MICROFLUIDIC DEVICE, METHOD, SYSTEM AND KIT

专利摘要:
microfluidic devices and methods for their production and use. The present invention relates to methods and systems for analyzing a sample. a microfluidic device for performing an analysis of a sample (eg, biological sample) is described having a sample application site, a porous component and a flow channel. the porous component provides uniform dissolution of a reagent and uniform mixing of sample and reagent without filtering the sample.
公开号:BR112016009958B1
申请号:R112016009958-3
申请日:2014-11-05
公开日:2021-08-03
发明作者:Scott Joseph Bornheimer；Jeffrey Sugarman；Wei Huang；Edward Michael Goldberg；Ming Tan
申请人:Becton, Dickinson And Company；
IPC主号:

专利说明:

[001] This application is related to United States Provisional Patent Application No. Serial 61/900,590, filed November 6, 2013, the disclosure of such application being incorporated herein by reference. INTRODUCTION
[002] Remote laboratory diagnosis includes the steps of obtaining a biological sample from a subject, performing sample analyzes to determine the presence or concentration of one or more target analytes, and providing a diagnosis to the subject at a single location. Remote laboratory diagnosis provides faster and often less costly results for the subject than diagnostic testing that requires obtaining a sample at one location and performing sample analysis at a different location.
[003] Rapid diagnosis of infectious diseases from a single drop of blood using a low-cost and easy technology available at the point of care would greatly improve global health initiatives. Flow cytometry based on microparticle immunoassays provides excellent accuracy and multiplexing, but is unsuitable for remote laboratory settings due to complex sample preparation and costly instrumentation. In view of the above, various fields of medicine and biotechnology would significantly advance with the availability of techniques capable of being used in a remote laboratory context, allowing for easy and flexible measurements of cell markers, particularly in biological fluids such as blood. SUMMARY
[004] Aspects of the present invention include a microfluidic device for analyzing a sample. Microfluidic devices in certain embodiments include a sample application site, a flow channel in fluid communication with the sample application site, and a porous component that contains porous matrix and an analysis reagent positioned between the sample application site. sample application and the flow channel. Suitable systems and methods for analyzing a sample, such as a biological sample, using the present microfluidic devices are also described.
[005] As summarized above, aspects of the present disclosure include a microfluidic device for analyzing a sample having a sample application site, a flow channel in fluid communication with the application site, and a porous component positioned between the site. of sample application and the flow channel. In embodiments, the porous component includes a porous matrix and an analysis reagent. In some cases, the porous matrix is a frit, such as a glass frit. In other cases, the porous matrix is a polymeric matrix. In some embodiments, the porous matrix is configured to be non-filtering with respect to sample components. In certain cases, the porous matrix is configured to provide mixing of the assay reagent with the sample flowing through the porous matrix. The porous matrix can have pores with diameters between 1 μm and 200 μm and pore volumes between 1 μL and 25 μL. For example, the pore volume can be between 25% and 75% of the porous matrix volume, such as between 40% and 60% of the porous matrix volume.
[006] The analysis reagent includes a reagent binding to one or more components of the sample. In some embodiments, the reagent is an analyte-specific binding member. For example, the analyte-specific binding member can be an antibody or antibody fragment. In certain cases, the analyte-specific binding member is an antibody that specifically binds to a compound, such as CD14, CD4, CD45RA, CD3 or a combination thereof. In some embodiments, the analyte-specific binding member is linked to a detectable label, such as an optically detectable label. For example, the optically detectable marker can be a fluorescent dye such as rhodamine, coumarin, cyanine, xanthene, polymethine, pyrene, boron difluoro-dipyrromethene, naphthalimide, phycobiliprotein, peridinin chlorophyll proteins or a combination thereof. In certain cases, the dye is phycoerythrin (PE), Phycoerythrin - Cyanine 5 (PE-Cy5) or allophycocyanin APC. In some embodiments, buffers include bovine serum albumin (BSA), trehalose, polyvinylpyrrolidone (PVP), or 2-(N-morpholino) ethanesulfonic acid or a combination thereof. For example, the buffer can include BSA, trehalose and PVP. Buffers can also include one or more chelating agents, such as ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), 2,3- 1-sulfonic dimercaptopropane (DMPS), and 2,3-dimercaptosuccinic acid (DMSA). In certain embodiments, the buffer includes EDTA. The assay reagent can be present in the porous matrix as a liquid. In other cases, the assay reagent is dry. In still other cases, the assay reagent is lyophilized.
[007] In some modalities, the flow channel is configured to receive a sample with a volume ranging from 1 mL to 1000 mL. In certain cases, the flow channel is a capillary channel configured to transport the sample through the flow channel by capillary action. In certain embodiments, the flow channel includes one or more optically transmissive walls. In one example, the flux channel is optically transmissive to ultraviolet light. In another example, the flux channel is optically transmissive to visible light. In yet another example, the flux channel is optically transmissive to near-infrared light. In yet another example, the flux channel is transmissive to ultraviolet light and visible light. In yet another example, the flux channel is transmissive to visible light and near-infrared light. In yet another example, the flux channel is transmissive to ultraviolet light, visible light and near-infrared light.
[008] Microfluidic devices according to certain modalities include a porous frit that contains microchannels that define a tortuous path that is of sufficient length for mixing a reagent and a sample. The pore volume can be 40 to 60% of the total volume of the porous frit, such as 2 µL or more, such as 5 µL, 10 µL and including 20 µL or more. In some embodiments microchannels provide flow through substantially all components of the sample. In some embodiments the microchannels have an average diameter through the pores between 5 μm and 200 μm such as between 5 μm and 60 μm or between 30 μm and 60 μm.
[009] The analysis mix includes a reagent and buffer. In some cases, the assay mixture provides for substantially uniform dissolution of the reagent in the sample over a predetermined period of time. The predetermined period of time may be between 5 seconds and 5 minutes, such as between 20 seconds and 3 minutes, or between 50 seconds and 2 minutes. In some embodiments, buffer components include bovine serum albumin (BSA), trehalose, and polyvinylpyrrolidone (PVP). The weight ratio of BSA:Trehalose:PVP can be 21:90:1. The total weight of the buffer components can range from 0.01 g/μL to 2 g/μL of the pore volume of the porous matrix. In some embodiments the buffer components include ethylenediaminetetraacetic acid (EDTA). In certain embodiments, the buffer components comprise 2-(N-morpholino) ethanesulfonic acid (MES). In some cases, the reagent includes one or more antibodies or antibody fragments conjugated to a detectable marker. Antibodies or antibody fragments can bind to a target, such as a target selected from CD14, CD4, CD45RA, CD3 or a combination thereof. In some cases, the detectable marker is a fluorescent dye. For example, the dye can be a compound such as rhodamine, coumarin, cyanine, xanthene, polymethine, pyrene, boron difluoro-dipyrromethene, naphthalimide, phycobiliprotein, peridinin chlorophyll proteins, their conjugates, and combinations thereof. In some embodiments the dye can be phycoerythrin (PE), Phycoerythrin -Cyanine 5 (PE-Cy5) or allophycocyanin APC. In embodiments of the present invention, the assay mixture can include enzymes, substrates, catalysts, nucleic acids, or a combination thereof. In certain cases, microfluidic devices can further include a biological sample such as blood, urine, saliva or a tissue sample.
[0010] Aspects of the present invention also include a method for analyzing a sample for an analyte, where the method includes contacting a sample with a sample application site of a microfluidic device having a flow channel in fluid communication with the sample application site and a porous component positioned between the sample application site and the flow channel, illuminating the sample in the flow channel with a light source, and detecting the light from the sample to determine the presence or concentration of one or more components in the sample.
[0011] In some embodiments, the sample is mixed with an analysis reagent present in the porous matrix of the porous component by moving the sample through the porous matrix. The movement of the sample through the porous matrix is, in certain embodiments, non-filtering with respect to the components of the sample. In some embodiments, the flow channel is a capillary channel and the sample is moved through the porous matrix by capillary action. Mixing the sample with the assay reagent can include labeling one or more components of the sample with a detectable label. In some cases, labeling includes contacting one or more components of the sample with an analyte-specific binding member, such as an antibody or antibody fragment. In certain cases, the analyte-specific binding member is an antibody that specifically binds to a compound, such as CD14, CD4, CD45RA, CD3 or a combination thereof. In some embodiments, the analyte-specific binding member is coupled to a detectable label, such as an optically detectable label. Examples of optically detectable labels include fluorescent dyes such as rhodamine, coumarin, cyanine, xanthene, polymethine, pyrene, boron difluoro-dipyrromethene, naphthalimide, phycobiliprotein, peridinin chlorophyll proteins, their conjugates, and combinations thereof. In some embodiments, the dye is phycoerythrin (PE), Phycoerythrin -Cyanine 5 (PE-Cy5) or allophycocyanin APC.
[0012] Methods according to some modalities include illuminating the sample in the flux channel with a broad spectrum light source. In some embodiments, the broad spectrum light source is an ultraviolet light source, a visible light source or an infrared light source, or a combination thereof. In certain embodiments, the sample is illuminated with light having a wavelength between 200 nm and 800 nm.
[0013] In some embodiments, the methods include detecting light from the sample in the flow channel. The light detected from the sample can include fluorescence, transmitted light, scattered light, or a combination thereof. In some cases, detection methods include sample fluorescence. In certain cases, detecting light from the sample includes capturing an image of the sample in the flow channel.
[0014] Methods for analyzing a sample, such as a biological sample, with the microfluidic devices concerned are also provided. In some embodiments, the methods include applying a liquid sample to a sample application site that is in fluid communication with a porous element and a capillary channel, directing the sample flow from the sample application site, through the porous element, for the capillary channel. The capillary channel may include an optically transmissive wall and the porous element comprises at least one optically active reagent and one or more buffer components.
[0015] The methods may further include dissolving the reagent in the sample where the dissolution of the reagent is substantially constant over a predetermined period of time, such as between 5 seconds and 5 minutes, or between 20 seconds and 3 minutes or between 1 minute and 2 minutes. In some embodiments, mixing of sample and reagent is done on a porous frit that provides a series of microchannels that define a tortuous path that is long enough to mix sample and reagent. Mixing can facilitate binding of the reagent to one or more components of the sample and is followed by optically evaluating the sample through the optically transmissive wall. The mixture can be passive (diffusive), convective, active or any combination thereof. The sample can flow by capillary action force through the porous element and through the capillary channel. In certain embodiments, optical evaluation includes obtaining an image of the sample through a transmissive wall, determining a background signal that corresponds to the unbound reagent and sample, and subtracting the background signal from the sample image. In some embodiments, the background signal is substantially constant (varies by 75% or less, such as 50%) along the transmissive wall. In some cases, the sample flows through the porous element substantially unfiltered. In embodiments, the sample can be a biological sample, such as blood, urine, tissue, saliva, or the like. In some embodiments, the optically active reagent includes a fluorescently labeled antibody or antibody fragment and provides the mixture to form one or more fluorescently labeled components in the biological sample.
[0016] Aspects of the present disclosure also include systems for practicing the present methods. Systems in accordance with certain embodiments include a light source, an optical detector for detecting one or more wavelengths of light, and a microfluidic device for analyzing a sample having a sample application site, a flow channel at fluid communication with the application site and a porous component positioned between the sample application site and the flow channel. DEFINITION OF SELECTED TERMINOLOGY
[0017] In general, terms used herein, unless otherwise defined, have meanings corresponding to their conventional use in fields relating to the invention, including analytical chemistry, biochemistry, molecular biology, cell biology, micros copying, image analysis, and the like, as depicted in the following treatises: Alberts et al., Molecular Biology of the Cell, Fourth Edition (Garland, 2002); Nelson and Cox, Lehninger Principles of Biochemistry, Fourth Edition (W.H. Freeman, 2004); Murphy, Fundamentals of Light Microscopy and Electronic Imaging (Wiley-Liss, 2001); Shapiro, Practical Flow Cytometry, Fourth Edition (Wiley-Liss, 2003); Owens et al. (Editors), Flow Cytometry Principles for Clinical Laboratory Practice: Quality Assurance for Quantitative Immunophenotyping (Wiley-Liss, 1994); Ormerod (Editor) Flow Cytometry: A Practical Approach (Oxford University Press, 2000); and the like.
[0018] "Antibody" or "immunoglobulin" means a protein, naturally or synthetically produced by recombinant or chemical means, which is capable of specifically binding to a particular antigen or antigenic determinant. Antibodies are typically heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. "Antibody fragment", and all grammatical variants thereof as used herein are defined as a portion of an intact antibody comprising the antigen binding site or variable region of the intact antibody, where the portion is free of the constant domains of the chain. heavy (i.e., CH2, CH3 and CH4, depending on antibody isotype) from the Fc region of the intact antibody. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments. The term "monoclonal antibody" (mAb) as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies making up the population are identical except for possible naturally occurring mutations that may be present in smaller quantities. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized by hybridoma culture, uncontaminated by other immunoglobulins. Guidance on the production and selection of antibodies for use in immunoassays can be found in readily available texts and manuals, for example, Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, 1988); Howard and Bethell, Basic Methods in Antibody Production and Characterization (CRC Press, 2001); Wild (editor) The Immunoas Say Handbook (Stockton Press, New York, 1994), and the like.
[0019] "Microfluidic device" means an integrated system of one or more chambers, ports and channels that are interconnected and in fluid communication and designed to carry out an analytical reaction or process, either alone or in co-operation with a device or instrument that provides support functions, such as sample introduction, fluid and/or reagent conduction means, temperature control, detection systems, data collection and/or integration systems, and the like. Microfluidic devices may further include valves, pumps, and specialized functional coatings on interior walls, for example, to prevent adsorption of sample components or reagents, facilitate reagent movement by electroosmosis, or the like. Such devices are generally manufactured on or as a solid substrate, which may be glass, plastic, or other solid polymeric materials, and typically have a planar shape to facilitate detection and monitoring of sample and reagent movement, especially through optical methods. or electrochemicals. Characteristics of a microfluidic device typically include cross-sectional dimensions of less than a few hundred square micrometers, and passageways typically have capillary dimensions, for example, with maximum cross-sectional dimensions from about 500 µm to about 0.1 µm. Microfluidic devices typically have volume capacities in the range of 1 µL to less than about 10 nL, eg 10100 nL. The fabrication and operation of microfluidic devices are well known in the art, as exemplified by the following references which are incorporated by reference: Ramsey, US Patent Publication 6,001,229; 5,858,195; 6,010,607; and 6,033,546; Soane et al., US Patent Publication 5,126,022 and 6,054,034; Nelson et al., US Patent Publication 6,613,525; Maher et al., US Patent Publication 6,399,952; Ricco et al., International Patent Publication WO 02/24322; Bjornson et al., International Patent Publication WO 99/19717; Wilding et al., US Patent Publication 5,587,128; 5,498,392; Sia et al., Electrophoresis, 24: 3563-3576 (2003); Unger et al., Science, 288: 113-116 (2000); Enzelberger et al., US Patent Publication 6,960,437.
[0020] "Sample" means an amount of material of biological, environmental, medical, or patient origin in which the predetermined detection or measurement of cells, particles, spheres and/or analytes is sought. A sample can comprise material from natural sources or from artificial sources, such as tissue cultures, fermentation cultures, bioreactors and the like. Samples can comprise animal, including human, fluid, solid (eg feces) or tissue, as well as liquid and solid food products and feed and ingredients such as dairy products, vegetables, meat and meat by-products, and waste . Samples may include materials taken from a patient, including, but not limited to, cultures, blood, saliva, spinal cerebral fluid, pleural fluid, milk, lymph, sputum, semen, needle aspiration, and the like. Samples can be obtained from all the various families of domestic animals, as well as wild or feral animals, including but not limited to animals such as ungulates, bear, fish, rodents, etc. Samples may include environmental materials such as surface matter, soil, water and industrial samples, as well as samples obtained from food and dairy processing instruments, appliances, equipment, utensils, disposables and non-disposable items. These examples are not to be interpreted as limiting the types of samples applicable to the present invention. The terms "sample", "biological sample" and "specimen" are used interchangeably. BRIEF DESCRIPTION OF THE FIGURES
[0021] The invention can be better understood from the following detailed description when read in conjunction with the accompanying drawings. Included in the drawings are the following figures:
[0022] Figure 1 shows an illustration of a top view of a microfluidic device according to certain embodiments.
[0023] Figure 2A shows an illustration of a top view of a microfluidic device according to certain embodiments.
[0024] Figure 2B represents a schematic diagram showing the side view of a microfluidic device according to certain embodiments.
[0025] Figure 3A represents an illustration of detection components of a sample in the microfluidic device according to certain embodiments.
[0026] Figure 3B represents an illustration of image enhancement of components of a sample in the microfluidic device according to certain embodiments. DETAILED DESCRIPTION
[0027] The microfluidic device and method for using it are described. The device may include a sample application site in communication with a porous component and a flow channel. The dimensions of the device can provide a capillary action to be the main force for transmitting a sample through the porous element and the flow channel. The device can be used to evaluate analytes or components of a sample that have been labeled with a detectable marker. The porous component is formed by a porous matrix, such as a frit and an analysis reagent. The porous component can provide a matrix for the assay reagent and be of sufficient dimensions to provide a tortuous path for mixing the sample and an assay reagent. Mixing can be passive or convective and requires no additional force other than capillary force to provide a sample that is substantially uniformly mixed with an assay reagent upon exiting the porous matrix. The assay reagent can provide uniform dissolution of a reagent such as a detectable label in the sample over a defined period of time.
