the flexible-plate-waveform component. DNA amplification takes place in the reaction chamber using the following thermal cycling program. The reaction mixture is heated at 95 ° C. for 3 minutes. The subsequent amplification cycle comprises the first step of aging at 95 ° C. for 1 minute; A second step of cooling the chamber at 37 ° C. for 1 minute; And a third step of heating the chamber at 72 ° C. for 3 minutes. This amplification cycle is repeated 20 cycles in total, and the reaction is terminated by aging at 72 ° C. for 5 minutes.
The amplified DNA fragment is analyzed by transfer to the capillary electrophoresis unit (H) by opening the valve of the reaction chamber (G) which rotates the disk at a speed of 1 to 30,000 rpm and directs it to the capillary electrophoresis unit (H), Thus causing a transfer of the desired amount of reaction mixture to the electrophoretic unit. The amount of reaction mixture, typically 10 μL, is determined by the combination of the length of time the valve of this reaction chamber G is opened and the speed at which the disk rotates. Capillary electrophoresis is performed as in Example 11 described below, and the fractionated DNA species are detected using optical or other means as described in Example 2 above. This method provides an integrated amplification and analysis apparatus that is preferably used for the performance of PCR and other amplification reactions of samples under limited sample conditions.
Example 5
DNA restriction and assimilation and analysis
Restriction enzyme assimilation and restriction fragment analysis are performed using disks and devices as described in Example 1 above. Double-chain DNA fragments are assimilated using endonucleases and subsequently analyzed by capillary electrophoresis. Reagent mixture, DNA assimilation and restriction fragment analysis are performed on disk. A schematic diagram of the disk structure is shown in FIG.
The disk includes a sample inlet port A; Three reagent chambers (B), (C) and (D); A reaction chamber E configured to mix reagents as described above in Example 5; And capillary electrophoresis unit (F). The reagent chamber comprises 1-2 μL of restriction enzyme such as HindIII at a concentration of 20 Units / μL in the reagent chamber B; 4 μL of a solution of 100 mM Tris-HCI (pH 7.9), 100 mM MgCl 2 and 10 mM dithiotriitol in the reagent chamber (C); Contains 30 μL distilled water in the reagent chamber (D). The disc is also provided to contain a number of sets of reaction components A through E.
Restriction enzyme assimilation of DNA is initiated by placing 4-5 μL of solution (typically 10 mM Tris-HCI, 1 mM EDTA, pH 8) containing 4 μgdml bacteriophage lambda DNA inside the sample inlet port (A). DNA samples and reagents in the reagent chambers (B), (C) and (D) rotate the disc at rotational speeds of 1 to 30,000 rpm and open valves to control the reagent chambers (B), (C) and (D). It is moved to the reaction chamber E by doing so. After mixing, the reaction is aged for 1 hour at 37 ° C. in the reaction chamber (E), which is heated by providing a Peltier heating element, which is located on the disk or located in the apparatus, in particular to heat the reaction chamber. After assimilation, a predetermined amount of assimilation DNA enters the capillary electrophoresis unit (F) by opening the valve of the reaction chamber (E) which rotates the disk at a speed of 1 to 30,000 rpm and directs the capillary electrophoresis unit (F). Analysis by transfer, thus causing the transfer of the desired amount of reaction mixture to the electrophoretic unit. The amount of reaction mixture, typically 10 μL, is determined by the combination of the length of time the valve of this reaction chamber E is opened and the speed at which the disk rotates. Capillary electrophoresis is performed as in Example 11 described below, and the fractionated DNA species are detected using optical or other means as described in Example 2 above.
Example 6
DNA synthesis
Oligonucleotide DNA synthesis is performed using disks and devices as described in Example 1 above. Synthesis is accomplished by stepwise movement of controlled pore glass (CPG) through a continuum of the reaction chamber containing the reagents required for phosphoamidite DNA synthesis. Reagents and CPG are continuously delivered to the reaction chamber using single-use valves that connect the reaction chambers with each other. Each disc has a number of synthetic reaction chambers for producing oligonucleotides having a length approximately equal to the length of the oligonucleotides produced by commercially available DNA synthesizers (ie, 100-150 bases). A schematic of the disk structure is shown in FIG. 23A.
CPGs carrying the first base of the chain (and thus defining the 3 'range of oligonucleotides) are mounted to the sample inlet port (A) by the user or by automated means. CPG is then sent to the reaction chamber containing trichloroacetic acid (TCA) in acetonitrile (CH₃CN) by rotating the disk at a speed of 1 to 30,000 rpm. Detritilation of the nucleotides is carried out at room temperature for a set amount of time, only in the case of bumps. The reagent is then quietly followed by opening a valve that is small enough to not allow the passage of CPG from the first reaction chamber and that has enough holes to drain the TCA-containing mixture into the decantation chamber. Deprotection of the base by detritilization is known to produce a colored product (orange) whose strength indicates a measure of the degree of reaction, and is therefore preferably optical for determining the absorbance of this effluent. Means are provided for recording on the device microprocessor / memory. After tilting the reaction mixture, the CPG is rotated into a rinse chamber containing CH 3 CN, which chamber optionally includes mixing means as described above. After the rinse, CH3CN is tilted into the outlet reservoir controlled by the size-selective valve as described above, and the CPG is rotated towards the second reaction chamber. The solution containing one of the four phosphoramidite bases (G, A, T, or C) corresponding to the next position of the oligonucleotide chain is mixed with CPG in the second reaction chamber. The reaction mixture in the second reaction chamber is allowed to mix and react for a defined time, usually on the order of 3 minutes. The reaction mixture is then decanted as described above and the CPG is rotated towards the rinse chamber containing CH 3 CN and mixing means. After rinsing, CH₃CN is decanted into the outflow reservoir, and CPG is rotated toward a third reaction chamber containing an oxidized mixture of iodine, water, pyridine, and tetrahydrofuran, where the reaction mixture is typically about one minute. It is aged for a fixed time. The reaction mixture is decanted into the outlet reservoir and the CPG is rotated towards the rinse chamber containing CH 3 CN. After the rinse, CH 3 CN is tilted into the outlet reservoir and the CPG is rotated towards the fourth reaction chamber with a 2-component “finishing” reagent. After the reaction is complete, the reaction mixture is decanted as described above with the outflow reservoir and the CPG is rotated toward the rinse chamber containing CH3CN. CH 3 CN is tilted into the outlet reservoir, and the CPG is rotated towards the fifth reaction chamber containing TCA, and begins another cycle. The cycle is first repeated by transferring CPG through the interconnected continuum of the four reaction chambers until the planned chain is fully synthesized. Thereafter, the CPG is rotated into a reaction chamber containing concentrated ammonium hydroxide and heated at 60 ° C. for a fixed time, usually six hours, during which time the DNA molecules are deprotected and cleaved from the CPG support. Finished oligonucleotides are removed by the user or by automated means.
The disc provides a continuum of reaction chambers comprising four reaction and rinse chambers per nucleotide that are interconnected and added to the oligonucleotide chain. The disc may be mounted to produce a particular oligonucleotide, or each reaction chamber 2 may be in contact with a reagent chamber containing each of the four nucleotide bases and connected to the reaction chamber by individually controlled valves. In this embodiment, actuating an appropriate valve at each stage of the cycle is controlled by signals from the device. Disks contain multiplexing of these integrated arrays. It is also provided to allow the synthesis of a plurality of oligonucleotides simultaneously. A schematic of a disk configured for multiple oligonucleotide synthesis is shown in FIG. 23B.