[0028] The practice of the present invention may utilize, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), cell biology, immunoassay technology, microscopy, image analysis and analytical chemistry, which are within the scope of competence in the technique. Such conventional techniques include, but are not limited to, fluorescent signal detection, image analysis, selecting light sources and optical signal detection components, tagging biological cells, and the like. Such conventional techniques and descriptions can be found in reference laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press); Murphy, Fundamentals of Light Microscopy and Electronic Imaging (Wiley-Liss, 2001); Shapiro, Practical Flow Cytometry, Fourth Edition (Wiley-Liss, 2003); Herman et al., Fluorescence Microscopy, 2nd Edition (Springer, 1998); the disclosures of which are incorporated herein in their entirety by reference for all purposes.
[0029] Before the present invention is described in greater detail, it is to be understood that this invention is not limited to the particular embodiments described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.
[0030] When a range of values is provided, it is understood that each intermediate value, to the tenth of a unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other indicated value or intermediate in the specified range is encompassed within the scope of the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the scope of the invention, subject to any specifically excluded limit in the indicated range. When the indicated range includes one or both of the limits, the ranges excluding either or both of these included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. While any methods and materials similar or equivalent to those described herein may also be used in the practice or analysis of the present invention, representative illustrative methods and materials are now described.
[0032] All publications and patents cited in this specification are hereby incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are hereby incorporated by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. Citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention has no right to predate such publication by virtue of a prior invention. In addition, the publication dates provided may differ from the actual publication dates which may need to be independently confirmed.
It should be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include the plural references unless the context clearly dictates otherwise. It should also be noted that claims may be worded to exclude any optional elements. As such, this statement is intended to serve as an antecedent basis for the use of such exclusive terminology as "exclusively", "only" and the like in connection with the recitation of claim elements, or the use of a "negative" limitation.
[0034] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has components and features that can be easily separated from or combined with features of any of the other various embodiments without if it departs from the scope or essence of the present invention. Any enumerated method can be performed in the order of the enumerated events or in any other order that is logically possible.
[0035] As summarized above, aspects of the present disclosure include a microfluidic device for analyzing a sample. In further describing more embodiments of the present description, microfluidic devices of interest are first described in greater detail. Next, methods for analyzing samples using object microfluidic devices are described. Suitable systems for practicing said methods of analyzing a sample for an analyte are described. Kits are also provided. MICROFLUID DEVICES
[0036] As summarized above, aspects of the present invention include a microfluidic device for analyzing a sample for one or more analytes. The term "analysis" is used herein in its conventional sense to refer to qualitatively assessing the presence or quantitatively measuring an amount of a target analyte species in the sample. As described in more detail below, a variety of different samples can be tested with the subject microfluidic device. In some cases, the sample is a biological sample. The term "biological sample" is used in its conventional sense to include an entire organism, plants, fungi or a subset of animal tissues, cells or component parts which may, in certain cases, be found in blood, mucus, lymph fluid, fluid synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen. As such, a "biological sample" refers to both the native organism and a subset of its tissues, as well as a homogenized, lysate or prepared extract of the organism or a subset of its tissues, including, but not limited to, by example, plasma, serum, spinal fluid, lymph fluid, sections of skin, respiratory tract, gastrointestinal, cardiovascular, and genitourinary, tears, saliva, milk, blood cells, tumors, organs. Biological samples can be any tissue type of organisms, including both normal and diseased tissue (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, the biological sample is a liquid sample, such as whole blood or its derivatives, for example, plasma, tears, urine, semen, etc., where in some cases the sample is a blood sample, including whole blood, such as blood obtained from venipuncture or fingerstick (where the blood may or may not be combined with any reagents prior to testing, such as preservatives, anticoagulants, etc.).
[0037] In certain embodiments the source of the sample is a "mammalian", where this term is used broadly to describe organisms that are found within the Mammalia class, including the carnivorous order (eg dogs and cats), Rodentia (eg, mice, guinea pigs, and rats), and primates (eg, humans, chimpanzees, and monkeys). In some cases, the subjects are human beings. Biological samples of interest can be obtained from human subjects of both sexes and at any stage of development (ie, neonates, infant, juvenile, adolescent, adult), where in certain modalities the human subject is a young, adolescent or adult. While the present disclosure can be applied to samples from a human subject, it is to be understood that the methods can also be performed on samples from other non-human animal subjects, such as, but not limited to, birds, mice, rats. , dogs, cats, cattle and horses.
[0038] In embodiments of the present invention, microfluidic devices include a sample application site, a flow channel in fluid communication with the sample application site, and a porous component that contains a porous matrix and a reagent. analysis positioned between the sample application site and the flow channel. The microfluidic device sample application site is a structure configured to receive a sample that has a volume ranging from 5 μL to 1000 μL, such as 10 μL to 900 μL, such as 15 μL to 800 μL, such as from 20 μL to 700 μL, such as from 25 μL to 600 μL, such as from 30 μL to 500 μL, such as from 40 μL to 400 μL, such as from 50 μL to 300 μL and including from 75 μL to 250 μL . The sample application site can be any convenient way, as long as it provides fluid access, either directly or through an intermediate component that provides fluid communication, to the flow channel. In some modalities, the sample application site is flat. In other embodiments, the sample application site is concave, such as in the form of an inverted cone that ends at the sample inlet hole. Depending on the amount of sample applied and the shape of the sample application site, the sample application site may have a surface area ranging from 0.01 mm2 to 1000 mm2, such as 0.05 mm2 to 900 mm2, such as from 0.1 mm2 to 800 mm2, such as from 0.5 mm2 to 700 mm2, such as from 1 mm2 to 600 mm2, such as from 2 mm2 to 500 mm2, and including from 5 mm2 to 250 mm2.
[0039] The microfluidic device inlet is in fluid communication with the sample application site and flow channel and may be of any suitable shape, where inlet cross-sectional shapes of interest include, but are not limited to: straight shapes in cross section, eg squares, rectangles, trapezoids, triangles, hexagons, etc., curvilinear shapes in cross section eg circular, oval, etc., as well as irregular shapes eg a portion of parabolic bottom coupled to a flat upper portion, etc. The dimensions of the nozzle orifice may vary, in some embodiments ranging from 0.01 mm to 100 mm, such as 0.05 mm to 90 mm, such as 0.1 mm to 80 mm, such as 0.5 mm to 70 mm, such as 1 mm to 60 mm, such as 2 mm to 50 mm, such as 3 mm to 40 mm, such as 4 mm to 30 mm and including 5 mm to 25 mm. In some embodiments, the inlet is a circular hole and the inlet diameter ranges from 0.01 mm to 100 mm, such as 0.05 mm to 90 mm, such as 0.1 mm to 80 mm, such as 0.5mm to 70mm, such as 1mm to 60mm, such as 2mm to 50mm, such as 3mm to 40mm, such as 4mm to 30mm, and including 5mm to 25 mm. Hence, depending on the shape of the inlet, the sample inlet orifice may have an opening ranging from 0.01 mm2 to 250 mm2, such as from 0.05 mm2 to 200 mm2, such as from 0.1 mm2 to 150 mm2 such as from 0.5 mm2 to 100 mm2 such as from 1 mm2 to 75 mm2 such as from 2 mm2 to 50 mm2 and including from 5 mm2 to 25 mm2.
[0040] In embodiments, the sample inlet is in fluid communication with a porous component that contains a porous matrix and an analysis reagent positioned between the sample application site and the flow channel. By "porous matrix" is meant a substrate that contains one or more pore structures configured for the permeation of liquid components therethrough. In some embodiments, the porous matrix contains a network of interconnected pores that provide a means for mixing an applied sample (eg, a biological sample as discussed in greater detail below) with an analysis reagent present in the porous matrix. In other embodiments, the porous matrix contains an interconnected pore network that is non-filtering for the sample. By "non-filtering" it is meant that the network of interconnected pores does not substantially restrict the passage of sample components through the porous matrix (i.e., into the flow channel), such as where the passage of 1% or less of the components of the sample is restricted by the pores of the porous matrix, such as 0.9% or less, such as 0.8% or less, such as 0.7% or less, such as 0.5% or less, such as 0.1 % or less, such as 0.05% or less, such as 0.01% or less, such as 0.001% or less, and including where 0.0001% or less of the sample components are constrained by the matrix pores porous. In other words, 1% or less of the sample remains in the porous matrix after the sample has passed, such as 0.9% or less, such as 0.8% or less, such as 0.7% or less, such as 0 .5% or less, such as 0.1% or less, such as 0.05% or less, such as 0.01% or less, such as 0.001% or less, and including 0.0001% or less of the sample remains in the porous matrix after the passage of the sample. In other words, the porous matrices of interest include a network of interconnected pores that are configured to pass substantially the entire sample through the porous matrix, such that 99% or more of the sample passes through the porous matrix, such as 99.5% or more, such as 99.9% or more, such as 99.99% or more, such as 99.999% or more, and including passing 99.9999% or more of the sample through the porous matrix . In certain embodiments, all (ie, 100%) of the sample passes through the porous matrix.
[0041] The porous matrix positioned between the sample application site and the flow channel can be of any suitable shape, such as planar polygonal shapes, including but not limited to a circle, oval, semicircle, crescent-shaped, shaped like a star, square, triangle, diamond, pentagon, hexagon, heptogon, octagon, rectangle or other suitable polygon. In other embodiments, the porous matrices of interest are three-dimensional, such as in the shape of a cube, cone, half-sphere, star, triangular prism, rectangular prism, hexagonal prism, or other suitable polyhedron. In certain embodiments, the porous matrix is disk-shaped. In other embodiments, the porous matrix is cylindrical. The dimensions of the porous matrix may vary, in some embodiments ranging from 0.01mm to 100mm, such as 0.05mm to 90mm, such as 0.1mm to 80mm, such as 0.5mm to 70mm, such as from 1mm to 60mm, such as from 2mm to 50mm, such as from 3mm to 40mm, such as from 4mm to 30mm and including from 5mm to 25mm. In some embodiments, the porous matrix is circular and the diameter of the porous matrix ranges from 0.01 mm to 100 mm, such as 0.05 mm to 90 mm, such as 0.1 mm to 80 mm, such as d 0 .5mm to 70mm, such as 1mm to 60mm, such as 2mm to 50mm, such as 3mm to 40mm, such as 4mm to 30mm and including 5mm to 25mm and has a height of 0.01 mm to 50 mm, such as 0.05 mm to 45 mm, such as 0.1 mm to 40 mm, such as 0.5 mm to 35 mm, such as 1 mm to 30 mm, such as 2 mm to 25 mm, such as 3 mm to 20 mm, such as 4 mm to 15 mm, and including 5 mm to 10 mm.
[0042] The pore size of the porous matrix may also vary depending on the biological sample and analysis reagents present and may range from 0.01 μm to 200 μm, such as from 0.05 μm to 175 μm, such as 0 ,1 μm to 150 μm, such as 0.5 μm to 125 μm, such as 1 μm to 100 μm, such as 2 μm to 75 μm and including 5 μm to 50 μm. In embodiments, the porous matrix can have a sufficient pore volume to contain all or part of the sample applied as desired. For example, 50% or more of the sample volume can fit within the porous matrix, such as 55% or more, such as 60% or more, such as 65% or more, such as 75% or more, such as 90% or more, such as 95% or more, such as 97% or more and including 99% or more of the sample volume can fit within the porous matrix. In certain embodiments, the porous matrix has a pore volume that is sufficient to contain the entire (ie, 100%) sample. For example, the pore volume of the porous matrix can range from 0.01 µL to 1000 µL, such as 0.05 µL to 900 µL, such as 0.1 µL to 800 µL, such as 0.5 µL to 500 μL, such as 1 μL to 250 μL, such as 2 μL to 100 μL and including 5 μL to 50 μL. In embodiments, the void fraction (i.e., the ratio of the void volume within the pores to the total volume) of porous matrices of ranges of interest from 0.1 to 0.9, such as 0.15 to 0, 85, such as from 0.2 to 0.8, such as from 0.25 to 0.75, such as from 0.3 to 0.7, such as from 0.35 to 0.65 and including from 0. 4 to 0.6. In other words, the pore volume is 10% and 90% of the total volume of the porous matrix, such as between 15% and 85%, such as between 20% and 80%, such as between 25% and 75%, such as between 30% and 70%, such as between 35% and 65% and including a pore volume between 40% and 60% of the total volume of the porous matrix.
[0043] In some embodiments, the porous matrices of interest are configured to provide a predetermined sample flow rate through the porous matrix. As discussed above, the sample can be mixed with an analytical reagent within the porous matrix pores and flow through the porous matrix into the flow channel by capillary action. In certain cases, the porous matrix is configured to provide a flow rate through the porous matrix to the flow channel that is 0.0001 µL/min or more, such as 0.0005 µL/min or more, such as 0.001 µL /min or more, such as 0.005 µL/min or more, such as 0.01 µL/min or more, such as 0.05 µL/min or more, such as 0.1 µL/min or more, such as 0 .5 μL/min or more, such as 1 μL/min or more, such as 2 μL/min or more, such as 3 μL/min or more, such as 4 μL/min or more, such as 5 μL/min or more, such as 10 μL/min or more, such as 25 μL/min or more, such as 50 μL/min or more, such as 100 μL/min and including a flow rate through the porous matrix of 250 μL/ min or more. For example, the porous matrix can be configured to pass the sample through the porous matrix (where the sample is mixed with the analysis reagent) at a rate ranging from 0.0001 µL/min to 500 µL/min, such as from 0.0005 μL/min to 450 μL/min, such as from 0.001 μL/min to 400 μL/min, such as from 0.005 μL/min to 350 μL/min, such as from 0.01 μL/min to 300 μL/min, such as from 0.05 μL/min to 250 μL/min, such as from 0.1 μL/min to 200 μL/min, such as from 0.5 μL/min to 150 μL/min and including pass the sample through the porous matrix at a rate of 1 µL/min to 100 µL/min.
[0044] In some embodiments, the subject porous matrices are configured to pass the sample through the porous matrix over a predetermined period of time. For example, the porous matrix can have a pore structure where the sample passes through the porous matrix over a period of time, such as over a period of 5 seconds or more, such as over a period of 10 seconds or more, such as over 30 seconds or more, such as over 60 seconds or more, such as over 2 minutes or more, such as over 3 minutes or more, such as over 5 minutes or more, such as over 10 minutes or more and including passing the sample through the porous matrix over a period of 30 minutes or more. In certain cases, the porous matrix is configured to have a pore structure in which the sample passes through the porous matrix over a period ranging from 1 second to 60 minutes, such as 2 seconds to 30 minutes, such as 5 seconds to 15 minutes, such as 10 seconds to 10 minutes, such as 15 seconds to 5 minutes and including 20 seconds to 3 minutes.
[0045] The porous matrix can be any suitable macroporous or microporous substrate and includes, but is not limited to, ceramic matrices, frits, such as sintered glass, polymeric matrices, as well as metal-organic polymeric matrices. In some embodiments, the porous matrix is a frit. The term "frit" is used herein in its conventional sense to refer to the porous composition formed from a sintered solid granulate, such as glass. Frits may have a chemical constituent that varies depending on the type of sintered granulate used to prepare the frit and may include, but not limited to, frits composed of aluminum silicate, boron trioxide, borophosphosilicate glass, borosilicate glass , ceramic enamel, cobalt glass, cranberry glass, fluorophosphate glass, fluorosilicate glass, molten quartz, germanium dioxide, metal borosilicate and embedded sulfide, lead glass, phosphate glass, phosphorus pentoxide glass, phosphosilicate glass , potassium silicate, soda lime glass, sodium hexametaphosphate glass, sodium silicate, tellurite glass, uranium glass, vitrite and combinations thereof. In some embodiments, the porous matrix is a glass frit, such as a borosilicate, aluminosilicate, fluorosilicate, potassium silicate or borophosphosilicate glass frit.
[0046] In some embodiments, the porous matrix is a porous organic polymer. Porous organic polymers of interest vary depending on sample volume, sample components, as well as analytical reagent present and may include, but are not limited to porous polyethylene, polypropylene, polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), acetate of ethyl vinyl (EVA), polycarbonate, polycarbonate alloys, polyurethane, polyethersulfone, copolymers and combinations thereof. For example, porous polymers of interest include homopolymers, heteropolymers and copolymers composed of monomeric units such as ethyl styrene, allylene monoalkylene monomers such as ethyl styrene, α-methyl styrene, vinyl toluene, benzene and vinyl acetate; methacrylic esters such as methyl methacrylate, ethyl methacrylate, butyl methacrylate, isobutyl methacrylate, isodecyl methacrylate, 2-ethylhexyl methacrylate, lauryl methacrylate, stearyl methacrylate, cyclohexyl methacrylate and benzyl methacrylate; chlorine-containing monomers such as vinyl chloride, vinylidene chloride and chloromethylstyrene; acrylonitrile compounds such as acrylonitrile and methacrylonitrile; and vinyl acetate, vinyl propionate, n-octadecyl acrylamide, ethylene, propylene, and butane, and combinations thereof.
[0047] In some embodiments, the porous matrix is a metal organic polymer matrix, for example an organic polymer matrix that has a base structure that contains a metal such as aluminum, barium, antimony, calcium, chromium, copper, erbium, germanium, iron, lead, lithium, phosphorus, potassium, silicon, tantalum, tin, titanium, vanadium, zinc or zirconium. In some embodiments, the porous metal organic matrix is an organosiloxane polymer including, but not limited to, methyltrimethoxysilane, dimethyldimethoxysilane, tetraethoxysilane, methacryloxypropyltrimethoxysilane, bis(triethoxysilyl)ethane, bis(triethoxysilyl)pentoxysilane, bis(triethoxy)silane polymers butane, bis(triethoxysilyl)hexane, bis(triethoxysilyl)heptane, bis(triethoxysilyl)octane, and combinations thereof.
[0048] In embodiments of the present disclosure, the porous component also includes an analysis reagent. In some embodiments, analytical reagents are present within the pores of the porous matrix and are configured to mix with the components of the applied sample as the sample passes through the porous matrix. Analysis reagents of interest present in the porous component can include analyte-specific binding members, such as enzymes, antibodies, substrates, oxidants, among other analyte-specific binding members. In certain cases, the analyte-specific binding member includes a binding domain. By "specific binding" or "specifically binds" is meant preferential binding of one domain (e.g., one binding pair member to the other binding pair member of the same binding pair) over other molecules or moieties in a solution or reaction mixture. The specific binding domain can bind (e.g., covalently or non-covalently) to a specific epitope of an analyte of interest. In certain instances, the specific binding domain non-covalently binds to a target. For example the binding between the analyte-specific binding member and the target analyte can be characterized by a dissociation constant, such as a dissociation constant of 10-5 M or less, 10-6 M or less, such as 10-7 M or less, including 10-8 M or less, for example 10-9 M or less, 10-10 M or less, 10-11 M or less, 10-12 M or less, 10-13 M or less, 10-14M or less, 10-15M or less and including 1016M or less.
Analyte specific binding members may vary depending on the type of biological sample and components of interest and may include, but are not limited to, antibody binding agents, proteins, peptides, haptens, nucleic acids, oligonucleotides. From. In some embodiments, the analyte-specific binding member is an enzyme. Examples of enzymes may include, but are not limited to, horseradish peroxidase, pyruvate oxidase, oxaloacetate decarboxylase, creatinine amidohydrolase, creatine amidinohydrolase, sarcosine oxidase, malate dehydrogenase, lactate dehydrogenase, FAD, TPP, P-5-P, NADH and their combinations.
In certain embodiments, the analyte-specific binding member is an antibody binding agent. The term "antibody binding agent" is used herein in the same conventional sense to refer to polyclonal or monoclonal antibodies or antibody fragments which are sufficient to bind an analyte of interest. Antibody fragments can be, for example, monomeric Fab fragments, monomeric Fab' fragments or dimeric F(ab')2 fragments. Also within the scope of the term "antibody binding agent" are molecules produced by antibody engineering, such as single chain antibody (scFv) molecules or humanized or chimeric antibodies produced from monoclonal antibodies by substitution of the constant regions of the chains. heavy and light to produce chimeric antibodies or substitution of both the constant regions and the framework portions of the variable regions to produce humanized antibodies. In certain embodiments, the analyte-specific binding member is an antibody or antibody fragment that specifically binds to a compound such as cluster of differentiation 14 (CD14), cluster of differentiation 4 (CD4), cluster of differentiation 45 RA (CD45RA ) and differentiation cluster 3 (CD3) or a combination thereof.
In some embodiments, the analyte-specific binding member is coupled to a detectable label. Any suitable detectable label can be used, including, but not limited to radioactive labels, labels detectable by spectroscopy techniques such as nuclear magnetic resonance, as well as optically detectable labels such as labels detectable by UV-vis spectrometry, infrared spectroscopy, transient absorption spectroscopy and emission spectroscopy (eg fluorescence, phosphorescence, chemiluminescence). In certain embodiments, the analyte-specific binding member is coupled to an optically detectable label. In one example, the optically detectable marker is a fluorophore. Examples of fluorophores may include, but are not limited to, 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine and derivatives such as acridine, acridine orange, acridine yellow, acridine red and acridine isothiocyanate; 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Yellow Lucifer VS); N-(4-anilino-1-naphthyl)maleimide; anthranilamide; Bright yellow; coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumaran 151); cyanine and its derivatives, such as cyanosine, Cy3, Cy5, Cy5.5, and Cy7; 4',6-diamidino-2-phenylindole (DAPI); 5',5''-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylaminocoumarin; diethylenetriamine penta acetate; 4,4'-diisothioyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride), 4-(4'-dimethylaminophenylazo)-benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosine and derivatives such as erythrosine B and erythrosine isothiocyanate; ethidium; fluorescein and its derivatives, such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)amine fluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6 -carboxyfluorescein (JOE), fluorescein isothiocyanate (FITC), fluorescein chlorotriazinyl, naphthofluorescein, and QFITC (XRITC); fluorescamine; IR144; IR1446; Green Fluorescent Protein (GFP); Reff Coral Fluorescent Protein (RCFP); Lissamine™; Rhodamine Lyssamine, Lucifer yellow; Malachite Green Isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Nile Red; Green Oregon; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron™ Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lyssamine 4,7-dichlororhodamine, sulfonyl chloride, rhodamine B, rhodamine (Rhod), rhodamine B, rhodamine 123, isothiocyanate rhodamine X, sulphorodamine B, sulphorodamine 101, sulfonyl chloride derivative of sulphorodamine 101 (Texas Red), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine, and tetramethyl isothiocyanate rhodamine (TRITC); riboflavin; rosolic acid derivatives and terbium chelate; xanthene or combinations thereof, among other fluorophores. In certain embodiments, the fluorophore is a fluorescent dye, such as rhodamine, coumarin, cyanine, xanthene, polymethine, pyrene, boron difluoro-dipyrromethene, naphthalimide, phycobiliprotein, peridinin chlorophyll proteins, their conjugates, or a combination thereof. As described in more detail below, fluorophores can be detected by emission maxima, light scattering, extinction coefficient, fluorescence polarization, fluorescence lifetime, or a combination of these.
[0052] The amount of analyte-specific binding member present in the assay reagent may vary depending on the volume and type of sample applied. In some cases, the amount of analyte-specific binding member is sufficient to provide a concentration of analyte-specific binding member in the sample present in the flow channel from 0.0001 µg/mL to 250 µg/mL, such as from 0.0005 μg/ml to 240 μg/ml, such as from 0.001 μg/ml to 230 μg/ml, such as from 0.005 μg/ml to 220 μg/ml, such as from 0.01 μg/ml to 210 μg/ml, such as from 0.05 μg/ml to 200 μg/ml, such as from 0.1 μg/ml to 175 μg/ml, such as from 0.5 μg/ml to 150 μg/ml and including an amount of analyte-specific binding member sufficient to provide a concentration of analyte-specific binding member in the sample present in the flow channel from 1 µg/ml to 100 µg/ml. For example, the dry weight of the analyte-specific binding member present in the porous component can range from 0.001 ng to 500 ng, such as 0.005 ng to 450 ng, such as 0.01 ng to 400 ng, such as 0 .05 ng to 350 ng, such as 0.1 ng to 300 ng, such as 0.5 ng to 250 ng and including an analyte-specific binding member dry mass of 1 ng to 200 ng.
[0053] In some embodiments, the porous component also includes one or more plugs. The term "buffer" is used in its conventional sense to refer to a compound that helps to stabilize (i.e., maintain) the composition, such as for example during dissolution of the assay reagent in the applied sample. Buffers of interest can include, but are not limited to, proteins, polysaccharides, salts, chemical binders and combinations thereof. Encompassed by the invention are both liquid and dry buffer formats, e.g. aqueous compositions which include the below components or dehydrated versions thereof.
[0054] In some embodiments, buffers include polysaccharides, such as for example glucose, sucrose, fructose, galactose, mannitol, sorbitol, xylitol, among other polysaccharides. In some cases, buffers include a protein such as BSA. In still other examples, buffers of interest in a chemical paste, including but not limited to low molecular weight dextrans, cyclodextrin, polyethylene glycol, polyethylene glycol polyvinylpyrrolidone (PVP) esters or other hydrophilic polymers selected from the group consists of hyaluronic acid, polyvinylpyrrolidone (PVP), N-vinylpyrrolidone copolymers, hydroxyethyl cellulose, methyl cellulose, carboxymethyl cellulose, dextran, polyethylene glycol (PEG), PEG/PPG block copolymers, acrylic acid homo and copolymers and methacrylic, polyurethanes, polyvinyl alcohol, polyvinyl ethers, maleic anhydride-based copolymers, polyesters, vinylamines, polyethyleneimines, polyethylene oxides, poly(carboxylic acids), polyamides, polyanhydrides, polyphosphazenes, and mixtures thereof.
In certain embodiments, buffers of interest include a biological buffer, including, but not limited to, N-(2-acetamido)-aminoethanesulfonic acid (ACES), acetate, N-(2-acetamido)iminodiacetic acid (ADA) , 2-aminoethanesulfonic acid (AES), ammonia, 2-amino-2-methyl-1-propanol (AMP), 2-amino-2-methyl-1,3-propanediol (AMPD), N-(1,1-) acid -dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), bicarbonate, N,N'-bis-(2 -hydroxyethyl)-glycine, [Bis-(2-hydroxyethyl)-imino]-tris-(hydroxymethylmethane) (BIS-Tris), 1,3-Bis[tris(hydroxymethyl)methylamino]propane (BIS-Tris-propane), boric acid, dimethylarsinic acid, bovine serum albumin (BSA), 3-(cyclohexylamino)-propanesulfonic acid (CAPS), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO), cyclohexylaminoethanesulfonic acid (CHES), citrate, 3-[N-Bis(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), formate, glycine, glycol lglycine, N-(2-hydroxyethyl)-piperazine-N'-ethanesulfonic acid (HEPES), N-(2-hydroxyethyl)-piperazine-N'-3-propanesulfonic acid (HEPPS, EPPS), N-(2-hydroxyethyl) acid Hydroxyethyl)-piperazine-N'-2-hydroxypropanesulfonic acid (HEPPSO), imidazole, malate, maleate, 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), 3- (N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO), phosphate, Piperazine-N,N'-bis(2-ethanesulfonic) (PIPES), Piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) (POPSO), pyridine, polyvinylpyrrolidone (PVP), succinate, 3-{[Tris(hydroxymethyl)-methyl]-amino}-propanesulfonic acid (TAPS), 3-[N-Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (TAPSO), acid 2-Aminoethanesulfonic acid, AES (taurine), trehalose, triethanolamine (TEA), 2-[Tris(hydroxymethyl)methylamino]ethanesulfonic acid (TES), N-[Tris(hydroxymethyl)methyl]-glycine (tricine), Tris( hydroxymethyl)- aminomethane (Tris), glyceraldehydes, mannose, glucosamine, mannoeptulose, sorbose- 6-phosphate, trehalose-6-phosphate, maleimide, iodoacetates, sodium citrate, sodium acetate, sodium phosphate, sodium tartrate, sodium succinate, sodium maleate, magnesium acetate, magnesium citrate, magnesium phosphate, ammonium acetate, ammonium citrate, ammonium phosphate, among other buffers.
[0056] The amount of each buffer component present in the porous matrix may vary depending on the type and size of the sample and the type of porous matrix used (inorganic frit, porous organic polymer, as described above) and may vary from 0.001% to 99% by weight, such as from 0.005% to 95% by weight, such as from 0.01% to 90% by weight, such as from 0.05% to 85% by weight, such as from 0.1% to 80% by weight, such as from 0.5% to 75% by weight, such as from 1% to 70% by weight, such as from 2% to 65% by weight, such as from 3% to 60% by weight. weight, such as from 4% to 55% by weight and including from 5% to 50% by weight. For example, the dry weight of buffer present in the porous matrix can range from 0.001 μg to 2,000 μg, such as from 0.005 μg to 1900 μg, such as from 0.01 μg to 1800 μg, such as from 0.05 μg to 1700 μg, such as from 0.1 ng to 1500 μg, such as from 0.5 μg to 1000 μg and including a buffer dry weight of 1 μg to 500 μg.
[0057] In some embodiments, the total weight of buffer present in the porous matrix depends on the void volume (i.e., the volume within the pores) of the porous matrix and ranges from 0.001 g to 5 g of buffer per mL of void volume in the porous matrix, such as 0.005 g to 4.5 g, such as 0.01 g to 4 g, such as 0.05 g to 3.5 g, such as 0.1 g to 3 g, such as 0 .5 g to 2.5 g and including from 1 g to 2 g of buffer per mL of void volume in the porous matrix.
[0058] In one example, the buffer present in the porous matrix includes bovine serum albumin (BSA). When the buffer present in the porous matrix includes BSA, the amount of BSA varies, ranging from 1% to 50% by weight, such as from 2% to 45% by weight, such as from 3% to 40% by weight, such as between 4% and 35% by weight, and including between 5% and 25% by weight. For example, the dry weight of BSA in the buffer can range from 0.001 μg to 2,000 μg, such as from 0.005 μg to 1900 μg, such as from 0.01 μg to 1800 μg, such as from 0.05 μg to 1700 μg, such as from 0.1 ng to 1500 μg, such as from 0.5 μg to 1000 μg and including a dry weight of BSA from 1 μg to 500 μg.
[0059] In another example, buffer present in the porous matrix includes polyvinylpyrrolidone (PVP). When the buffer present in the porous matrix includes PVP, the amount of PVP varies, ranging from 0.01% to 10% by weight, such as from 0.05% to 9% by weight, such as from 0.1% to 8 % by weight, such as between 0.5% and 7% by weight, and including between 1% and 5% by weight. For example, the dry weight of PVP in the buffer can range from 0.001 μg to 2,000 μg, such as from 0.005 μg to 1900 μg, such as from 0.01 μg to 1800 μg, such as from 0.05 μg to 1700 μg, such as 0.1 ng to 1500 μg, such as 0.5 μg to 1000 μg and including a dry weight of PVP from 1 μg to 500 μg.
[0060] In yet another example, buffer present in the porous matrix includes trehalose. When the buffer present in the porous matrix includes trehalose, the amount of trehalose varies, ranging from 0.001% to 99% by weight, such as from 0.005% to 95% by weight, such as from 0.01% to 90% by weight. weight, such as from 0.05% to 85% by weight, such as from 0.1% to 80% by weight, such as from 0.5% to 75% by weight, such as from 1% to 70% by weight weight, such as from 2% to 65% by weight, such as from 3% to 60% by weight, such as from 4% to 55%, by weight, and including from 5% to 50% by weight. For example, the dry weight of trehalose in the buffer can range from 0.001 μg to 2,000 μg, such as from 0.005 μg to 1900 μg, such as from 0.01 μg to 1800 μg, such as from 0.05 μg to 1700 μg, such as 0.1 ng to 1500 μg, such as 0.5 to 1000 μg μg and including a dry weight of trehalose from 1 μg to 500 μg.
[0061] In certain embodiments, buffer present in the porous matrix includes BSA, trehalose and polyvinylpyrrolidone. For example, the buffer can include BSA, trehalose and polyvinylpyrrolidone in a weight ratio of BSA:trehalose:PVP ranging from 1:1:1 to 25:100:1 In certain cases, the weight ratio of BSA:trehalose: PVP is 21:90:1.
[0062] In some embodiments, buffers can further include one or more complexing agents. A "complexing agent" is used in its conventional sense to refer to an agent that aids in mixing the sample with the assay reagent and can also serve to bind ions (eg iron or other ions) and prevent formation of precipitates during mixing. A complexing agent can be an agent that is capable of complexing with a metal ion. In some cases, the complexing agent is a chelating agent such as ethylenediaminetetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), nitrolotriacetic acid (NTA), ethylenediamine diacetate (EDDA), ethylenediamine-di(o-hydroxyphenylacetic) acid (EDDHA), hydroxyethylethylenediaminetriacetic acid (HEDTA), cyclohexane diamine tetraacetic acid (CDTA), ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA), 2,3- dimercaptopropane-1-sulfonic acid (DMPS), and 2,3-dimercaptosuccinic acid (DMSA) and the like. Naturally occurring chelating agents can also be used. By naturally occurring chelating agent is meant that the chelating agent is a naturally occurring chelating agent, that is, not an agent that was first synthesized by human intervention. The naturally occurring chelating agent can be a low molecular weight chelating agent, wherein by low molecular weight of the chelating agent it is meant that the molecular weight of the chelating agent does not exceed about 200 daltons. In certain embodiments, the molecular weight of the chelating agent is greater than about 100 daltons. In some embodiments, analysis reagents of interest include ethylenediaminetetraacetic acid (EDTA). Whenever a chelating agent is present in the porous matrix, the amount of chelating agent can range from 0.001% to 10% by weight, such as from 0.005% to 9.5% by weight, such as from 0.01% to 9% by weight, such as from 0.05% to 8.5% by weight, such as from 0.1% to 8% by weight, such as from 0.5% to 7.5% by weight, and including from 1% to 7% by weight. For example, the dry weight of the chelating agent in the analysis reagent can range from 0.001 μg to 2000 μg, such as from 0.005 μg to 1900 μg, such as from 0.01 μg to 1800 μg, such as from 0.05 μg to 1700 μg, such as 0.1 ng to 1500 μg, such as 0.5 μg to 1000 μg and including a dry weight of chelating agent from 1 μg to 500 μg.