DNA synthesis may also be performed on CPG contained and first mounted in the reaction chamber toward the periphery of the disc and reagents delivered by the use of a multi-purpose two-way valve, as schematically shown in FIG. 23A. In such a disc, as many as 1250 reaction chambers can be produced, which can hold 100 nL and have a 150 μm spacing (measured from the center of one sphere to the center of the next sphere) at the center of a 120 mm diameter disc.
The reagent reservoir with sufficient volume to supply the reagent chamber on the disc is prefilled with four phosphoramidites, CH3CN, TCA, oxidant and finishing reagent. Trityl-bearing CPG or a connector directly attached to the reaction chamber is likewise first mounted on the disk. A volume of microliters of reagent is sufficient for each reaction. The TCA is rotated into each first reaction chamber and allowed to react for a defined time, typically on the order of one minute, and then to the outflow (disposal) chamber on the disk periphery. The CH 3 CN rinse is rotated into each reaction chamber and then to the waste chamber. By selective valve operation, A, C, G, or T phosphoramidite is rotated into a reaction chamber requiring a base and reacted for a predetermined time, typically three minutes, and then to a waste chamber. A CH 3 CN rinse is rotated into each reaction chamber and then to the waste chamber. The oxidant mixture is rotated into each reaction chamber and reacted for a set period of time, typically one minute, and then to the waste chamber. Another CH3CN rinse is rotated into each reaction chamber and then to the waste chamber. A two-component finishing reagent is rotated into each reaction chamber and reacted for a set period of time, typically one minute, and then to the waste chamber. For each cycle, the last CH3CN rinse is rotated into each reaction chamber and then to the waste chamber. The cycle is repeated for a predetermined number of cycles until each oligonucleotide is fully synthesized. The concentrated ammonium hydroxide is then rotated into each reaction chamber and reacted for a fixed time, usually six hours, at 60 ° C. to allow the finished DNA to be deprotected and cleaved from the support. The DNA can then be removed by manual or automated means. Conversely, the linkage of the oligonucleotide to the CPG support is chosen to interfere with the activity of ammonium hydroxide, so that the deprotected oligonucleotide remains in the reaction chamber while bound to the CPG.
Peptide synthesis discs are also provided, such that the arrangement of reagent chambers and reaction chambers as described above is adapted for synthetic reactions involving peptide synthesis governance.
Example 7
DNA chain by enzyme
Nucleotide chaining of DNA fragments is determined by a method of conjugation by Sanger, an enzyme using a disk prepared as described in Example 1 above (see FIG. 24). DNA template (200 pg in 250 mL) and 100 femtomol of suitable primers are transferred towards the sample inlet port either manually or by an automated process.
The DNA is then transferred to a mixing chamber containing a terminator solution (ie, a solution comprising a dideoxy foam of nucleotides G. A, T or C) by spinning the disk at a rotational speed of 1 to 30,000 rpm. Terminator solutions typically contain 5 picomoles of each deoxynucleotide, 0.5 picomoles of one dideoxynucleotide covalently bound to a fluorescent label, 100 nL containing 90 mM Tris-HCl- (pH7.5), 45 mM MgCl ₂ and 100 mM NaCl Solution. The contents of the mixing chamber were spun at a rotational speed of 1 to 30,000 rpm, producing a reaction mixture in the reaction chamber having a final concentration of buffer of 26 mm Tris-HCl (pH 7.5), 13 mM MgCl 2, 32 mM NaCl and 6 mM DTT. The reaction chamber is then transferred to a reaction chamber containing 0.1 units of polymerase of T7 DNA (or alternatively 0.1 units of Tag polymerase) and 20 nL 0.1M dithiothritol (DTT). The reaction chamber is heated to 37 ° C. (or alternatively 65 ° C. for Taq polymerase) by means of an integral heat resistant element with the disc or alternatively located in an apparatus for specially heating the reaction chamber, for a fixed time, typically 1 Incubate for minutes. The reaction was spun with an equal volume of 90% formamide / EDTA and capillary electrophoresis on disk at 90 ° C. for 1 minute. The set of dideoxynucleotide terminal DNA fragments containing the reaction mixture is then separated by capillary electrophoresis and the sequence of fragments determined by laser induced fluorescence detection as described above. Discs are also provided that include multiple synthetic sequences and allow for the simultaneous synthesis of multiple dideoxynucleotide terminal oligonucleotides. The subtracted nucleotide sequence is determined from the pattern of the detected fluorescence signal and the sequence is determined from the pattern of the detected fluorescence signal and the sequence derived by the device microprocess from these data.
Example 8
Liquid phase synthesis and analysis
Various collometric chemical analyzes are performed using discs as described in Example 1. For example, discs are provided for conducting solution analysis to determine iron concentrations in test solutions (such as industrial effluents) using standard collometric tests (see FIG. 25). The device was constructed with a reagent chamber containing 40 uL 12 N HCl, 100 uL 10% hydroxylamine hydrochloric acid, 100 uL 10% sodium citrate buffer (pH 4) and 50 uL 0.02% 1.10-phenanthroline. Since the reagent chambers are arranged as shown in FIG. 25, these reagents are added to the reaction chamber by continuously opening the valves controlling the flow from each reagent chamber. Transfer of reagents to the reaction chamber is accomplished by spinning the disk of Example 1 at 1-30,000 rpm, whereby centrifugal force transfers each reagent liquid from the reagent chamber to the reaction chamber. As shown in FIG. 25, the sample is introduced through the sample port and delivered to the reaction chamber under the action of centripetal force. The valve of the reagent chamber containing HCl (B) is opened and acid is added to this sample. The sample is incubated for 10 minutes to dissolve all iron oxide present. Hydroxyl amine hydrochloric acid (Reagent D) and citrate (Reagent E) are then added to the reaction mixture. The reaction mixture is incubated for 10 minutes to ensure complete reduction of iron trioxide to iron dioxide. Next, 1.10-phenanthroline is transferred from reagent chamber F and the color product, which synthesize iron dioxide. The solution is incubated at 30 ° C. for 30 minutes to complete the color. Photometry at 520 nm is made after the incubation process in a "read" cell (G) connected to the reaction chamber via a valve (G).
Example 9
Solid phase (surface / colloid) synthesis / analysis
Oligonucleotides, single-chain DNA or double-chain DNA are covalently bound to the reaction particles (beads or magnetic particles or chromatographic substrates) using a disk prepared as described in Example 1 and shown in FIG. 26. In the illustrated embodiment, 25 uL portions of carboxyl active magnetic particles (BioMag 4125, PerSeptive Diagnostics- Framingham. MA) are added to the disc through the sample introduction port. The particles are changed from the initial solution to 50uL 0.1M imidazole (pH 6) by gently pouring the stock solution into the effluent or waste container through the valve, so the valve is shaped to prevent the loss of magnetic particles from the reaction chamber. . The imidazole solution is then added to the particle reaction chamber from the imidazole chamber on the disc, and the transfer of the imidazole is controlled by a valve. Motive force to pour the atomic particle solution and transfer imidazole from the imidazole chamber to the particle reaction chamber is provided by spinning the disk at a rotational speed of 1 to 30,000 rpm. With particular reference to FIG. 26, when the disk spins, the dense magnetic particles are pelletized in a funnel at the end of the reaction chamber and settle into trash. The valve controlling the imidazole reagent chamber containing 50 uL of 0.1 M imidazole is then open at the above level of particles but not at below, and transfers the particles through the valve to the reaction chamber and the next still container. Used. This still step can be repeated several times to change the liquid phase to obtain the desired composition. Typically, three replacements are sufficient. Alternatively, the proper shape of the reagent chamber and reaction chamber may provide sufficient imidazole for a single reaction chamber, or, alternatively, for the entire cycle of replacement, by controlled addition and removal of imidazole from the clotor of the reagent chamber. Allow replacement in a single reagent chamber of sufficient size to obtain.