[0063] All or part of the porous matrix may contain the analysis reagent and buffer components. For example, 5% or more of the porous matrix may contain analysis reagent and buffer components, for example 10% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, such as 95% or more and including 99% or more. In certain embodiments, the entire porous matrix contains analytical reagent and buffer components. The assay reagent and buffer components can be homogeneously distributed throughout the porous matrix or can be positioned at specific locations within the porous matrix, or some combination thereof. For example, in one example, the assay reagent and buffer components are homogeneously distributed throughout the porous matrix. In another example, the assay reagent and buffer components are positioned at specific locations in the porous matrix, such as in specific increments of every 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more and which include positioning the porous matrix every 2 mm or more of the porous matrix. In yet another example, the assay reagent and buffer components can be homogeneously distributed over a first half of the porous matrix and in specific increments across a second half of the porous matrix. In certain embodiments, the assay reagent and buffer components are positioned in the porous matrix as a gradient, where the amount of assay reagent and buffer components increases from a proximal end (e.g., closer to the application site of the sample) to the distal end (eg closer to the flow channel). In one example, the amount of assay reagent increases linearly along the sample's flow path through the porous matrix. In another example, the amount of assay reagent and buffer components increases exponentially along the path of the sample's flow through the porous matrix.
Analysis reagents and buffer components can be present in the porous component in any suitable physical state, such as a liquid, dry solid, or can be lyophilized. In some embodiments, assay reagents and buffer components are present as a dry solid. In other embodiments, assay reagents and buffer components are lyophilized. All or part of the assay reagents and buffer components can be in the same physical state. For example, 5% or more of the assay reagents and buffer components may be present in the porous matrix as a dry solid, such as 10% or more, such as 25% or more, such as 50% or more, such as 75 % or more, such as 90% or more and including 95% or more of the assay reagents and buffer components. In some embodiments, 5% or more of the assay reagents and buffer components are lyophilized, such as 10% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more and including when 95% or more of the assay reagents and buffer components are lyophilized.
[0065] In embodiments of the present disclosure, a flow channel is positioned adjacent the porous component and in fluid communication with the sample mixed with the analysis reagent and buffer components in the porous matrix. As discussed in more detail below, the sample can pass through and be mixed with the analysis reagent in the porous matrix due to a force (eg, centrifugal force, electrostatic force, capillary action) and into the flow channel. In some embodiments, the flow channel is an elongated channel enclosed by one or more walls. Depending on sample size, the flow channel may vary. In some modalities, the flow channel is linear. In other embodiments, the flow channel is non-linear. For example, the flow channel can be curved, circular, tortuous, twisted, or have a helical configuration.
[0066] The length of the flow channel can vary, ranging from 10 mm to 1000 mm, such as from 15 mm to 950 mm, such as from 20 mm to 900 mm, such as from 20 mm to 850 mm, such as from 25mm to 800mm such as 30mm to 750mm such as 35mm to 700mm such as 40mm to 650mm such as 45mm to 600mm such as 50mm to 550mm and including from 100mm to 500mm.
[0067] In embodiments, the flow channel cross-sectional shape may vary, where examples of cross-sectional shapes include, but are not limited to, straight cross-sectional shapes, e.g., squares, rectangles, triangles, trapezoids, hexagons , etc., curvilinear cross-sectional shapes, eg circular, oval, etc., as well as irregular shapes, eg a parabolic bottom portion coupled to a flat top portion, etc. In embodiments, the cross-sectional dimensions of the flow channel may vary, ranging from 0.01 mm to 25 mm, such as 0.05 mm to 22.5 mm, such as 0.1 mm to 20 mm, such as. such as 0.5mm to 17.5mm, such as 1mm to 15mm, such as 2mm to 12.5mm, such as 3mm to 10mm, and including 5mm to 10mm. For example, when the flow channel is cylindrical, the diameter of the flow channel can range from 0.01 mm to 25 mm, such as 0.05 mm to 22.5 mm, such as 0.1 mm to 20 mm, such as from 0.5 mm to 15 mm, such as from 1 mm to 10 mm, and including from 3 mm to 5 mm.
[0068] The ratio between the length and height of the cross section may vary, ranging from 2 to 5000, such as 3 to 2500, such as 4 to 2000, such as 5 to 1500, such as 10 to 1000, such as from 15 to 750 and including from 25 to 500. In some cases, the ratio of the length to height of the cross section is 10. In other examples, the ratio of the length to height of the cross section is 15 In still other cases, the ratio of the length to the height of the cross section is 25.
[0069] In some embodiments, the flow channel is configured to have a cross-sectional height that is substantially equivalent to the dimensions of the target analyte. By "substantially equivalent" to the dimensions of the target analyte means that one or more of the height or width of the flow channel differs from the size of the target analyte by 5% or less, such as 4% or less, such as 3% or less, such as 2% or less, such as 1% or less, such as 0.5% or less, such as 0.1% or less and including 0.01% or less. In these modalities, the cross-sectional dimensions of the flow channel are substantially the same as the analyte size and the target analytes are configured to flow through the flow channel only one analyte at a time. In certain cases, the target analyte is cells, such as leukocytes or erythrocytes. In some embodiments, the flow channel is configured to have a cross-sectional height that is substantially equivalent to the diameter of an erythrocyte. In other embodiments, the flow channel is configured to have a cross-sectional height that is substantially equivalent to the diameter of a white blood cell.
[0070] In embodiments of the present invention, the flow channel is a structure configured to receive and retain a sample with a volume ranging from 5 µL to 5000 µL, such as from 10 µL to 4000 µL, such as from 15 µL to 3000 μL, such as from 20 μL to 2000 μL, such as from 25 μL to 1000 μL, such as from 30 μL to 500 μL, such as from 40 μL to 400 μL, such as from 50 μL to 300 μL and including 75 µL to 250 µL.
[0071] In some embodiments, the flow channel is a capillary channel and is configured to move a liquid sample through the flow channel by capillary action. The term "capillary action" is used herein in its conventional sense to refer to the movement of a liquid by intermolecular forces between the liquid (i.e., cohesion) and the adjacent walls (i.e., adhesion) of a narrow channel, without assistance. of (and sometimes in opposition to) gravity. In these modalities, the cross-sectional width of the flow channel is sufficient to provide the capillary action of the sample in the flow channel and can have a width ranging from 0.1 mm to 20 mm, such as 0.5 mm to 15mm, such as from 1mm to 10mm and including from 3mm to 5mm.
[0072] In some embodiments, the flow channel includes one or more optically transmissive walls. By "optically transmissive" it is meant that the walls of the flux channel allow the propagation of one or more wavelengths of light through it. In some embodiments, the flow channel walls are optically transmissive to one or more of ultraviolet light, visible light, and near-infrared light. In one example, the flux channel is optically transmissive to ultraviolet light. In another example, the flux channel is optically transmissive to visible light. In yet another example, the flux channel is optically transmissive to near-infrared light. In yet another example, the flux channel is transmissive to ultraviolet light and visible light. In yet another example, the flux channel is transmissive to visible light and near-infrared light. In yet another example, the flux channel is transmissive to ultraviolet light, visible light, and near-infrared light. Depending on the desired transmitting properties of the flow channel walls, the optically transmissive wall can be any suitable material, such as quartz, glass, or polymers including but not limited to optically transmissive polymers such as acrylics, acrylics. cos/styrenes, cycloolefin polymers, polycarbonates, polyesters and polystyrenes, among other optically transmissive polymers.
[0073] In embodiments of the present disclosure, the sample application site of the microfluidic device is a structure configured to receive a sample that has a volume ranging from 5 μL to 1000 μL, such as from 10 μL to 900 μL, such as such as from 15 μL to 800 μL, such as from 20 μL to 700 μL, such as from 25 μL to 600 μL, such as from 30 μL to 500 μL, such as from 40 μL to 400 μL, such as from 50 μL to 300 μL and including from 75 μL to 250 μL. The sample application site can be any convenient way, as long as it provides for fluid access, either directly or through an intermediate component that provides for fluid communication, to the flow channel. In some modalities, the sample application site is flat. In other embodiments, the sample application site is concave, such as in the form of an inverted cone that ends at the sample inlet hole. Depending on the amount of sample applied and the shape of the sample application site, the sample application site may have a surface area ranging from 0.01 mm2 to 1000 mm2, such as 0.05 mm2 to 900 mm2, such as from 0.1 mm2 to 800 mm2 such as from 0.5 mm2 to 700 mm2 such as from 1 mm2 to 600 mm2 such as from 2 mm2 to 500 mm2 and including from 5 mm2 to 250 mm2.
[0074] The inlet of the microfluidic device is in fluid communication with the sample application site and the flow channel and may be of any suitable shape, where inlet cross-sectional shapes of interest include, but are not limited to : Straight cross-sectional shapes, eg squares, rectangles, triangles, trapezoids, hexagons, etc., curvilinear cross-sectional shapes, eg circular, oval, etc., as well as irregular shapes, eg a portion of parabolic bottom coupled to a flat top portion. The dimensions of the nozzle orifice may vary, in some embodiments ranging from 0.01mm to 100mm, such as 0.05mm to 90mm, such as 0.1mm to 80mm, such as 0.5 mm to 70 mm, such as 1 mm to 60 mm, such as 2 mm to 50 mm, such as 3 mm to 40 mm, such as 4 mm to 30 mm and including 5 mm to 25 mm. In some embodiments, the inlet is a circular hole and the inlet diameter ranges from 0.01 mm to 100 mm, such as 0.05 mm to 90 mm, such as 0.1 mm to 80 mm, such as 0 .5mm to 70mm, such as 1mm to 60mm, such as 2mm to 50mm, such as 3mm to 40mm, such as 4mm to 30mm and including 5mm to 25mm . Therefore, depending on the shape of the inlet, the sample inlet hole may have an opening that varies, ranging from 0.01 mm2 to 250 mm2, such as from 0.05 mm2 to 200 mm2, such as from 0.1 mm2 to 150 mm2 such as from 0.5 mm2 to 100 mm2 such as from 1 mm2 to 75 mm2 such as from 2 mm2 to 50 mm2 and including from 5 mm2 to 25 mm2.
[0075] In some embodiments, the microfluidic devices in question include a vent channel. The vent channels of interest can have a variety of different configurations and are configured to couple in fluid communication a vent outlet (eg positioned adjacent to the sample application site) with the distal end of the flow channel ( that is, further away from the sample application site). The vent channel may be an elongated structure, similar to those described above for the flow channel, including a configuration having a length that is greater than its width. Although the length to width ratio may vary, in some cases the length to width ratio is in the range of 5 to 2000 such as 10 to 200 and includes 50 to 60. In some cases the length of the vent channel varies between 5 and 200, such as 10 to 100 and including 50 to 75 mm. In some cases, the ventilation channels of interest have a micrometer size in cross-sectional dimension, for example, a larger cross-sectional dimension (eg diameter in the case of the tubular channel) ranging from 0.1 to 10, such as 0.5 to 5 and including 1 to 2 mm. In some cases, the width of the ventilation channel ranges from 0.1 to 10, such as 0.5 to 5 and including 1 to 2 mm. In some cases, the height of the channel ranges from 0.5 to 5, such as 0.2 to 2 and including 0.5 to 1 mm. The cross sectional shape of the vent channels may vary, in some cases cross sectional shapes of the vent channels of interest include, but are not limited to: straight shapes in cross section, eg squares, rectangles, trapezoids, triangles - angles, hexagons, etc., curvilinear shapes in cross section, eg circular, oval, etc., as well as irregular shapes, eg a parabolic bottom portion coupled to a flat top portion. In embodiments, the cross-sectional dimensions of the vent channel may vary, ranging from 0.01 mm to 25 mm, such as 0.05 mm to 22.5 mm, such as 0.1 mm to 20 mm, such as. as from 0.5mm to 17.5mm, such as from 1mm to 15mm, such as from 2mm to 12.5mm, such as from 3mm to 10mm and including from 5mm to 10mm. For example, when the vent channel is cylindrical, the diameter of the vent channel may range from 0.01 mm to 25 mm, such as from 0.05 mm to 22.5 mm, such as from 0.1 mm to 20 mm, such as from 0.5 mm to 15 mm, such as from 1 mm to 10 mm and including from 3 mm to 5 mm.
[0076] Whenever the microfluidic devices in question include a vent channel, the flow channel can be separated from the vent channel by a hydrophobic region. By hydrophobic region is meant a region or domain that is resistant to being wetted by water, for example, that repels aqueous media. The hydrophobic region can be one that has a surface energy that is less than the surface energy of the capillary channel surfaces. The magnitude of the difference in surface energies can vary, ranging in some cases from 5 to 500, such as 10 to 30 dynes/cm. The surface energy of the hydrophobic region may also vary, ranging in some cases from 20 to 60, such as from 30 to 45 dynes/cm, for example, as measured using the protocol described in ASTM Std. D2578. The dimensions of the hydrophobic region are configured to at least partially, if not completely, impede liquid sample flow past the hydrophobic region. The dimensions of the hydrophobic region may vary, in some cases having a surface area ranging from 0.01 mm2 to 100 mm2, such as from 0.05 mm2 to 90 mm2, such as from 0.1 mm2 to 80 mm2, such as such as from 0.5 mm2 to 75 mm2 and including from 1 mm2 to 50 mm2.
[0077] Referring to Figure 1, a microfluidic device for analyzing a sample according to certain modalities, such as with an imaging apparatus as described in Goldberg, US Patent Publication 2008/0212069 is shown. Figure 1 represents an example of a microfluidic device with a sample application site (1), porous component (porous element 2), and a flow channel (eg, capillary channel 3). As shown in Figure 1, the microfluidic device also includes a hydrophobic junction (4) and a vent channel (5). To visualize the sample in the flow channel, this example represents a flow channel that has an optically transmissive wall (6). The sample application site is configured to receive a fluid sample, such as a biological fluid (e.g., blood, saliva, serum, semen, plasma or the like). In some modalities, the sample is a blood sample. As discussed above, the sample application site is in fluid communication with the porous component in a manner that directs a sample of a sample through the porous component. The porous component can be placed in a chamber or channel in such a way that the sample is directed through the porous element. The porous element can be flush with the walls of microfluidic devices disposed either in an equipped chamber in the device or along a capillary or other channel. In some embodiments, the sample application site and the porous component are configured in a way that provides for the flow of a sample from the sample application site through the porous matrix of the porous component and the capillary channel by capillary force, but other means of movement of the sample are possible. Centrifugal force, electrostatic force or any other force can be used alone or in conjunction with capillary force to transmit sample through the porous element. The sample application site can support application of a sample delivered by any means such as a pipette or directly from an organism such as through a blood sample from a human fingertip.
[0078] In some embodiments, the porous component includes a porous frit made of a plurality of microchannels that serve as a matrix for an analysis mix. As described above, microchannels can form a void volume in the frit that is between 40 and 60% of the total frit volume. In some embodiments, the frit can occupy a volume of about 10 µL and the total void volume can be between 4 and 6 µL. In some embodiments, the pores are as narrow as possible to provide a dry reagent suspension surface and tortuous path for mixing, without filtering cells or other objects down to 15-20 microns. The assay mixture can be dried or otherwise preserved within the void volume of the porous filter and can comprise the buffer components and one or more reagents, such as a detectable label that binds to one or more targets or analytes present. in the sample. Buffer components can provide for a uniform dissolution rate of the reagent in the sample over a defined period of time. The buffer components can comprise any combination of a protein, sugar and/or a chemical binder. The protein component can be an albumin such as bovine serum albumin (BSA). The sugar can be any sugar such as a mono, di or polysaccharide. For example, sucrose, mannitol, trehalose (such as trehalose D+) can stabilize biomolecules or other reagents in the frit and provide protection to reagents such as biomolecules. In the development of lyophilized or preserved reagents, proteins or sugars (saccharides and polyols) can be added to the formulation, in order to improve stability and provide uniform dissolution of reagents or other biomolecules and additionally to prolong the duration of stability of the reagents on the device.
[0079] Low molecular weight dextran, cyclodextrin, polyethylene glycol, polyethylene glycol polyvinylpyrrolidone (PVP) esters, or other hydrophilic polymers selected from the group consisting of hyaluronic acid, polyvinylpyrrolidone (PVP), N-vinylpyrrolidone copolymers, hydroxyethyl cellulose, methyl cellulose, carboxymethyl cellulose, dextran, Polyethylene glycol (PEG), PEG/PPG block copolymers, acrylic and methacrylic acid homo and copolymers, polyurethanes, polyvinyl alcohol, polyvinylethers, maleic anhydride-based copolymers , polyesters, vinylamines, polyethyleneimines, polyethylene oxides, poly(carboxylic acids), polyamides, polyanhydrides, polyphosphazenes, and mixtures thereof can be used to stabilize the reagent and aid in the continuous dissolution of the reagent in the sample.