After the replacement cycle is over, the magnetic particles are transferred to the next reaction chamber containing 250 ug dry 1-ethyl-3 (3-dimethyl aminopropyl) carbodiimide (EDAC). The reagent chamber contains a 170 OD (170 ng) 5-aminoated DNA oligonucleotide chamber in 50 uL of 0.1 M imidazole solution prior to addition of particles to dissolve the EDAC. The particles are then added in about 100 uL 0.1 M imidazole through the valve. Upon addition of magnetic particles to the reaction chamber, the device is stopped and incubated at 40 ° C. for 6 hours. It can be heated by a heat source (such as a Peltier heating device) installed in the disk itself or positioned in an instrument shaped to allow specific heating of the reaction chamber. In an alternative embodiment, the disk can be stopped at a predetermined position for the device to ensure the heating characteristics of the reaction chamber.
After incubation, the particles are rinsed and replaced with 100 uL of water as the disk spins as described above. Three replacements are usually done to purify the particles. The product thus made is collected in the distal end of the disc which is easily accessible for continued use. Discs are also provided that include multiple synthetic sequences and allow for the simultaneous synthesis of multiple particle-linked oligonucleotides.
Example 10
Micro-shrink system
The disks described in Example 1 (see FIG. 27) undergo micro-shrinkage of the solvents from the components or solutions of the mixture as altered HPLC or other conventional biochemical separation methods. In particular, the channels on the disc are coated with a compound (such as octanol) by standard procedures to provide a surface with affinity for the mixture, typically a complex chemical or biochemical mixture. In a silicon disk, for example, the surface of the channel is activated by filling the chamber with aqueous epoxysilane at 95 ° C. for 1 hour. The disc is washed with distilled water for about 5 hours to remove unreacted silane, added into the solvent and incubated at 95 ° C. for 1 hour to rinse the solvent next to remove the unreacted octane.
The sample mixture containing the components to be suspended is added to the injection port and transferred through the coating separation channel by rotating the disk at 1 to 30,000 rpm. The reagent chamber is used to suspend the sample opened at the inlet of the channel and held on the coating channel into a collection vessel. The isolated sample component is then collected at the outlet port.
Example 11
Freezone Moses Electrophoresis
Freezone capillary electrophoresis is performed on a disk fabricated as described in Example 1 above, and is approximately shown in FIG. In particular a 5 μm × 75 μm × 25 mm capillary (recognize that all dimensions are appropriate within the exact limits for producing a component such as a capillary in the disc) is lithographically etched on the glass disc. Electrical connections are made by plating platinum over the non-etched surfaces of the glass before sealing the top of the device, using standard methods. The separation channel is crossed by a 15 mm sample introduction channel located 3 mm away from the buffer vessel. At one end of the cross-channel there is a sample inlet port and at the other end there is an electrical connection that controls the application of the sample to the capillary.
In the execution of capillary electrophoresis on the disc, the separation channel is filled from the buffer vessel by rotating the disc at a speed of 1 to 30,000 rpm. Once the channel is filled, rotation stops until pressure needs to be applied to the channel again. The sample is introduced by applying a voltage between the cross assay inlet and the assay outlet channels on the chip (see FIG. 28). A 50V potential drop is applied between the sample inlet and outlet ports while the separation channel port is floating. The sample comprises a solution of 1 mM Tris-HCl (pH 8), 5 mM EDTA with 1 mM Mg ²⁺ (typically prepared from chloride) and 1 mM Ca ²⁺ . Running buffer consists of 10 mM Tris-HCl (pH 8), 5 mM EDTA. Separation to the cathode is then accomplished by floating a potential in the sample vessel and applying 250 V along the separation channel. Separation is monitored at a 2 cm position from the inlet port, for example by monitoring the UV absorbance of 254 nm using a photodiode detector positioned on the device of interest to the UV light source (mercury lamp) and the capillary channel.
Example 12
DNA electrophoresis
Gel electrophoresis is carried out on a prepared disc as described in Example 1 above. In this application, gel media is prepared in the separation channel; However, such gel media must be protected from shear forces that develop as the disk rotates during transfer of the sample or buffer to the electrophoretic channel. Therefore, the gel filled capillary is preferably arranged centrifugally on the disc as shown roughly in FIG. Eventually, the gel will receive shear force from centripetal force induced pressure during rotation only if the fluid container contacts the capillary during rotation of the disk. At rest, the planar geometry of the disk prevents capillary water pressure. This is good in standard capillary electrophoresis systems where water pressure is not easily controlled because the buffer volume is the vessel height that needs to be carefully adjusted before each run to prevent hydraulic flow. This is also excellent for capillary electrophoresis performed on discs of the present invention from electrophoresis performed on microchips, where the buffer vessel is located on the plane of the separation channel and is therefore sensitive to hydraulic-driven fluid flow.
Gel electrophoresis is performed on discs of the present invention to separate double chain PCR fragments, DNA fragments comprising oligonucleotides and single-standard dideoxynucleotide-terminal enzyme DNA sequence components, the system being shown in FIG. It is shaped like this. The prepared disks comprise polyacrylamide gels arranged centrifugally in microetched separation channels in the disks. Polyacrylamide gels were prepared from 7M urea (Urea), 45 mM Tris-borate buffer (pH 8.3), 1 mM EDTA, 9% acrylamide, 0.1% TEMED and 10% ammonium persulfate. The disc may be prepared in a separate channel by mixing the components (where the nonpolymerization, polymer polyacrylamide may be sensitive to the photocatalytic polymer on storage), in particular by introducing TEMED and ammonium persulfate into the mixture. Can be. The disc is stopped upon filling of the separation channel to allow gel polymerization. Immediately before completion of the polymerization, the outlet channel overflows the buffer from the large buffer vessel at the outlet side of the channel controlled by the valve, thereby removing droplets and non-polymerizable monomers. A similar process occurs on the inlet side of the gel.