[0080] Buffer components can be grouped in the appropriate ratio and concentration to provide for continuous dissolution of a reagent in a sample. The total amount of buffer components may depend on the void volume of the porous frit. In some embodiments the combined weight of the buffer components (eg BSA, trehalose and PVP) can be between 0.01 and 2 grams per μl frit void volume, such as 0.1 grams/μl volume of emptiness. In some embodiments, the buffer components of the present invention may contain a weight ratio of BSA:Trehalose:PVP that is on the order of 21:90:1. The weight ratio of the buffer components can vary as much as 5, 10 or 20%, as long as the uniform dissolution property of the reagent in a liquid sample over a predetermined period of time is maintained. The predetermined period of time may be on the order of seconds or minutes, such as between 5 seconds and 5 minutes, or between 20 seconds and 3 minutes, or between 1 and 2 minutes, during which a uniform dissolution of the reagent in the sample is maintained . This provides an improvement in the uniformity of distribution of unbound reagent in the sample through the capillary channel and the evaluation of the sample. The concentration of unreacted reagent normally may deviate by less than 1%, 5%, 10%, 20% or 50% along the capillary channel. In some embodiments the buffer components may contain components such as ethylenediaminetetraacetic acid (EDTA) or 2-(N-morpholino) ethanesulfonic acid (MES) or the like, or any other material useful for maintaining the stability of the sample or reagents during during the analysis. The analysis mixture can comprise enzymes, substrates, catalysts, or any combination thereof to react with the sample (e.g., horseradish peroxidase, pyruvate oxidase, oxaloacetate decarboxylase, creatinine amidohydrolase, creatine amidinohydrolase, sarcosine oxidase, malate dehydrogenase, lactate dehydrogenase, FAD, TPP, P-5-P, NADH, Amplex Red). Other components of the assay mix can be used to regulate the pH, dissolution rate, or stability of the sample and/or assay mix (eg, hydroxypropylmethylcellulose, hydroxypropylcellulose). As the sample passes through the porous element, the microchannels provide mixing of the sample and the reagent, while the uniform dissolution rate of the reagent provides for substantially uniform distribution of the unreacted reagent as it flows out of the porous matrix. and for channel flow.
[0081] As discussed above, analysis reagents can include any material capable of reacting or binding to an analyte in a biological sample as desired. In some embodiments, the reagent is an antibody or antibody fragment that binds to components in the sample, such as a specific target on the cell surface in the sample. There may be one or more different reagents in the assay mix. In some embodiments, the antibody or antibody fragments can specifically bind to cellular targets, such as CD14, CD4, CD45RA, CD3, or any combination thereof. Antibodies or antibody fragments can be conjugated to a dye or other detectable label such as a fluorescent dye or magnetic particles. In some embodiments, the detectable marker is a dye selected from the group comprising rhodamine, coumarin, cyanine, xanthene, polymethine, pyrene, boron difluoro-dipyrromethene, naphthalimide, phycobiliprotein, peridinin chlorophyll proteins, their conjugates, and their combinations. In some embodiments the dye may be phycoerythrin (PE), phycoerythrin -cyanin 5 (PE-Cy5) or allophycocyanin (APC). The detectable label can be magnetic, phosphorescent, fluorescent or optically active in any form.
[0082] As depicted in Figure 1, microfluidic devices of interest according to certain embodiments include a capillary chamber having a flat geometry with large dimensions in width and length and a height that is either (a) substantially equivalent to the depth of field. of an objective lens of a detector, or (b) only slightly larger than the cells to be analyzed in a sample. The sample can be optically evaluated through one or more transmissive walls in the microfluidic device. Uniform distribution of unreacted reagent in the sample provides improved observations of background signal along the length of the transmissive wall. This beneficially provides easier detection of bound reagent as concentrations of detectable signal above background signal are observed.
[0083] Another example of a microfluidic device 100 is illustrated in greater detail in Figures 2A and 2B and includes a sample application site 10 in fluid communication with a porous component 20 and flow channel 30. In this embodiment, the flow channel flux includes optically transmissive wall 40. The frit portion of the porous component can be prepared from any suitable material such as plastic (e.g. polyethylene, polypropylene, polytetrafluoroethylene, polyvinylidene fluoride, ethylvinyl acetate, polycarbonate, polycarbonate alloys, polyurethane, polyethersulfone or any combination thereof) as discussed above. In some embodiments, the porous matrix is high-density polyethylene. The porous matrix can be a solid of any size or shape that fills a region between the flow channel and the application site. The porous element can be placed in a separate chamber or merely occupying a region of the capillary channel. The external dimensions of the porous frit are designed in conjunction with the overall device so that the porous frit fits snugly into the overall device and essentially no sample passes around the porous frit. In some embodiments, the porous frit is integrated as part of the flow channel. The porous frit may be a solid material composed of a series of microchannels and having a void volume of between 25 and 75%, such as 4060% or 45-55%. Microchannels can provide the combination of the analysis mix and a sample through a plurality of tortuous paths. In some embodiments, the average pore diameter of the microchannels can be between 5 and 200 microns, such as between 30 and 60 microns; and the average void volume can be 40-60% of the total frit volume. The average diameter and tortuous path of the microchannels can beneficially provide mixing of sample and reagent, while allowing the sample to flow through the substantially unfiltered porous element. The device can use any force such as gravity or centrifugal force in addition to capillary force to provide sample movement through the flow channel.
[0084] Whenever the microfluidic devices in question use capillary action, the microfluidic devices do so because the flow surfaces are hydrophilic, and wetting the surfaces is energetically favorable. Such devices require the incoming sample to displace the air residing in the device. It is desirable that both the applied sample as well as the vented air are contained within the cartridge in order to protect users from potentially biohazardous materials. In some embodiments of the present disclosure, any combination of the following features can be used in the device. For example, the capillary channel or sample application site can include a mixing chamber where preserved reagents can be located separate from the capillary channel. Capillary channel dimensions can impact the image and sample flow in the device. In some embodiments the channel can be between 2 and 10 mm wide, such as between 3 and 5 mm or between 3 and 4 mm wide. In some embodiments, the capillary channel can be between 1 and 1000 microns deep, such as between 20 and 60 microns deep or between 40 and 60 microns deep. Depths less than 60 microns in depth can beneficially provide for imaging of leukocytes in a whole blood sample, minimizing the erythrocyte obscuration effects. The capillary channel can be of any length that provides capillary flow along a channel. In some embodiments the capillary channel can be between 10 and 100 mm in length.
[0085] As discussed above, the device is suitable for assays to detect analytes in a sample, comprising a biological fluid, such as urine, saliva, plasma, blood, in particular whole blood. Specific components of the sample can be distinctly labeled using fluorescent dyes that are distinguishable from one another. In this way, components can be distinguished by their fluorescent emissions. METHODS FOR ANALYZING A SAMPLE
[0086] Aspects of this description include methods for analyzing a sample. As discussed above, the term "analysis" is used herein in its conventional sense to refer to evaluating qualitatively or quantitatively by measuring the presence or amount of a species of the target analyte. A variety of different samples can be tested by the present methods. In some cases, the sample is a biological sample. The term "biological sample" is used in its conventional sense to include an entire organism, plants, fungi or a subset of animal tissues, cells or parts of components which may, in certain cases, be found in blood, mucus, lymph fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen. As such, a "biological sample" refers to both the native organism and a subset of its tissues, as well as a homogenized, lysate or prepared extract from the organism or a subset of its tissues, including, but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, sections of skin, respiratory tract, gastrointestinal, cardiovascular, and genitourinary, tears, saliva, milk, blood cells, tumors, organs. Biological samples can be any tissue type of organisms, including both normal and diseased tissue (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, the biological sample is a liquid sample, such as whole blood or its derivatives, plasma, tears, urine, semen, etc., where in some cases the sample is a blood sample, including whole blood, such as blood. obtained from venipuncture or fingerstick (where the blood may or may not be combined with any reagents prior to testing, such as preservatives, anticoagulants, etc.).
[0087] In certain embodiments the source of the sample is a "mammalian", where this term is used broadly to describe organisms that are found within the Mammalia class, including the carnivorous order (eg, dogs and cats), Rodentia ( for example, mice, guinea pigs, and rats), and primates (for example, humans, chimpanzees, and monkeys). In some cases, the subjects are human beings. Biological samples of interest can be obtained from human subjects of both sexes and at any stage of development (ie, neonates, infant, juvenile, adolescent, adult), where in certain modalities the human subject is a young, adolescent or adult. While the present disclosure may apply to samples from a human subject, it is to be understood that the methods can also be performed on samples from other animal subjects (i.e., on "non-human subjects"), such as, but not limited to, birds, mice, rats, dogs, cats, cattle and horses.
[0088] In modalities, the amount of sample analyzed in the methods in question may vary, for example, ranging from 0.01 μL to 1000 μL, such as from 0.05 μL to 900 μL, such as from 0.1 μL to 800 μL, such as from 0.5 μL to 700 μL, such as from 1 μL to 600 μL, such as from 2.5 μL to 500 μL, such as from 5 μL to 400 μL, such as from 7.5 μL to 300 μL and including from 10 μL to 200 μL of sample.
[0089] The sample can be applied to the sample application site using any convenient protocol, for example, through dropper, pipette, syringe and the like. The sample can be applied together with or incorporated in an amount of an appropriate liquid, eg buffer, to provide adequate fluid flow. Any suitable liquid can be used, including but not limited to buffers, cell culture media (e.g., DMEM), etc. Buffers include, but are not limited to: tris, tricine, MOPS, HEPES, PIPES, MES, PBS, TBS and the like. When desired, detergents can be present in the liquid, for example NP-40, TWEEN™ or TritonX100 detergents.
[0090] In some embodiments, the biological sample is preloaded into a microfluidic device (as described above) and stored for a predetermined period of time prior to evaluation of the biological sample in the flow channel. For example, the biological sample can be pre-loaded into the microfluidic device, as described in more detail below, for a period of time before the biological sample in the flow channel is evaluated in accordance with the present methods. The length of time the biological sample is stored after preloading can vary, such as 0.1 hours or more, such as 0.5 hours or more, such as an hour or more, such as two hours or more, such as 4 hours or more, such as 8 hours or more, such as 16 hours or more, such as 24 hours or more, such as 48 hours or more, such as 72 hours or more, such as 96 hours or more, such as such as 120 hours or more, such as 144 hours or more, such as 168 hours or more, and including preloading the biological sample into the container 240 hours or more before analyzing the biological sample or may vary, such as from 0, 1 hour to 240 hours before analyzing the biological sample, such as from 0.5 hours to 216 hours, such as from one hour to 192 hours and including from 5 hours to 168 hours before analyzing the biological sample.
[0091] In certain embodiments, the biological sample is preloaded into the microfluidic device and the sample in the flow channel is measured at a remote location (eg a laboratory for analysis according to the present methods). By "remote location" is meant a location other than the location where the sample is contained and preloaded within the container. For example, a remote location can be another location (eg office, laboratory, etc.) in the same city, another location in a different city, another location in a different state, another location in a different country, etc., with respect to the location of the processing device, for example, as described in greater detail below. In some cases, two locations are spaced apart from each other if they are separated from each other by a distance of 10 m or more, such as 50 m or more, including 100 m or more, eg 500 m or more, 1000 m or more, 10,000 m or more, etc.
[0092] In the practice of methods according to certain modalities, a sample is brought into contact with a sample application site of a microfluidic device (as described above), the sample passes from the sample application site through a porous component where the sample mixes with an analysis reagent in a porous matrix and into a flow channel. As summarized above, passing the sample through the porous component mixes the sample with an analytical reagent. In some embodiments, the sample passes through the porous matrix into the flow channel without losing any of the sample's components. By the term "lossless" is meant that the interconnected pore network of the porous matrix does not substantially restrict the passage of sample components through the flow channel, such as where 99% or more of the sample passes through the porous matrix to the flow channel, such as 99.5% or more, such as 99.9% or more, such as 99.99% or more, such as 99.999% or more, and including the passage of 99.9999% or more of the sample through the porous matrix. In certain embodiments, all (ie, 100%) of the sample passes through the porous matrix. In other words, 1% or less of the sample components are constrained by the pores of the porous matrix, such as 0.9% or less, such as 0.8% or less, such as 0.7% or less, such as 0 .5% or less, such as 0.1% or less, such as 0.05% or less, such as 0.01% or less, such as 0.001% or less and including where 0.0001% or less of the components of the sample are restricted by the pores of the porous matrix. In other words, 1% or less of the sample remains in the porous matrix after the sample passes into the flow channel, such as 0.9% or less, such as 0.8% or less, such as 0, 7% or less, such as 0.5% or less, such as 0.1% or less, such as 0.05% or less, such as 0.01% or less, such as 0.001% or less and including 0 .0001% or less of the sample remains in the porous matrix after the sample passes into the flow channel.
[0093] In embodiments, passing the sample through the porous matrix provides mixing of the sample with an analysis reagent in the porous matrix. In some embodiments, mixing the sample with the assay reagent includes binding one or more components of the sample with an analyte-specific binding member. By "bond" is meant that the sample component and the analyte-specific binding member form one or more physical or chemical bonds with each other, including, but not limited to, bonds by ionic, dipolar, hydrophobic, coordinative, covalent, van der Waals or hydrogen bonding to the sample component with the analyte-specific binding member. In some cases, binding the sample component to an analyte-specific binding member includes covalently binding the sample component to the analyte-specific binding member. In certain cases, binding of the sample component to an analyte-specific binding member includes non-covalent binding (e.g., via hydrogen bonding) of the sample component to the analyte-specific binding member. For example the binding between the analyte-specific binding member and the target analyte can be characterized by a dissociation constant, such as a dissociation constant of 10-5 M or less, 10-6 M or less, such as 10-7 M or less, including 10-8 M or less, for example 10-9 M or less, 10-10 M or less, 10-11 M or less, 10-12 M or less, 10-13 M or less, 10-14M or less, 10-15M or less and including 10-16M or less.
As discussed above, analyte-specific binding members may vary depending on the sample being analyzed and the target analytes of interest and may include, but are not limited to antibody binding agents, proteins, peptides, haptens, acids nucleic, oligonucleotides. In some embodiments, the analyte-specific binding member is an enzyme. Examples of enzymes may be horseradish peroxidase, pyruvate oxidase, oxaloacetate decarboxylase, creatinine amidohydrolase, creatine amidinohydrolase, sarcosine oxidase, malate dehydrogenase, lactate dehydrogenase, FAD, TPP, P-5-P, NADH and its Redplex combinations.
[0095] In certain embodiments, the methods include passing the sample through the porous component to bind one or more sample components to an antibody binding agent. The antibody binding agent can be, for example, a polyclonal or monoclonal antibody or a fragment thereof sufficient to bind the analyte of interest. Antibody fragments may in some cases be monomeric Fab fragments, monomeric Fab' fragments or dimeric F(ab')2 fragments. Also within the scope of the term "antibody binding agent" are antibody engineered molecules, such as single chain antibody (scFv) molecules or humanized or chimeric antibodies produced from monoclonal antibodies by substitution of heavy chain constant regions and light to produce chimeric antibodies or substitution of both the constant regions and the framework portions of the variable regions to produce humanized antibodies. In certain embodiments, one or more components of the sample are coupled to an antibody or antibody fragment that specifically binds to a compound, such as CD14, CD4, CD45RA and CD3 or a combination thereof.
In embodiments, the analyte-specific binding agent can be coupled to a detectable label, such as radioactive labels, labels detectable by spectroscopy techniques such as nuclear magnetic resonance, as well as optically detectable labels. In some embodiments, mixing the sample with the assay reagent in the porous matrix includes binding one or more components of the sample to an analyte-specific binding member conjugated to an optically detectable label. In certain cases, the optically detectable marker is detectable by emission spectroscopy, such as by fluorescence spectroscopy. In these cases, the optically detectable label is a fluorophore such as 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine and derivatives such as acridine, acridine orange, acridine yellow, acridine red and acridine isothiocyanate; 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Yellow Lucifer VS); N-(4-anilino-1-naphthyl)maleimide; anthranilamide; Bright yellow; coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumaran 151); cyanine and its derivatives, such as cyanosine, Cy3, Cy5, Cy5.5, and Cy7; 4',6-diamidino-2-phenylindole (DAPI); 5',5''-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylaminocoumarin; diethylenetriamine penta acetate; 4,4'-diisothioyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride), 4-(4'-dimethylaminophenylazo)-benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosine and derivatives such as erythrosine B and erythrosine isothiocyanate; ethidium; fluorescein and its derivatives, such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)amine fluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6 -carboxyfluorescein (JOE), fluorescein isothiocyanate (FITC), fluorescein chlorotriazinyl, naphthofluorescein, and QFITC (XRITC); fluorescamine; IR144; IR1446; Green Fluorescent Protein (GFP); Reff Coral Fluorescent Protein (RCFP); Lissamine™; Rhodamine Lyssamine, Lucifer yellow; Malachite Green Isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Nile Red; Green Oregon; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron™ Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lyssamine 4,7-dichlororhodamine, sulfonyl chloride, rhodamine B, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulphorodamine B, sulphorodamine 101, sulfonyl chloride derivative of sulphorodamine 101 (Texas Red), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine, and isothiocyanate tetramethyl rhodamine (TRITC); riboflavin; rosolic acid derivatives and terbium chelate; xanthene or combinations thereof, among other fluorophores. In certain embodiments, the fluorophore is a fluorescent dye, such as rhodamine, coumarin, cyanine, xanthene, polymethine, pyrene, boron difluoro-dipyrromethene, naphthalimide, phycobiliprotein, peridinin chlorophyll proteins, their conjugates, or a combination thereof.