To introduce the DNA sample, the valve is opened from the inlet port holding the solution of DNA debris or, alternatively, pipettes the sample directly to the disc. The sample is applied to the separation channel by spinning the disk at 1 to 30,000 rpm and pressing the sample and buffer into the channel filled with buffer on the gel. Upon introduction of the sample into the separation channel and the sample inlet channel, the sample is concentrated at the gel / buffer interface before entering the separation matrix, similar to sample concentration during conventional slab gel electrophoresis. Electrophoresis is performed at 250 V / cm to achieve separation of DNA fragments, and the cathode (positive electrode) is positioned at the outlet end of the channel end from the sample inlet channel. A laser induced fluorescence detector is positioned at the outlet of the gel filled capillary chamber to detect labeled DNA fragments as described above in Example 2.
Example 13
Extending Spectrophotometer Pathway Length
Spectrophotometric measurements in the rotating structure of the present invention can be limited by the fairly small path length provided by spectrophotometric irradiation across the longitudinal dimension of the disc. The intensity of absorbance of the solution depends not only on the depth of the absorbing layer, but also on the concentration of absorbing molecules (as described in Lambert-Beer Law).
The measuring cell in the rotating microsystem platform of the present invention exhibits a short longitudinal passage length and the side passage length through the disk can be extended (ie, centimeters to millimeters). Certain measurements may reinforce lateral dimensions by introducing light into the detection chamber.
One apparatus for providing longitudinal irradiation in lateral dimensions is shown in FIG. 16. Light exits vertically towards the disc. The mirror is positioned at an angle of 45 degrees to the direction of the irradiation beam, so that light is guided laterally through the detection chamber. Light penetrates the detection cell and is directed back to another 45 ° mirror on a photosensitive detector such as a photodiode or photomultiple tube. These mirrors may be inserted into the disk, molded integrally with the disk, or secured in a plastic or other substrate comprising the disk.
Example 14
Cell counting, checking and monitoring
Provided are methods for identifying a particular cell or cell morphology of a biological sample. For example, the microplatforms of the present invention are prepared by a surface coated with an antibody which is a monocell effective against E. coli. Here the remaining space is blocked by the BSA. The milk sample is placed in contact with the reaction chamber that is introduced onto the disc and includes the surface coated with the antibody. Milk is incubated in the chamber for 30 minutes. The microsystem platform is then rotated to remove unwanted material. A suitable amount of buffer for washing the microsystem chamber is then added to the surface or chamber through the microchannels from the vessel containing the wash buffer, which is released by centrifugal force and opening of the microvalve. In a useful embodiment, the wash buffer comprises an antibody that is an E. coli-specific monocell crosslinked to an enzyme (such as a peroxide). Therefore, the incubation proceeds for 5 minutes. The disc again contains an enzymatic substrate (tetramethylbenzidine and hydrogen peroxide), which is double-spinned with the opening of the appropriate microvalve to remove the wash from the chamber and held in a reagent chamber connected to the reaction chamber by a microvalve-controlled microchannel. Add solution. The amount of E. coli. Bound in the reaction chamber is measured quantitatively with respect to the detection enzyme activity, where activity is determined by spectrophotometric measurement by the appearance of the light absorbing product or the survival of the light absorbing substrate.
It is to be understood that the foregoing has highlighted specific embodiments of the invention and therefore all such improvements and modifications are within the scope of the invention.
The present invention provides an integrated microanalysis and / or microsynthetic chemistry system for performing various biological, biochemical, and chemical analyzes on a micro scale. The present invention provides an apparatus and method for performing such a microscale process on a microplatform such that the fluid on the platform flows in a given channel operated by centrifugal forces resulting from the rotation of this platform.
According to one embodiment of the present invention there is provided a microanalysis and / or microsynthetic chemistry system comprising a combination of two components. The first component is a microplatform, which is a rotatable structure most preferably consisting of a disk, which comprises a sample inlet, a fluid microchannel, a reagent reservoir, a reaction chamber, a detection chamber, and a sample outlet. It is included. The disk is rotated at a speed in the range of about 1 to 30,000 rpm to produce centrifugal acceleration that enables fluid flow. The disc of the invention also preferably comprises a fluid inlet, an air outlet, and an air flow channel. The fluid inlet introduces the sample into the disk for processing and / or analysis. The air outlet, in particular the air flow outlet, provides a means to move air with respect to the fluid so that the fluid can flow on the disk without any obstruction. In addition, the particular area on the disc is preferably provided with components for which the fluid is to be analyzed, ie heat sources, light sources, in particular monochromatic light sources, and sound sources, as well as detectors for each need. Alternatively, some or all of these components may be received on a second disk that is in optical or direct contact with the first disk.
The second component of the present invention is a micromanipulating device corresponding to a disc player and / or a reading device for controlling the function of the disc. Such devices include a mechanism or a motor capable of operating the disk in a loaded or rotating state. In addition, such a device preferably provides a means by which a user can operate a microsystem in a disk, access information or analyze data using a keyboard and computer display.
The present invention provides methods and apparatus for operating a sample consisting of fluids, cells, and / or particles containing or containing an analyte. The microplatform disks of the present invention include sample inlets, microchannels, chambers, valves, heaters, coolers, electrophoresis, and detection systems on the disks and, of course, are not limited to include only these components. Movement of the sample is facilitated by appropriately providing air holes or air movement channels that allow the flow of air and at the same time prevent loss of fluid and / or particles during acceleration.
Preferred embodiments of the disc of the present invention preferably include mechanical, optical, and rheological control structures (or “systems”) microfabricated on substrates made of plastic, silica, quartz, metal, or ceramic. These structures are constructed on a sub-millimeter scale by photolithography, etching, stamping, and other suitable means.
The movement of the sample is controlled by centrifugal or linear acceleration and the selective operation of the valve on the disc.
In a preferred embodiment of the invention, part of the disc is allocated for information processing by standard readers and / or base digital technologies. Data according to the processing and analysis is recorded on the disk surface using digital recording means. In another embodiment, read memory (ROM) on disk includes disk information, instructions, experimental protocols, data analysis, and statistical methods available by the user operating the disk.
Methods of fluid flow by centrifugal acceleration and micromanipulation devices that enable such processes are used in the field of synthesis and analysis of a wide range of fluids and in the detection of analytes, including fluids, particularly biological fluids. Depending on the selective opening of adjacent chambers by capillary, mechanical, or thermally reactive valve mechanisms, chemical and biochemical reactions are performed in the reaction chamber on the disc. The contents of these chambers enter the reaction chamber under the action of centrifugal acceleration. The reaction product can then be used as a reagent for the next reaction and then recovered by the signal of the detection system.

权利要求:
Claims (84)
[1" claim-type="Currently amended] In the centrifugal force micromanipulator,
The apparatus includes a microsystem platform comprising a first flat plane and a second flat plane opposite the first flat plane;
It consists of a base, a rotating means, a power source and a combination of a local operating device including a user interface and operation control means,
The first plane includes a plurality of embedded microchannels and sample input means, the sample input means and microchannels being connected in fluid contact, and the second flat plane facing the first flat plane of the platform Encoded in a set of electromagnetically readable instruments for controlling rotation speed, duration or orientation of the platform,
The rotating means of the micromanipulator is connected to the microsystem platform and rotatably connected thereto.
And move the fluid volume in the microchannel of the platform into the microchannel by centripetal force resulting from the rotational motion of the platform for a time and for a time sufficient to move fluid into the microchannel.
[2" claim-type="Currently amended] In the centrifugal force micromanipulator,
The apparatus includes a microsystem platform comprising a first flat plane and a second flat plane opposite the first flat plane;
It consists of a base, a rotating means, a power source and a combination of a local operating device including a user interface and operation control means,
The first plane includes a plurality of embedded microchannels, a reaction chamber and a reagent chamber, and sample input means, wherein the sample input means, the microchannel, the reaction chamber and the reagent chamber are in fluid contact, and the platform of the platform The second flat plane facing the one flat plane is encoded with a set of electromagnetically readable instruments for controlling rotational speed, duration or direction of the platform,
The rotating means of the micromanipulator is connected to the microsystem platform and rotatably connected thereto.
And move the fluid volume in the microchannel of the platform into the microchannel by centripetal force resulting from the rotational motion of the platform for a time and for a time sufficient to move fluid into the microchannel.
[3" claim-type="Currently amended] In the centrifugal force micromanipulator,
The apparatus includes a microsystem platform comprising a first flat plane and a second flat plane opposite the first flat plane;
It consists of a base, a rotating means, a power source and a combination of a local operating device including a user interface and operation control means,
The first plane includes a plurality of embedded microchannels, a reaction chamber and a reagent chamber, and sample input means, wherein the sample input means, the microchannel, the reaction chamber and the reagent chamber are in fluid contact, and the microchannel, Fluid motion from the reaction chamber and the reagent chamber is controlled by a microvalve connected thereto and the second flat plane facing the first flat plane of the platform is electronic for controlling the speed, duration or direction of the platform. Encoded as a set of miraculously readable instruments,
The rotating means of the micromanipulator is connected to the microsystem platform and rotatably connected thereto.
And move the fluid volume in the microchannel of the platform into the microchannel by centripetal force resulting from the rotational motion of the platform for a time and for a time sufficient to move fluid into the microchannel.
[4" claim-type="Currently amended] 2. The apparatus of claim 1, wherein the first and second flat planes of the microsystem platform form a disk.
[5" claim-type="Currently amended] The rotational motion of the microsystem platform as recited in claim 1, wherein the first and second flat planes of the microsystem platform form centrifugally positioned holes coupled to the spindle on the micromanipulator. Device which is delivered to.
[6" claim-type="Currently amended] The apparatus of claim 1, wherein the microsystem platform is comprised of a material selected from the group consisting of organic materials, inorganic materials, crystals, and amorphous materials.
[7" claim-type="Currently amended] 7. The apparatus of claim 6, wherein the microsystem platform is also comprised of a material selected from the group consisting of silicon, silica, quartz, ceramic, metal or plastic.
[8" claim-type="Currently amended] 5. The apparatus of claim 4, wherein the microsystem platform consists of a disk having a radius of about 1 to 25 cm.
[9" claim-type="Currently amended] The microsystem platform of claim 1, wherein the microsystem platform has a thickness of about 0.1 to 100 mm, the cross sectional dimension of the microchannel between the first and second flat planes is less than 500 μm, and 1 of the cross sectional dimension of the platform. To 90%.
[10" claim-type="Currently amended] 11. The microsystem platform of claim 10, wherein the microsystem platform has a thickness of about 0.1 to 100 mm, the cross sectional dimension of the reaction chamber or reagent chamber between the first and second flat planes is less than 500 micrometers, and the thickness of the platform Device, characterized in that from 1 to 75% of.
[11" claim-type="Currently amended] The apparatus of claim 1, wherein the microsystem platform rotates at a rotational speed of about 1 to 30,000 rpm.
[12" claim-type="Currently amended] 2. The microsystem platform of claim 1, wherein the microsystem platform comprises a plurality of simple input means, a reagent chamber, a reaction chamber, and a plurality of microchannels connected to and inserted into the reaction chamber, wherein the volume of fluid containing the sample is from the sample input means. Wherein the volume of reagent is moved in and out of the reaction chamber from and within the reaction chamber by centripetal forces generated by rotation of the microsystem platform.
[13" claim-type="Currently amended] 4. The microsystem platform of claim 1, wherein the microsystem platform has a detection chamber inserted into the first flat plane of the platform and connected to the microchannels, and the micromanipulation device includes a detection means such that the detection chamber is provided by the detection means. Wherein the device is analyzed to yield.
[14" claim-type="Currently amended] 14. The apparatus of claim 13, wherein the means for detection on the micromanipulator is aligned with the detection chamber on the platform by rotational movement of the microsystem platform.
[15" claim-type="Currently amended] 14. An apparatus according to claim 13, wherein said detection means consists of a light source and a photodetector.
[16" claim-type="Currently amended] 16. The apparatus of claim 15, wherein the light source projects a detection chamber and is reflected transversely through the detection chamber and detected by a photodetector.
[17" claim-type="Currently amended] 17. The apparatus of claim 16, wherein the detection chamber on the microsystem platform is optically transparent.
[18" claim-type="Currently amended] 15. The device according to claim 14, wherein the detection means is stationary and tested at a frequency such as the rotation frequency of the platform or other members of the platform.
[19" claim-type="Currently amended] 19. An apparatus according to claim 18, wherein said detection means is comprised of a stroboscopic light source.
[20" claim-type="Currently amended] 20. The apparatus according to claim 19, wherein said detection means consists of a monochromatic light source.
[21" claim-type="Currently amended] 14. An apparatus according to claim 13, wherein said detection means detects absorbance, fluorescence, chemiluminescence, light scattering or radioactivity.
[22" claim-type="Currently amended] The device of claim 1, further comprising a temperature control factor in thermal contact with the microplatform.
[23" claim-type="Currently amended] 2. The apparatus of claim 1, further comprising a heat detection unit in thermal contact with the microplatform.
[24" claim-type="Currently amended] 2. An apparatus according to claim 1, wherein the microsystem platform comprises filtering means connected to a microchannel.
[25" claim-type="Currently amended] 2. The apparatus of claim 1, wherein the microsystem platform has a mixing factor connected to the reaction chamber or microchannel.