[0097] In the practice of the present methods, after the sample has been mixed with the analysis reagent in the porous matrix and passes into the flow channel (for example, by capillary action), the sample is illuminated in the flow channel with a source of light. Depending on the type of sample and the target analytes to be analyzed, the sample can be illuminated in the flow channel immediately after the sample has passed through the porous matrix and flow channel. In other embodiments, the sample is illuminated after a predetermined period of time after the sample is brought into contact with the analysis reagents in the porous matrix, such as a time period in the range of 10 seconds to 1 hour, such as 30 seconds to 30 minutes, for example, 30 seconds to 10 minutes, including 30 seconds to 1 minute. The sample can be illuminated with one or more light sources. In some embodiments, the sample is illuminated with one or more broadband light sources. The term "broadband" is used herein in its conventional sense to refer to a light source that emits light over a wide range of wavelengths, such as, for example, spanning 50 nm or more, such as 100 nm or more, such as 150 nm or more, such as 200 nm or more, such as 250 nm or more, such as 300 nm or more, such as 350 nm or more, such as 400 nm or more and including covering 500 nm or more . For example, a suitable broadband light source emits light having wavelengths from 400 nm to 700 nm. Another example of a suitable broadband light source includes a light source that emits light having wavelengths from 500 nm to 700 nm. Any suitable broadband light source protocol can be used, such as a halogen lamp, deuterium arc lamp, xenon arc lamp, stabilized fiber coupled broadband light source, a continuous spectrum wideband LED , super-luminescent emitting diode, semiconductor light-emitting diode, wide-spectrum LED white light source, an integrated multi-LED white light source, among other broadband light sources, or any combination thereof.
[0098] In other embodiments, the sample is illuminated with one or more narrowband light sources that emit a particular wavelength or narrow range of wavelengths. The term "narrowband" is used herein in its conventional sense to refer to a light source that emits light that has a narrow range of wavelengths, such as for example, 50 nm or less, such as 40 nm or less. , such as 30 nm or less, such as 25 nm or less, such as 20 nm or less, such as 15 nm or less, such as 10 nm or less, such as 5 nm or less, such as 2 nm or less and including light sources that emit a specific wavelength of light (ie, monochromatic light). Any suitable narrowband light source protocol can be used, such as a narrow wavelength LED, laser diode or a wideband light source coupled to one or more bandpass filters, optical diffraction gratings , monochromators, or any combination thereof.
[0099] In certain embodiments, methods include irradiating the sample in the flow channel with one or more lasers. The type and number of lasers will vary depending on the sample as well as the desired emitted light collected and can be a gas laser such as a helium-neon laser, argon laser, krypton laser, xenon laser, nitrogen laser, CO laser, CO laser, argon fluoride excimer laser (ArF), krypton fluoride excimer laser (KrF), xenon chloride excimer laser (XeCl) or xenon fluoride excimer laser (XeF) or a combination thereof. In other cases, methods include irradiating the sample in the flow channel with a dye laser, such as a stilbene, coumarin or rhodamine laser. In still other cases, the methods include irradiating the sample in the flow channel with a metal vapor laser, such as a helium-cadmium laser (HeCd), helium-mercury laser (HeHg), helium-selenium laser ( HeSe), helium-silver laser (HeAg), strontium laser, neon-copper laser (NeCu), copper laser or gold laser and combinations thereof. In still other cases, the methods include irradiating the sample in the flow channel with a solid state laser, such as a ruby laser, an Nd:YAG laser, NdCrYAG laser, Er:YAG laser, Nd:YLF laser, laser Nd:YVO4 laser, Nd:YCa4O(BO3) laser, Nd:YCOB laser, titanium sapphire laser, thulium YAG laser, ytterbium YAG laser, ytterbium2O3 laser or doped cerium laser and combinations thereof.
[00100] Depending on the analyte to be analyzed, as well as interferents present in the biological sample, the biological sample can be illuminated using one or more light sources, such as two or more light sources, such as three or more light sources, such as four or more light sources, such as five or more light sources and including ten or more light sources. Any combination of light sources can be used as desired. For example, a first light source can be a wideband white light source (eg, wideband white LED) and the second light source can be a near-infrared wideband light source (eg, broadband LED near IR). In other examples, where two light sources are used, a first light source may be a wideband white light source (eg, wideband white light LED) and the second light source may be a white light source. narrow-spectrum light (for example, a narrow-band or near-IR visible light LED). In still other examples, the light source is a plurality of narrowband light sources each emitting specific wavelengths, such as an array of two or more LEDs, such as an array of three or more LEDs, such as an array of five or more LEDs, including an array of ten or more LEDs.
[00101] When using more than one light source, the sample can be illuminated with the light sources simultaneously or sequentially, or a combination thereof. For example, where the sample is illuminated with two light sources, the methods in question may include simultaneously illuminating the sample with both light sources. In other modalities, the sample can be sequentially illuminated by two light sources. When the sample is sequentially illuminated with two or more light sources, the time each light source illuminates it can independently be 0.001 seconds or more, such as 0.01 seconds or more, such as 0.1 seconds or more, such as 1 second or more, such as 5 seconds or more, such as 10 seconds or more, such as 30 seconds or more, and including 60 seconds or more. In modalities where the sample is sequentially illuminated by two or more light sources, the time the sample is illuminated by each light source can be the same or different.
[00102] The period of time between illumination by each light source may also vary, as desired, being independently separated by a delay of 1 second or more, such as 5 seconds or more, such as for 10 seconds or more, such as such as for 15 seconds or more, such as for 30 seconds or more and including for 60 seconds or more. In modalities where the sample is sequentially illuminated by more than two light sources (i.e., three or more), the delay between illumination by each light source can be the same or different.
[00103] Depending on the analysis protocol, the illumination of the sample can be continuous or at specific intervals. For example, in some modalities, the sample can be illuminated continuously throughout the entire time the sample is being analyzed. When the light includes two or more light sources, the sample can be continuously illuminated by all light sources simultaneously. In other cases, the sample is illuminated continuously with each light source sequentially. In other embodiments, the sample may be illuminated at regular intervals, such as illuminating the sample every 0.001 microseconds, every 0.01 microseconds, every 0.1 microseconds, every 1 microsecond, every 10 microseconds, every 100 microseconds and including each 1000 microseconds. The sample may be illuminated with the light source one or more times in any given measurement period, such as 2 or more times, such as 3 or more times, including 5 or more times in each measurement period.
[00104] Depending on the light source and the characteristics of the flow channel (eg the width of the flow channel), the flow channel can be radiated from a distance that varies, such as 1 mm or more from the flow channel , such as 2 mm or more, such as 3 mm or more, such as 4 mm or more, such as 5 mm or more, such as 10 mm or more, such as 15 mm or more, such as 25 mm or more, and including 50mm or more of the flow channel. In addition, the angle at which the flow channel is irradiated may also vary, ranging from 10° to 90°, such as 15° to 85°, such as 20° to 80°, such as 25° to 75 ° and including from 30° to 60°. In certain embodiments, the flow channel is radiated by the light source at an angle of 90° to the axis of the flow channel.
[00105] In certain embodiments, irradiation of the flux channel includes moving one or more light sources (eg lasers) along the longitudinal axis of the flux channel. For example, the light source can be moved upstream or downstream along the longitudinal axis of the flow channel by radiating the flow channel along a predetermined length of the flow channel. For example, methods may include moving the light source along the longitudinal axis of the flow channel by 1 mm or more, such as 2.5 mm or more, such as 5 mm or more, such as 10 mm or more, such as 15mm or more, such as 25mm or more and including 50mm or more of the flow channel. The light source can be moved continuously or at specific intervals. In some modalities, the light source is moved continuously. In other embodiments, the light source is moved along the longitudinal axis of the flow channel at specific intervals, such as, for example, in increments of 0.1 mm or more, such as increments of 0.25 mm or more, and including 1mm and larger increments.
[00106] In practicing the methods according to aspects of the present disclosure, the light emitted from the sample in the flow channel is measured at one or more wavelengths. In embodiments, emitted light is measured at one or more wavelengths, such as at 5 or more different wavelengths, such as at 10 or more different wavelengths, such as at 25 or more different wavelengths, such as such as at 50 or more different wavelengths, such as at 100 or more different wavelengths, such as at 200 or more different wavelengths, such as at 300 or more different wavelengths, and including measurement of light emitted from the sample in the flow channel at 400 or more different wavelengths.
[00107] In some embodiments, measuring the light emitted from the sample in the flow channel includes measuring the light emitted over a range of wavelengths (eg, 200 nm - 800 nm). For example, the methods may include measuring the light emitted from the sample in the flow channel over one or more of the wavelength ranges: 200 nm - 800 nm; 400 nm - 500 nm; 500 nm - 600 nm; 600 nm - 700 nm; 700 nm - 800 nm; 550nm - 600nm; 600 nm - 650 nm; 650 nm - 700 nm and any one portion or combinations thereof. In one example, the methods include measuring the light emitted from the sample in the flow channel over wavelengths ranging from 200 nm - 800 nm. In another example, methods include measuring the light emitted from the sample in the flow channel over wavelengths ranging from 500 nm - 600 nm and 650 nm - 750 nm. In certain cases, the methods include measuring the light emitted from the sample in the flow channel at 575 nm, 660 nm and 675 nm, or a combination thereof.
[00108] The measurement of light emitted from the sample in the flow channel over a range of wavelengths, in certain cases, includes collecting spectra of light emitted over the range of wavelengths. For example, the methods may include collecting spectra of light emitted from the sample in the flow channel over one or more of the wavelength ranges: 200 nm - 800 nm; 400 nm - 500 nm; 500 nm - 600 nm; 600 nm - 700 nm; 700 nm - 800 nm; 550nm - 600nm; 600 nm - 650 nm; 650 nm - 700 nm and any portion or combinations thereof. In one example, the methods include collecting spectra of light emitted from the sample in the flow channel over wavelengths ranging from 400 nm - 800 nm. In another example, the methods include collecting spectra of light emitted from the sample in the flow channel over wavelengths ranging from 500 nm - 700 nm.
[00109] In certain embodiments, light emitted from the sample in the flux channel is detected at one or more specific wavelengths. For example, the methods may include detecting light emitted from the sample in the flow channel at 2 or more specific wavelengths, such as at 3 or more specific wavelengths, such as at 4 or more specific wavelengths, such as such as at 5 or more specific wavelengths, such as at 10 or more specific wavelengths and including detecting light emitted from the sample in the flow channel at 25 or more specific wavelengths. In certain embodiments, emitted light is detected at 575 nm. In other embodiments, emitted light is detected at 660 nm. In still other embodiments, emitted light is detected at 675 nm.
[00110] Depending on the specific analysis protocol, the light emitted from the sample in the flow channel can be measured continuously or at specific intervals. For example, in some modalities, the measurement of emitted light is continuous throughout the entire time the sample is being analyzed. When the measurement of emitted light includes measurement of two or more wavelengths or ranges of wavelengths, the wavelengths or ranges of wavelengths can all be measured simultaneously, or each wavelength or range of wavelengths. waveform can be measured sequentially.
[00111] In other modalities, the emitted light is measured at specific intervals, such as measuring the light emitted from the sample in the flow channel every 0.001 microseconds, every 0.01 microseconds, every 0.1 microseconds, every 1 microsecond, every 10 microseconds, every 100 microseconds and including every 1000 microseconds. The light emitted from the sample in the flow channel can be measured one or more times during the subject methods, such as two or more times, such as 3 or more times, such as 5 or more times and that includes 10 or more times.
[00112] The light emitted from the sample in the flow channel can be measured by any convenient light detection protocol, including but not limited to optical sensors or photodetectors, such as active pixel sensors (APS), avalanche photodiodes, sensors of imaging, charge-coupled devices (CCD), charge-coupled intensified devices (ICCD), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes, phototransistors, photocon- quantum dot conductors or photodiodes and their combinations, among other photodetectors. In certain embodiments, emitted light is measured with a charge-coupled device (CCD), charge-coupled semiconductor devices (CCD), active pixel sensors (APS), complementary metal oxide semiconductor image sensors (CMOS) or sensors N-type image of metal oxide semiconductor (NMOS). In certain embodiments, light is measured with a charge-coupled device (CCD). When transmitted light is measured with a CCD, the active sensing surface area of the CCD can vary, such as 0.01 cm2 to 10 cm2, such as 0.05 cm2 to 9 cm2, such as 0.1 cm2 to 8 cm2, such as from 0.5 cm2 to 7 cm2 and including from 1 cm2 to 5 cm2.
[00113] In some embodiments, the methods include optically adjusting the light emitted from the flow channel. For example, emitted light can be passed through one or more lenses, mirrors, holes, slits, grids, light refractors, and any combinations thereof. In some cases, emitted light passes through one or more focusing lenses in order to reduce the profile of light propagated over the active surface of the detector. In other cases, emitted light passes through one or more reduction lenses, such as to increase the profile of light propagated over the active surface of the detector. In still other cases, methods include collimating light. For example, emitted light can be collimated by passing light through one or more collimating lenses or collimated mirrors or a combination thereof.
[00114] In certain embodiments, the methods include passing emitted light collected from the flow channel through optical fibers. Suitable fiber optic protocols for propagating light from the flux channel to the active surface of a detector include, but are not limited to, fiber optic protocols such as those described in U.S. Pat. 6,809,804, the disclosure of which is incorporated herein by reference.
[00115] In certain embodiments, the methods include passing the emitted light through one or more wavelength separators. Wavelength separation, in certain embodiments, may include selectively passing or blocking wavelengths or wavelength ranges of polychromatic light. To separate wavelengths of light, transmitted light can be passed through any suitable wavelength separation protocol, including, but not limited to, colored glass, bandpass filters, interference filters, dichroic mirrors, gratings. diffraction, monochromators and their combinations, among other wavelength separation protocols.
[00116] In other embodiments, the methods include separating the wavelengths of light by passing light emitted from the flow channel through one or more optical filters, such as one or more bandpass filters. For example, optical filters of interest may include bandpass filters having minimum bandwidths ranging from 2 nm to 100 nm, such as 3 nm to 95 nm, such as 5 nm to 95 nm, such as 10 nm at 90 nm, such as from 12 nm to 85 nm, such as from 15 nm to 80 nm and including bandpass filters with minimum bandwidths ranging from 20 nm to 50 nm.
[00117] In certain embodiments, the subject fluorescence assay may include methods of imaging samples in capillary channels, such as those described in US Patent Application Nos. 8,248,597; 7,927,561 and 7,738,094, as well as those described in co-pending US Patent Application Nos. 13/590,114 filed August 20, 2012, 61/903,804 filed November 13, 2013 and 61/949,833 filed March 7, 2014, the descriptions of which are incorporated herein by reference.
[00118] In certain embodiments, the methods include capturing an image of the stream channel. Capturing one or more images of the flow channel may include illuminating the flow channel with one or more light sources (as described above) and capturing the image with a charge-coupled device (CCD), semiconductor charge-coupled devices ( CCD), active pixel sensors (APS), complementary metal oxide semiconductor image sensors (CMOS) or metal oxide semiconductor (NMOS) N-type image sensors. Stream channel images can be captured continuously or at specific intervals. In some cases, the methods include capturing images continuously. In other cases, the methods include capturing images at specific intervals, such as capturing an image of the flow channel every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 milliseconds, every 10 milliseconds, every 100 milliseconds and including every 1000 milliseconds, or some other interval. Where flow channel images are captured with a CCD camera detector, the active sensing surface area of the CCD can vary, such as from 0.01 cm2 to 10 cm2, such as from 0.05 cm2 to 9 cm2. such as from 0.1 cm2 to 8 cm2 such as from 0.5 cm2 to 7 cm2 and including from 1 cm2 to 5 cm2.
[00119] All or part of the flow channel can be captured in each image, such as 5% or more of the flow channel, such as 10% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, such as 95% or more and including 99% or more of the flow channel can be captured in each image. In certain modalities, the entire stream channel is captured in each image. One or more images can be captured, as desired, such as 2 or more images, such as 3 or more images, such as 5 or more images, such as 10 or more images, such as 25 or more images and including 100 or more images. When more than one image is captured from the stream channel, the plurality of images can be automatically collated or averaged by a processor with digital image processing algorithm.
[00120] Flow channel images can be captured at any suitable distance from the flow channel as long as a useful flow channel image is captured. For example, flow channel images can be captured at 0.01 mm or more from the flow channel, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more , such as 1 mm or more, such as 2.5 mm or more, such as 5 mm or more, such as 10 mm or more, such as 15 mm or more, such as 25 mm or more and including 50 mm or more of the flow cytometry flow channel. Flow channel images can also be captured at any angle to the flow channel. For example, flow channel images can be captured at an angle to the longitudinal axis of the flow channel ranging from 10° to 90°, such as 15° to 85°, such as 20° to 80°, such as from 25° to 75°, including from 30° to 60°. In certain embodiments, images of the flow channel are captured at a 90° angle to the longitudinal axis of the flow channel.