[26" claim-type="Currently amended] 27. The apparatus of claim 25, wherein the microsystem platform comprises a static mixer having a smooth side of the reaction chamber or microchannel.
[27" claim-type="Currently amended] 4. The microsystem platform of claim 3, wherein the microsystem platform comprises a plurality of microvalve operatively connected to the microchannel, reaction chamber, reagent chamber, sample input means, sample outlet port, etc., wherein fluid flow on the microsystem platform comprises a microvalve. Device controlled by opening and closing.
[28" claim-type="Currently amended] 28. The apparatus of claim 27, wherein the microsystem consists of a capillary microvalve connected to the reaction chamber or microchannel.
[29" claim-type="Currently amended] 2. The apparatus of claim 1, wherein the microsystem platform comprises a plurality of air channels, exhaust air ports, and air displacement channels.
[30" claim-type="Currently amended] The device according to claim 1, wherein the rotating means of the device consists of an electric motor.
[31" claim-type="Currently amended] The apparatus of claim 1, wherein the apparatus comprises rotational motion control means for controlling rotational acceleration and speed of the microsystem platform.
[32" claim-type="Currently amended] 10. The device of claim 1, wherein the device comprises a user interface comprised of an alphanumeric keypad and coordinator.
[33" claim-type="Currently amended] The apparatus of claim 1 wherein the apparatus comprises an alternating current or direct current power source.
[34" claim-type="Currently amended] 10. The apparatus of claim 1, wherein the microsystem platform includes an electrical connector in contact with an electrical connector connected to a micromanipulator.
[35" claim-type="Currently amended] The device of claim 1, wherein the device consists of a memory and a microprocessor coupled to the device.
[36" claim-type="Currently amended] An apparatus according to claim 1, wherein said apparatus comprises reading or writing means.
[37" claim-type="Currently amended] 37. An apparatus according to claim 36, wherein said reading means comprises small disk laser reading means.
[38" claim-type="Currently amended] An apparatus according to claim 36, wherein said writing means is comprised of small disk writing means.
[39" claim-type="Currently amended] 2. The apparatus of claim 1, wherein the second flat plane of the microsystem platform is coded in machine instruction language.
[40" claim-type="Currently amended] 40. The apparatus of claim 39, wherein the machine instruction language controls the acquisition or analysis of data from the platform, the storage and retrieval of data, the transfer to another device, the direct diagnostics of the device, or the operation of the platform.
[41" claim-type="Currently amended] 2. The apparatus of claim 1, wherein the micromanipulator comprises a permanent storage memory or a read only memory encoded in a machine instruction language.
[42" claim-type="Currently amended] 42. The apparatus of claim 41, wherein the machine instruction language controls the acquisition or analysis of data from the platform, the storage and retrieval of data, the transfer to another device, the diagnostic performance of the device directly, or the operation of the platform.
[43" claim-type="Currently amended] 2. The apparatus of claim 1, further comprising first and second microsystem platforms in contact with each other across one plane of each microsystem platform.
[44" claim-type="Currently amended] The apparatus of claim 1, wherein the microsystem platform is rotated at a speed of about 1 to 30,000 rpm.
[45" claim-type="Currently amended] The apparatus of claim 1, wherein the fluid on the microsystem platform is moved within the microchannel of the platform at a fluid velocity of about 0.1 cm / sec to 1,000 cm / sec.
[46" claim-type="Currently amended] The device of claim 1 for measuring an analyte in a biological sample,
The microsystem platform,
Multiple sample inlet ports, each operatively connected to the platform, arranged concentrically about the center of the platform,
A plurality of microchannels operatively connected to the platform and arranged radially spaced from the center of the platform,
A number of reagent chambers containing reagent properties for the analyte to be measured,
A plurality of analyte chambers arranged circumferentially around the outer edge of the microplatform,
Discharge of reagents from each reagent chamber is controlled by a microvalve and the plurality of microchannels are also operatively connected to the reagent chamber,
The movement of the biological sample through the microchannels from the sample inlet port, and the movement of the reagents through the microchannels from the reagent chamber are driven by centripetal forces generated by the rotational movement of the microsystem platform.
[47" claim-type="Currently amended] 49. The device of claim 46, wherein the biological sample consists of blood, urine, sap, plasma, saliva, semen, amniotic fluid.
[48" claim-type="Currently amended] 47. The apparatus of claim 46, wherein the analyte detection chamber is optically transparent.
[49" claim-type="Currently amended] 47. The apparatus of claim 46, wherein the apparatus further comprises an electrical wiring between the electrical controller unit and each microvalve, the opening and closing of the valve being controlled by an electrical signal from the controller unit.
[50" claim-type="Currently amended] 47. The apparatus of claim 46, wherein the microchannels are arranged linearly from the center to the perimeter of the platform.
[51" claim-type="Currently amended] 49. The apparatus of claim 46, wherein the microchannels are arranged concentrically from the center to the circumference of the platform.
[52" claim-type="Currently amended] 47. The apparatus of claim 46, wherein the micromanipulator comprises detection means.
[53" claim-type="Currently amended] 49. The apparatus of claim 46, wherein the detection means is stationary and tests the analyte detection chamber at a frequency equal to the rotational frequency of multiple components of the platform or platform.
[54" claim-type="Currently amended] 49. An apparatus according to claim 46, wherein said detection means consists of a stroboscopic light source.
[55" claim-type="Currently amended] 47. An apparatus according to claim 46, wherein said detection means consists of a monochromatic light source.
[56" claim-type="Currently amended] 49. The apparatus of claim 46, wherein said detecting means detects fluorescence, chemiluminescence, light scattering or radioactivity.
[57" claim-type="Currently amended] In a method of measuring an analyte in a biological sample,
Applying a biological sample to the sample inlet port of the microsystem platform of claim 46;
Positioning the microsystem platform on the micromanipulator;
Providing rotational motion to the microsystem platform for a period of time at a speed sufficient to operate a biological sample containing the analyte from the sample inlet port through the microchannel;
Opening each microvalve controlling the discharge of the reagent from the reagent chamber by a signal generated from the control unit continuously for a predetermined time so that the reagent is transferred into the microchannel and mixed with the biological sample;
Observing the mixture of reagent and biological sample in an analyte detection chamber such that a detector comprising the device detects a signal proportional to the analyte provided in the biological sample;
Recording the measurement of the analyte in the biological sample.
[58" claim-type="Currently amended] 58. The method of claim 57, wherein said biological sample consists of blood, urine, sap, plasma, saliva, semen, amniotic fluid.
[59" claim-type="Currently amended] 59. The method of claim 57, wherein measurements of the analyte in the sample are recorded on the device or microplatform on the microplatform and on the device.
[60" claim-type="Currently amended] 59. The method of claim 57, wherein the analyte detection chamber on the microsystem platform is optically transparent.
[61" claim-type="Currently amended] 59. The method of claim 57, wherein the signal detected in the analyte detection chamber is detected at a frequency equal to the rotational frequency of the platform or multiple components of the platform.
[62" claim-type="Currently amended] 59. The method of claim 57, wherein said detected signal consists of a monochromatic light source.
[63" claim-type="Currently amended] 63. The method of claim 62, wherein the detected signal is comprised of a fluorescent signal, chemiluminescent signal, or color signal.
[64" claim-type="Currently amended] The apparatus of claim 1 for detecting particulates or gases having an environmental sample,
The microsystem platform,
A plurality of sample inlet ports having air inlet vents and connected funnel channels, each operatively connected to the platform, arranged concentrically about the center of the platform,
A plurality of microchannels operatively connected to the platform and arranged radially spaced from the center of the platform,
A plurality of reagent chambers containing reagent properties for the particulate or gas to be measured,
Having a plurality of particulate or gas detectors arranged circumferentially around the outer edge of the microplatform,
The discharge of reagent from each reagent chamber is controlled by a microvalve, the microvalve is in electrical contact with a controller unit, the plurality of microchannels are also operatively connected to the reagent chamber,
The movement of the environmental sample through the microchannels from the sample inlet port, and the movement of the reagents through the microchannels from the reagent chamber are driven by centripetal forces generated by the rotational movement of the microsystem platform.
[65" claim-type="Currently amended] 65. The device of claim 64, wherein the environmental sample consists of air, water, soil, or destroyed biological material.
[66" claim-type="Currently amended] 65. The apparatus of claim 64, wherein the detector consists of a gas sensor chip.
[67" claim-type="Currently amended] 65. The apparatus of claim 64, wherein the detector comprises an optically transparent particulate collection chamber.
[68" claim-type="Currently amended] 68. The apparatus of claim 67, wherein the detector also comprises a coherent light source.
[69" claim-type="Currently amended] 69. The apparatus of claim 68, wherein the particulates are detected by light scattering.
[70" claim-type="Currently amended] 65. The apparatus of claim 64, wherein the detector comprises a particulate collection chamber operatively microchanneled in a reagent chamber with reagents for chemically testing the particulates.
[71" claim-type="Currently amended] A method of detecting a gas or particulate having an environmental sample,
Contacting the environmental sample with a sample inlet port of the microsystem platform of claim 46;
Positioning the microsystem platform on the micromanipulator;
Providing rotational motion to the microsystem platform for a period of time at a speed sufficient to operate a gaseous or particulate environmental sample from the sample inlet port through the microchannel;
Opening each microvalve controlling the discharge of the reagent from the reagent chamber by a signal generated from the control unit continuously at a predetermined time so that the reagent is transferred into the microchannel and mixed with the environmental sample;
Detecting a gaseous or particulate component or mixture of environmental sample and environmental sample directly in the gas or particulate detection chamber such that the detector detects a signal proportional to the gas or particulate provided to the environmental sample;
Recording a measurement of a gas or particulate in said environmental sample.
[72" claim-type="Currently amended] 72. The method of claim 71, wherein the environmental sample consists of air, water, soil, or destroyed biological material.
[73" claim-type="Currently amended] 72. The method of claim 71 wherein the gas is detected by a gas sensor chip.
[74" claim-type="Currently amended] 72. The method of claim 71, wherein the particulates are detected in an optically transparent particulate collection chamber.
[75" claim-type="Currently amended] 72. The method of claim 71, wherein the particulates are also detected by coherent light scattering.
[76" claim-type="Currently amended] 72. The method of claim 71, wherein the particulates are detected in a particulate collection chamber operatively microchanneled to a reagent chamber with reagents for chemically testing the particulates, wherein the particulates are reagents by rotation of the platform and operation of the microvalve. After the discharge of the method characterized in that the reaction is mixed in the reagents in the microchannel.
[77" claim-type="Currently amended] 2. The microsystem platform of claim 1, wherein the microsystem platform consists of a stack of thin film disks having a sample inlet port, a reactant reservoir, a reaction chamber, a sample outlet port, and a microchannel, each of the membrane disks of the stack having its own inclusion. And provide a platform of the invention.
[78" claim-type="Currently amended] The apparatus of claim 1, wherein the red blood cell volume fraction value is determined from a blood sample.
The microsystem platform consists of a radial alignment of microchannels with a diameter of about 100 μm, the microchannels being treated with heparin to prevent solidification and opening at ends near the center of the disc,
The apparatus has a micromanipulating device, has recording means and a coherent light source operably connected,
The movement of the blood sample through the microchannels is driven by centripetal forces generated by the rotational movement of the microsystem platform.
[79" claim-type="Currently amended] 80. The apparatus of claim 78, wherein the coherent light source is mounted on a mobile truck radially aligned from the center of rotation of the platform.
[80" claim-type="Currently amended] 79. The apparatus of claim 78 further comprising a crake electrode operatively connected to each of the microchannels of the microsystem platform,
The electrode is in contact with a blood sample in the microchannel.
[81" claim-type="Currently amended] 79. The apparatus of claim 78, further comprising: a severing electrode operatively connected to each of the microchannels of the microsystem platform,
The electrode is in contact with a blood sample in the microchannel.
[82" claim-type="Currently amended] A method for determining an erythrocyte volume fraction value from a blood sample,
Applying a blood sample to the distal end of the microchannel of the microsystem platform of claim 78,
Positioning the microsystem platform on the micromanipulator;
Providing rotational motion to the microsystem platform for a period of time at a speed sufficient to operate a red blood cell having a blood sample to be moved along a region of the microchannel;
Scanning the microchannel along the length of the microchannel with a coherent light source,
Detecting changes in light scattering at locations along the microchannels forming a boundary between red blood cells and plasma,
Recording a boundary position for each microchannel;
Comparing said boundary position for each microchannel with a standard curve representing an erythrocyte volume fraction value for said boundary position and recording the erythrocyte volume fraction determined thereby.
[83" claim-type="Currently amended] A method of determining blood oxygenation from a blood sample,
Applying a blood sample to the distal end of the microchannel of the microsystem platform of claim 80;
Positioning the microsystem platform on the micromanipulator;
Providing rotational motion to the microsystem platform for a period of time at a rate sufficient to operate the blood sample to contact the crake electrode connected to the microchannel;
Means for detecting blood oxygenation values for blood samples,
Recording the blood oxygenation determined thereby.
[84" claim-type="Currently amended] The method of claim 1, wherein the microsystem platform comprises a plurality of simple input means, a reactant reservoir, a reaction chamber, a microvalve and a microchannel connected to and inserted into the reaction chamber,
The microsystem platform consists of an aligned stack structure,
The first layer of the laminated structure consists of a simple input means, a reactant reservoir, a reaction chamber, and a microvalve, the second layer consists of a microvalve, and the third layer consists of electrical connections from the microvalve to the electrical control unit. And the fourth layer consists of a sealing layer, the layers being stacked on top of the solid substrate of the microsystem platform and integrated.