[00121] In some embodiments, capturing flow channel images includes moving one or more image sensors along the flow path. For example, the image sensor can be moved upstream or downstream along with the image capture stream in a plurality of sensing fields. For example, the methods can include capturing images of the flow stream in two or more different detection domains, such as 3 or more detection domains, such as 4 or more fields and including 5 or more detection detection fields. The image sensor can be moved continuously or at specific intervals. In some modes, the image sensor is moved continuously. In other embodiments, the image sensor can be moved along the flow path at specific intervals, such as, for example, in increments of 1 mm or more, such as increments of 2 mm or more, and including increments of 5 mm or more. most.
[00122] In certain embodiments, the methods include reducing the background signal of images captured from the stream channel. In these embodiments, the methods include capturing an image of the flow channel with unbound optically labeled analyte-specific binding members (i.e., assay reagent not mixed with the sample) and reducing (e.g., subtracting) the signal. background of the captured images of the sample in the flow channel. In some cases, the methods include capturing an image of the sample in the flow channel, determining the background of unbound optically tagged analyte-specific binding members, and reducing the background of the captured image of the sample in the flow channel. . In embodiments of the present disclosure, background signal may be determined one or more times, such as two or more times, such as 3 or more times, such as 5 or more times, and including 10 or more times. When desired, the background signal can be averaged to provide a background signal average. In certain embodiments, determining background signal includes capturing one or more images of the flow channel in the absence of sample.
[00123] Depending on the analysis reagents, the unbound reagent in the flow channel is substantially constant. In other words, the distribution of unbound reagent present in the flow channel is homogeneous and the variation in the amount of unbound reagent in different regions of the flow channel varies by 10% or less, such as by 5% or less, such as by 4% or less, such as by 3% or less, such as by 2% or less, such as by 1% or less, such as by 0.5% or less and which includes by 0.1% or less. Thus, the background signal varies along the longitudinal axis of the flow channel by 10% or less, such as by 5% or less, such as by 4% or less, such as by 3% or less, such as by 2 % or less, such as by 1% or less, such as by 0.5% or less and including by 0.1% or less. In certain embodiments, the methods include reducing the background of the captured image of the sample in the flow channel, where the background varies by 10% or less, such as by 5% or less, such as by 4% or less , such as by 3% or less, such as by 2% or less, such as by 1% or less, such as by 0.5% or less, and including by 0.1% or less along the longitudinal axis of the flow channel.
[00124] As illustrated in Figures 1 and 2A-B, the microfluidic devices of interest can be used to detect serological concentrations of human antibodies in finger puncture volumes (5-50 μL) of whole blood in an unwashed format. In some specific embodiments, the methods include applying a liquid sample to the sample application site and directing the sample flow through capillary force to the porous element. As the sample enters the porous element a preparation of reagents dissolves in the sample at a substantially continuous rate. The assay mix may comprise an optically active reagent for specific sample component labeling and a set of buffer components that provide for continuous dissolution of the reagent in the sample. In some embodiments the buffer components can comprise bovine serum albumin (BSA), trehalose (such as trehalose D+), polyvinylpyrrolidone (PVP) or any combination thereof. The optically active reagent can be any detectable marker such as a fluorescently labeled antibody conjugate. Buffer and sample can be mixed in the porous element through passive mixing through a network of tortuous paths in the porous element resulting in reagent that is bound to sample components and unbound reagent. The sample labeled with a detectable marker can then be evaluated as discussed above, such as optically or magnetically along the capillary channel of the microfluidic device. In some embodiments, the sample can be evaluated by obtaining a signal or image of the sample through a transmissive wall. Signal processing can include subtracting a background signal from the unbound reagent. The amount of unbound reagent along the transmitting wall can be substantially constant. In some embodiments the amount of unbound reagent varies less than 50%, 40%, 30%, 20%, or 10% along the transmissive wall, beneficially providing improved detection for reagent bound to sample components. Detection may comprise subtraction of background optical signals and observing the number, optical properties, morphological or configuration of the signals in addition to the background signal. SYSTEMS FOR ANALYSIS OF A SAMPLE FOR AN ANALYTE
Aspects of the present invention further include systems for practicing the present methods. In embodiments, systems that include one or more of the present microchannels and optical evaluation system with a light source, and a detector for detecting one or more wavelengths of light emitted by the sample in the flow channel are provided. In certain embodiments, the systems additionally include one or more of the present microchannels directly integrated into the optical evaluation system.
[00126] As summarized above, aspects of the present invention include analyzing a sample for one or more analytes. The systems include one or more light sources for evaluating a flow channel that contains a sample of interest mixed with an analysis reagent. In some embodiments, the light source is a wideband light source, emitting light that has a wide range of wavelengths, such as, for example, spanning 50 nm or more, such as 100 nm or more, such as 150 nm or more, such as 200 nm or more, such as 250 nm or more, such as 300 nm or more, such as 350 nm or more, such as 400 nm or more and including spanning 500 nm or more. For example, a suitable broadband light source emits light with wavelengths from 200 nm to 800 nm. Any suitable broadband light source protocol can be used, such as a halogen lamp, deuterium arc lamp, xenon arc lamp, stabilized fiber coupled broadband light source, a continuous spectrum wideband LED , superluminescent emitting diode, semiconductor light emitting diode, wide-spectrum LED white light source, an integrated multiLED white light source, among other broadband light sources, or any combination thereof.
[00127] In other embodiments, the light source is a narrowband light source that emits a particular wavelength or narrow range of wavelengths. In some cases, narrowband light sources emit light with a narrow range of wavelengths, such as for example, 50 nm or less, such as 40 nm or less, such as 30 nm or less, such as 25 nm or less, such as 20 nm or less, such as 15 nm or less, such as 10 nm or less, such as 5 nm or less, such as 2 nm or less and including light sources that emit a specific wavelength of light (ie, monochromatic light). Any suitable narrowband light source protocol can be used, such as a narrow wavelength LED, laser diode or a wideband light source coupled to one or more bandpass filters, optical diffraction gratings , monochromators, or any combination thereof. In certain embodiments, the narrowband light source is a laser, such as a gas laser, such as a helium-neon laser, argon laser, krypton laser, xenon laser, nitrogen laser, CO2 laser, CO laser, argon fluoride excimer laser (Arf), krypton fluoride excimer laser (KrF), xenon chloride excimer laser (XeCl) or xenon fluoride excimer laser (XeF) or one thereof combination. In other cases, methods include irradiating the sample in the flow channel with a metal vapor laser, such as a helium-cadmium laser (HeCd), helium-mercury laser (HeHg), helium-selenium laser (HeSe ), helium-silver laser (HeAg), strontium laser, neon-copper laser (NeCu), copper laser or gold laser, or a solid state laser such as a ruby laser, an Nd laser :YAG, NdCrYAG laser, Er:YAG laser, Nd:YLF laser, Nd:YVO4 laser, Nd:YCa4O(BO3) laser, Nd:YCOB laser, titanium sapphire laser, thulium YAG laser, ytterbium YAG laser, ytterbium2O3 laser or doped cerium laser and combinations thereof.
[00128] The subject systems may include one or more light sources, as desired, such as two or more light sources, such as three or more light sources, such as four or more light sources, such as five or more light sources and including ten or more light sources. In embodiments, the light sources emit light having wavelengths ranging from 200 nm to 1000 nm, such as 250 nm to 950 nm, such as 300 nm to 900 nm, such as 350 nm to 850 nm, and including from 400 nm to 800 nm.
[00129] As summarized above, the present systems are configured to receive a microfluidic device with a sample application site, a flow channel in fluid communication with the sample application site, and a porous component having a porous matrix and an analysis reagent positioned between the sample application site and the flow channel. In these embodiments, the systems can also include a cartridge holder for receiving the microfluid in the present system. For example, the cartridge holder can include a holder for receiving the cartridge microfluidic device and one or more cartridge retainers for holding the cartridge microfluidic device in the cartridge holder. In some cases, the cartridge holder includes vibration dampeners to reduce agitation of the cartridge microfluidic device positioned in the cartridge holder, as well as one or more cartridge presence references configured to indicate that a cartridge microfluidic device is present. in the cartridge holder.
[00130] In some embodiments, the systems include a transport cartridge coupled to the cartridge holder to move the microfluidic device in and out of the evaluation system. In some embodiments, the transport cartridge is coupled to one or more conversion or lateral movement protocols to move the microfluidic device. For example, the transport cartridge can be coupled to a mechanically driven conversion platform, mechanical lead screw assembly, mechanical slide device, mechanical lateral movement device, mechanically operated adapted conversion device, a motor driven conversion platform , mechanical lead screw assembly, mechanical slide device, such as those employing a stepper motor, servo motor, brushless electric motor, brushed DC motor, micro-step motor drive, high-resolution stepper motor, among other types of engines. The systems may also include an assembly of cartridge positioning transport rails to facilitate lateral movement of the cartridge holder.
[00131] As described above, the light emitted by the sample in the flow channel is collected and detected using one or more photodetectors. In certain embodiments, the systems include one or more objective lenses to collect light emitted from the flux channel. For example, the objective lens can be a magnifying glass with a nominal magnification ranging between 1.2 and 5, such as a nominal magnification of 1.3 to 4.5, such as a nominal magnification of 1.4 to 4 , such as a nominal magnification of 1.5 to 3.5, such as a nominal magnification of 1.6 to 3, including passing light transmitted through a magnifying lens with a nominal magnification of 1.7 to 2, 5. Depending on the configuration of the light source, sample chamber, and detector, objective lens properties may vary. For example, the numerical aperture of the objective lens in question can also vary, ranging from 0.01 to 1.7, such as 0.05 to 1.6, such as 0.1 to 1.5, such as 0.2 to 1.4, such as 0.3 to 1.3, such as 0.4 to 1.2, such as 0.5 to 1.1 and including a numerical aperture ranging from 0.6 to 1.0. Likewise, the focal length of the objective lens varies, ranging from 10mm to 20mm, such as from 10.5mm to 19mm, such as from 11mm to 18mm and including from 12mm to 15mm.
[00132] In some embodiments, the objective lens is coupled to a focusing module to focus the slit beam projection transmitted through the sample chamber to the detector for detection. For example, an autofocus module suitable for focusing the slit beam projection transmitted through the sample may include, but is not limited to, that described in U.S. Patent No. 6,441,894, filed October 29, 1999 , the disclosure of which is incorporated herein by reference.
[00133] Systems of the present disclosure may also include one or more wavelength splitters. The term "wavelength splitter" is used herein in its conventional sense to refer to an optical protocol for separating polychromatic light into its wavelength elements such that each wavelength can be conveniently detected. Examples of suitable wavelength separators in such systems may include, but are not limited to, colored glass, bandpass filters, interference filters, dichroic mirrors, diffraction gratings, monochromators and their combinations, among other protocols. wavelength separation. Depending on the light source and the sample being analyzed, systems may include one or more wavelength splitters, such as two or more, such as three or more, such as four or more, such as five or more and including 10 or more wavelength separators. In one example, systems include two or more bandpass filters. In another example, the systems include two or more bandpass filters and a diffraction grating. In yet another example, the systems include a plurality of bandpass filters and a monochromator. In certain embodiments, the systems include a plurality of bandpass filters and diffraction gratings configured in a filter wheel configuration. When systems include two or more wavelength splitters, the wavelength splitter can be used individually or in series to separate polychromatic light into component wavelengths. In some embodiments, the wavelength splitters are arranged in series. In other embodiments, the wavelength splitters are arranged individually.
[00134] In some embodiments, the systems include one or more diffraction gratings. Diffraction gratings of interest may include, but are not limited to, broadcast, dispersive or reflective diffraction gratings. Appropriate diffraction grating spacings can range from 0.01 μm to 10 μm, such as from 0.025 μm to 7.5 μm, such as from 0.5 μm to 5 μm, such as from 0.75 μm to 4 μm , such as from 1 μm to 3.5 μm and including 1.5 μm and 3.5 μm.
[00135] In some embodiments, the systems include one or more optical filters. In certain cases, systems include bandpass filters with minimum bandwidths ranging from 2 nm to 100 nm, such as 3 nm to 95 nm, such as 5 nm to 95 nm, such as 10 nm to 90 nm, such as 12 nm to 85 nm, such as 15 nm to 80 nm and including bandpass filters with minimum bandwidths ranging from 20 nm to 50 nm.
[00136] The systems of the present disclosure also include one or more detectors. Examples of suitable detectors may include, but are not limited to optical sensor or photodetectors, such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCD), charge-coupled devices. (ICCD), light emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes, phototransistors, photoconductors or quantum dot photodiodes and their combinations, among other photodetectors. In certain embodiments, transmitted light is measured with a charge-coupled device (CCD). When transmitted light is measured with a CCD, the active sensing surface area of the CCD can vary, such as 0.01 cm2 to 10 cm2, such as 0.05 cm2 to 9 cm2, such as 0.1 cm2 to 8 cm2, such as from 0.5 cm2 to 7 cm2 and including from 1 cm2 to 5 cm2.
[00137] In some embodiments, the systems include one or more cameras or camera sensors to capture an image of the flow channel. Suitable cameras for capturing a flow image include, but are not limited to charge-coupled device (CCD), charge-coupled semiconductor (CCD) devices, active pixel sensors (APS), complementary oxide semiconductor image sensors metal (CMOS) or metal oxide semiconductor (NMOS) N-type image sensors.
[00138] In embodiments of the present invention, the detectors of interest are configured to measure light emitted from the flow channel at one or more wavelengths, such as at 2 or more wavelengths, such as at 5 or more wavelengths. different waves, such as 10 or more different wavelengths, such as 25 or more different wavelengths, such as 50 or more different wavelengths, such as 100 or more different wavelengths, such as 200 or more different wavelengths, such as at 300 or more different wavelengths and including measuring light transmitted through the sample chamber at 400 or more different wavelengths.
[00139] In modalities, the detector can be configured to measure light continuously or at specific intervals. In some cases, the detectors of interest are configured to measure light continuously. In other cases, the detectors of interest are configured to take measurements at specific intervals, such as measuring light every 0.001 millisecond, every 0.01 millisecond, every 0.1 millisecond, every 1 millisecond, every 10 millisecond - do, every 100 milliseconds and including every 1000 milliseconds, or some other interval.
[00140] In certain embodiments, the light emitted by the sample in the flow channel is measured with an imaging system such as those described in US Patent Application Nos. 8,248,597; 7,927,561; 7,738,094 and copending US Patent Application Nos. 13/590,114 filed August 20, 2012, 61/903,804 filed November 13, 2013 and 61/949,833 filed March 7, 2014, the descriptions of which are incorporated herein by reference.
[00141] In certain cases, the systems of interest include one or more of the present microfluidic devices (as described above) integrated into the imaging system. Thus, in these embodiments, the systems in question are not configured to receive a microfluidic device described above, but rather are configured to receive the fluidic sample directly, which is subsequently removed after analyzing the sample. By "removed" it is meant that no amount of sample remains in contact with the systems concerned, including any flow channel, sample application site, inlet, as well as porous matrix. In other words, when the sample is removed, all traces of the sample are cleaned from the system components. In some embodiments, the systems can additionally include one or more washing devices for cleaning the integrated microfluidic device. For example, washing devices can include microconducts with or without a spray nozzle for delivering wash buffer to clean the microfluidic device. In certain embodiments, these systems include a reservoir for storing one or more wash buffers. KITS
Aspects of the invention further include kits, wherein the kits include one or more microfluidic analysis devices. In some cases, kits may include one or more analysis components (eg, labeled reagents, buffers, etc., as described above). In some cases, kits may further include a sample collection device, for example, a lance or needle configured to pierce the skin to obtain a whole blood sample, a pipette, etc., as desired. The kits' various analysis components may be present in separate containers, or some or all of them may be pre-combined. For example, in some cases one or more kit components, eg microfluidic devices, are present in a sealed pouch, eg a foil pouch or sterile wrap.
[00143] In addition to the above components, the kits in question may additionally include (in certain embodiments) instructions for practicing the present methods. These instructions may be present in the concerned kits in a variety of ways, one or more of which may be present in the kit. One form in which instructions can be presented is as information printed on a suitable medium or substrate, for example, a piece or pieces of paper on which the information is printed, on kit packaging, on an information sheet, and the like. Yet another form of these instructions is a computer medium, eg floppy disk, compact disk (CD), portable flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address that can be used over the internet to access information in a secluded location. UTILITY
[00144] The methods, devices, and kits of the present disclosure find use in a variety of different applications and can be used to determine whether an analyte is present in a multiplicity of different sample types from a multiplicity of possible sources. Depending on the application and desired result of the methods described herein, an analyte can be detected qualitatively ("present" versus "absent"; "yes, above a predetermined limit" versus "no, not above a predetermined limit" ; etc.) or a quantitative form, for example, as an amount in a sample (such as the concentration in the sample). Many different types of analytes can be analytes of interest, including, but not limited to: proteins (including both free proteins and proteins bound to the surface of a structure such as a cell), nucleic acids, viral particles, and the like. In addition, the samples can be of in vitro or in vivo origin, and the samples can be diagnostic samples.