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同族专利:

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公开号 | 公开日

---

NO982563D0|1998-06-04|

---

CN1208464A|1999-02-17|

---

AT291225T|2005-04-15|

---

AU702403B2|1999-02-18|

---

WO1997021090A1|1997-06-12|

---

JP2002503331A|2002-01-29|

---

EP0865606A1|1998-09-23|

---

AU1283397A|1997-06-27|

---

MX9804463A|1998-12-31|

---

DE69634490D1|2005-04-21|

---

EP0865606B1|2005-03-16|

---

NO982563L|1998-08-05|

---

CA2239613A1|1997-06-12|

---

DE69634490T2|2006-03-02|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1995-12-05|Priority to US821595P

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1995-12-05|Priority to US60/008,215

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1995-12-06|Priority to US826795P

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1995-12-06|Priority to US60/008,267

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1995-12-18|Priority to US881995P

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1995-12-18|Priority to US60/008,819

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1996-08-12|Priority to US2375696P

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1996-08-12|Priority to US60/023,756

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1996-12-05|Application filed by 피.리차드 이글링, 가메라 바이오사이언스 코포레이션

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1996-12-05|Priority to PCT/US1996/019514

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1999-09-27|Publication of KR19990072013A

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2001-11-15|Application granted

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2001-11-15|Publication of KR100306951B1

优先权:

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申请号 | 申请日 | 专利标题

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US821595P| true| 1995-12-05|1995-12-05|

---

US60/008,215|1995-12-05|

---

US826795P| true| 1995-12-06|1995-12-06|

---

US60/008,267|1995-12-06|

---

US881995P| true| 1995-12-18|1995-12-18|

---

US60/008,819|1995-12-18|

---

US2375696P| true| 1996-08-12|1996-08-12|

---

US60/023,756|1996-08-12|

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PCT/US1996/019514|WO1997021090A1|1995-12-05|1996-12-05|Devices and methods for using centripetal acceleration to drive fluid movement in a microfluidics system with on-board informatics|