[00145] By putting into practice the methods of the present disclosure, samples may be obtained from in vitro origin (for example, extract from a laboratory cultured cell culture) or from in vivo origin (for example, a mammalian subject, a human subject, a research animal, etc.). In some embodiments, the sample is obtained from an in vitro source. In vitro origins include, but are not limited to, prokaryotic (eg bacterial) cell cultures, eukaryotic (eg mammalian, fungal) cell cultures (eg established cell line cultures, eukaryotic cell line cultures). known or acquired cells, immortalized cell cultures, primary cell cultures, laboratory yeast cultures, etc.), tissue cultures, column chromatography eluents, cell lysates/extracts (eg lysates/extracts containing protein, nucleic acid-containing lysates/extracts, etc.), viral packaging supernatants, and the like. In some embodiments, the sample is obtained from an in vivo source. In vivo sources include living multicellular organisms and can give rise to diagnostic samples.
[00146] In some embodiments, the analyte is a diagnostic analyte. A "diagnostic analyte" is an analyte from a sample that has been obtained from or derived from a living multicellular organism, eg mammal, in order to make a diagnosis. In other words, the sample was taken to determine the presence of one or more disease analytes in order to diagnose a disease or condition. Therefore, methods are diagnostic methods. Since methods are "diagnostic methods", they are methods that diagnose (ie, determine the presence or absence of) a disease (eg, disease condition, diabetes, etc.) or condition (eg, pregnancy) in a living organism, such as a mammal (eg, a human being). As such, certain embodiments of the present disclosure are methods that are used to determine whether a living subject has a particular disease or condition (e.g., diabetes). "Diagnostic methods" also include methods that determine the severity or status of a particular disease or condition.
[00147] In certain embodiments, methods are methods of determining whether an analyte is present in a diagnostic sample. As such, methods are methods of evaluating a sample where the analyte of interest may or may not be present. In some cases, it is not known whether the analyte is present in the sample prior to performing the analysis. In other cases, prior to performing the assay, it is unknown whether the analyte is present in the sample in an amount that is greater than (exceeds) a predetermined threshold amount. In such cases, methods are methods of evaluating a sample in which the analyte of interest may or may not be present in an amount that is greater than (exceeds) the predetermined limit.
[00148] Diagnostic samples include those obtained from in vivo sources (eg, a mammalian subject, a human subject, and the like) and may include samples obtained from tissues or cells from a subject (eg, biopsies, tissue samples, whole blood, fractionated blood, hair, skin and the like). In some cases, cells, fluids or tissues derived from a subject are cultured, stored or manipulated prior to evaluation and such a sample may be considered a diagnostic sample if the results are used to determine the presence, absence, status, or severity of a disease (eg disease condition, diabetes, etc.) or condition (eg pregnancy) in a living organism.
[00149] In some cases, a diagnostic sample is a tissue sample (eg whole blood, fractionated blood, plasma, serum, saliva and the like) or is obtained from a tissue sample (eg whole blood, blood fractionated, plasma, serum, saliva, skin, hair and the like). An example of a diagnostic sample includes, but is not limited to, cell and tissue cultures derived from a subject (and their derivatives, such as supernatants, lysates, and the like); tissue and body fluid samples; non-cellular samples (eg, column eluents; cell biomolecules such as proteins, lipids, carbohydrates, nucleic acids; synthesis reaction mixtures; nucleic acid amplification reaction mixtures; enzymatic reactions; or in vitro biochemical or test solutions; or products of other in vitro and in vivo reactions, etc.); etc.
[00150] The present methods can be used with samples from a variety of different types of subjects. In some embodiments, a sample is from a subject within the Mammalia class, including the order carnivore (eg, dogs and cats), Rodentia (eg, mice, guinea pigs, and rats), Lagomorpha (eg, rabbits), and primates (eg, humans, chimpanzees, and apes), and the like. In some cases, the animals or hosts, that is, the subjects are human beings. EXPERIMENTAL
[00151] The following example is given by way of illustration and not by way of limitation. The example is provided for illustrative purposes only, and is not intended to limit the scope of the present disclosure in any way. Efforts have been made to ensure accuracy with respect to the numbers used (eg quantities, temperatures, etc.), but some experimental errors and deviations must, of course, be allowed for.
[00152] A finger puncture quantity (5-50 µL) of whole blood is loaded into a sample application site of a capillary device of the present invention (shown in Figs. 2A and B) where it is drawn into the element porous through capillary force. The porous element is a porous frit and associated analysis mixture. The composition of the reaction is a preserved buffer including BSA, MES, trehalose D+, EDTA, PVP and a mixture of reagents. The BSA:Trehalose:PVP ratio in a dry weight is 21:90:1. The reagent mix consists of a set of antibody-dye conjugates, specific for CD14, CD4, CD45RA, and CD3 antigens in the blood sample. Once loaded, a cap is placed over the sample application site, sealing the sample application site and a capillary channel vent outlet. Capillary blood flow follows through the porous element and along the channel, unhindered by the capillary sealing cap from the outside environment. The flux can end at a hydrophobic junction. Anti-CD14, CD4, CD45RA and CD3 antibodies present in the porous element dissolve in the blood sample at a substantially constant rate as the sample passes through the porous element and along the capillary channel for about 2 minutes from the time. time the sample was applied. The blood sample flows through the porous element substantially unimpeded and unfiltered. The specific components in the blood sample will bind to the antibody-dye conjugates, enabling the detection and quantification of analytes in the sample. Detection is performed using an LED to illuminate the cartridge where the transmissive wall region is located. The optical signal is imaged through the optically transmissive wall of the capillary channel using a low-power microscope with a CCD-camera detector and an appropriate filter. A schematic diagram of the image is shown in Fig. 3A through the transmissive wall 50 of the capillary channel 60. A schematic diagram of the image analysis results (Fig. 3B) shows that, after processing, the signal distribution of the dye conjugates - antibody bound to analyte in cells is measurably superior to free conjugate in the sample stream. Image processing allows the reduction of the background signal 70 in order to form a clearer image of cells labeled with the dye-antibody conjugates and determine the number of cells that test positive for the antibodies CD14, CD4, CD45RA, CD3 .
[00153] Notwithstanding the attached clauses, the disclosure set forth herein is also defined by the following clauses:
[00154] 1. Microfluidic device comprising:
[00155] a sample application site;
[00156] a flow channel in fluid communication with the sample application site; and
[00157] a porous component positioned between the sample application site and the flow channel, wherein the porous component comprises:
[00158] a porous matrix; and
[00159] an analysis reagent.
[00160] 2. The microfluidic device according to clause 1, wherein the porous matrix is configured to be non-filtering with respect to the sample for which the device is configured for testing.
[00161] 3. The microfluidic device according to clauses 1 or 2, wherein the porous matrix is configured to supply the assay reagent mixture with a sample flowing therethrough.
[00162] 4. The microfluidic device according to any one of the preceding clauses, wherein the porous matrix comprises pores with diameters between 1 μm and 200 μm.
[00163] 5. The microfluidic device according to any of the preceding clauses, wherein the porous matrix comprises a pore volume between 1 µL and 25 µL.
[00164] 6. The microfluidic device according to any of the preceding clauses, in which the pore volume is between 25% and 75% of the volume of the porous matrix.
[00165] 7. The microfluidic device of clause 6, in which the pore volume is between 40% and 60% of the porous matrix volume.
[00166] 8. The microfluidic device according to any of the preceding clauses, in which the porous matrix is a frit.
[00167] 9. The microfluidic device according to any of the preceding clauses, wherein the porous matrix comprises glass.
[00168] 10. The microfluidic device according to any one of the preceding clauses, wherein the porous matrix comprises a porous polymer.
[00169] 11. The microfluidic device according to any one of the preceding clauses, wherein the porous component further comprises a plug.
[00170] 12. The microfluidic device according to any one of the preceding clauses, wherein the reagent comprises an analyte-specific binding member.
13. The microfluidic device according to clause 12, wherein the analyte-specific binding member comprises an antibody or an analyte-binding fragment thereof.
14. The microfluidic device according to any one of clauses 12 to 13, wherein the analyte-specific binding member is coupled to a detectable label.
15. The microfluidic device according to any one of clauses 12 to 14, wherein the analyte-specific binding member specifically binds to a target selected from CD14, CD4, CD45RA, CD3 or a combination thereof.
[00174] 16. The microfluidic device according to any one of clauses 14 to 15, wherein the detectable label is an optically detectable label.
[00175] 17. The microfluidic device according to clause 16, wherein the optically detectable label comprises a fluorescent dye.
[00176] 18. The microfluidic device according to clause 17, wherein the fluorescent dye comprises a compound selected from the group consisting of rhodamine, coumarin, cyanine, xanthene, polymethine, pyrene, boron difluoro-dipyrromethene, naphthalimide, phycobiliprotein, peridinin chlorophyll proteins, their conjugates or a combination thereof.
[00177] 19. The microfluidic device according to any one of clauses 11 to 18, wherein the buffer comprises bovine serum albumin (BSA), trehalose, polyvinylpyrrolidone (PVP) or 2-(N-morpholino) ethanesulfonic acid or a your combination.
[00178] 20. The microfluidic device according to clause 19, wherein the buffer comprises BSA, trehalose and PVP.
[00179] 21. The microfluidic device according to clause 20, wherein the amount of BSA in the buffer is between 1% to 50% by weight.
[00180] 22. The microfluidic device according to any one of clauses 20 to 21, wherein the amount of trehalose in the buffer is between 1% to 99% by weight.
[00181] 23. The microfluidic device according to any one of clauses 20 to 22, wherein the amount of PVP in the buffer is between 0.01% and 10% by weight.
[00182] 24. The microfluidic device according to any one of the preceding clauses, wherein the assay mixture comprises a chelating agent.
[00183] 25. The microfluidic device according to clause 24, wherein the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis-(beta-aminoethyl ether) N,N,N', acid N'-tetraacetic acid (EGTA), 2,3-dimercaptopropane-1-sulfonic acid (DMPS), and 2,3-dimercaptosuccinic acid (DMSA).
[00184] 26. The microfluidic device according to clause 25, wherein the chelating agent is EDTA.
[00185] 27. The microfluidic device of any of the preceding clauses, wherein the flow channel comprises an optically transmissive wall.
[00186] 28. The microfluidic device according to clause 27, wherein the walls of the flow channel are optically transmissive to one or more of ultraviolet light, visible light and near infrared light.
[00187] 29. The microfluidic device according to any of the preceding clauses, wherein the sample application site is configured to receive a sample that has a volume ranging from 5 µL to 2000 µL.
[00188] 30. The microfluidic device according to any of the preceding clauses, wherein the device is configured for portable.
[00189] 31. A method comprising:
[00190] contacting a sample with a sample application site of a microfluidic device, the microfluidic device comprising:
[00191] a flow channel in fluid communication with the sample application site; and
[00192] a porous component positioned between the sample application site and the flow channel, wherein the porous component comprises a porous matrix and an analysis reagent;
[00193] illuminate the sample in the flow channel with a light source; and
[00194] detect light from the sample.
[00195] 32. The method according to clause 31, in which the sample mixes with the analysis reagent by following the sample through the porous matrix.
[00196] 33. The method according to clause 31, wherein mixing the sample with the assay reagent comprises labeling one or more components of the sample with a detectable label.
[00197] 34. The method according to clause 33, wherein the labeling comprises binding one or more components of a specific binding member to the analyte.
[00198] 35. The method according to clause 34, wherein the analyte-specific binding member is conjugated to an optically detectable label.
[00199] 36. The method according to any one of clauses 34 to 35, wherein the analyte-specific binding member is an antibody or antibody fragment.
37. The method according to clause 36, wherein the antibody or antibody fragment specifically binds to a target selected from CD14, CD4, CD45RA, CD3 or a combination thereof.
38. The method according to any one of clauses 35 to 37, wherein the optically detectable label comprises a fluorescent dye.
[00202] 39. The method according to clause 38, wherein the fluorescent dye comprises a compound selected from the group consisting of rhodamine, coumarin, cyanine, xanthene, polymethine, pyrene, boron difluoro-dipyrromethene, naphthalimide, phycobiliprotein, peridinin chlorophyll proteins, their conjugates or a combination thereof.
[00203] 40. The method according to any one of clauses 32 to 39, wherein 95% or more of the sample passes through the porous matrix into the flow channel.
[00204] 41. The method according to any one of clauses 32 to 40, wherein the method comprises illuminating the sample with a broad spectrum light source.
[00205] 42. The method according to clause 41, wherein the broad spectrum light source comprises an ultraviolet light source and a visible light source.
43. The method according to any one of clauses 41 to 42, wherein the method comprises illuminating the sample with light having a wavelength between 200 nm and 800 nm.
[00207] 44. The method according to any one of clauses 31 to 43, wherein detecting light from the sample comprises capturing an image of the sample in the capillary channel.
[00208] 45. The method according to any one of clauses 31 to 44, wherein the sample is a biological fluid.
[00209] 46. The method according to clause 45, wherein the biological fluid is whole blood.
[00210] 47. The method according to clause 45, wherein the biological fluid is plasma.
[00211] 48. A microfluidic device comprising:
[00212] a sample application site;
[00213] a flow channel in fluid communication with the sample application site;
[00214] a porous component positioned between the sample application site and the flow channel, wherein the porous component comprises:
[00215] a porous matrix; and
[00216] an analysis reagent; and
[00217] an amount of biological sample present in the microfluidic device.
[00218] 49. The microfluidic device according to clause 48, in which the biological sample is whole blood.
[00219] 50. The microfluidic device according to clause 49, wherein the biological sample is plasma.
[00220] 51. A system comprising:
[00221] a light source;
[00222] an optical detector for detecting one or more wavelengths of light; and
[00223] a microfluidic device comprising:
[00224] a sample application site;
[00225] a flow channel in fluid communication with the sample application site; and
[00226] a porous component positioned between the sample application site and the capillary channel, wherein the porous component comprises a porous matrix and an analysis reagent.
[00227] 52. A kit comprising:
[00228] a microfluidic device comprising:
[00229] a sample application site;
[00230] a flow channel in fluid communication with the sample application site; and
[00231] a porous component positioned between the sample application site and the flow channel, wherein the porous component comprises a porous matrix and an analysis reagent; and
[00232] a container housing the device.
[00233] 53. The kit of clause 52, wherein the container comprises a pouch.
[00234] 54. A microfluidic device for analyzing a sample comprising a sample application site in communication with a porous element and a capillary channel,
[00235] wherein the porous element comprises an analysis mixture and a porous frit; and
[00236] in which the frit provides a series of microchannels that define a tortuous path that is of sufficient length for the combination of the analysis and sample mixture and in which the microchannels provide the flow through substantially all the components of the sample.
[00237] 55. The device of clause 54, wherein the porous frit has an average void volume between 40-60% of the total frit volume.
[00238] 56. The device of clause 54, wherein the assay mixture comprises a reagent and a set of buffer components and wherein the set of buffer components provides for substantially continuous dissolution of the reagent in the sample over a period of time of predetermined time.
[00239] 57. The device of clause 54, wherein the set of buffer components is selected from the group comprising bovine serum albumin, trehalose, polyvinylpyrrolidone and or any combination thereof.
58. The device of clause 54, wherein the set of buffer components comprises bovine serum albumin, trehalose and polyvinylpyrrolidone.
[00241] 59. The device of clause 54, wherein the total weight of the buffer components is between 0.01 and 2 grams per µL of frit void volume in the frit.
[00242] 60. The device of clause 54, wherein the set of buffer components comprises 2-(N-morpholino) ethanesulfonic acid.
61. The device of clause 54, wherein the assay mixture comprises ethylenediaminetetraacetic acid (EDTA).
62. The device of clause 54, wherein the reagent comprises one or more antibodies or antibody fragments conjugated to one or more detectable labels.
63. The device of clause 62, wherein the antibody or antibody fragment is specific for a target selected from CD14, CD4, CD45RA, CD3 or any combination thereof.
[00246] 64. The device of clause 62, wherein the detectable marker is a fluorescent dye selected from the group comprising rhodamine, coumarin, cyanine, xanthene, polymethine, pyrene, boron difluoro-dipyrromethene, naphthalimide, phycobiliprotein, chlorophyll proteins peridinine, its conjugates, and its combinations.
[00247] 65. The device of clause 54, wherein the microchannels have an average passage pore diameter between 5 and 200 microns.
66. The device of clause 54, further comprising a sample.
[00249] 67. The provision of clause 66, wherein the sample is blood.
[00250] 68. The provision of clause 66, wherein the sample is plasma.
[00251] 69. The device of clause 54, further comprising an optically transmissive wall along at least a portion of the capillary channel.
[00252] 70. A method for analyzing a sample of a liquid comprising:
[00253] applying a liquid sample to a sample application site, wherein the sample application site is in fluid communication with a porous element and a channel;
[00254] direct the sample flow from the sample application site through the porous element to the channel, wherein the channel comprises an optically transmissive wall and wherein the porous element comprises an optically active reagent and a set of buffer components ;
[00255] dissolving the reagent in the sample wherein the dissolution of the reagent is substantially constant over a predetermined period of time;
[00256] mixing the sample and the reagent in the porous element, wherein the porous element comprises a porous frit that provides a series of microchannels that define a tortuous flow path that is of sufficient length for the mixture of sample and reagent and that the mixture provides for the binding of the reagent to the sample; and
[00257] optically evaluate the sample through the transmissive wall.
[00258] 71. The method of clause 70, wherein the sample flows by a capillary action force through the porous element and through the channel.
[00259] 72. The method of clause 70, wherein the predetermined period of time is between 5 seconds and 5 minutes.
[00260] 73. The method of clause 72, in which the optical evaluation comprises:
[00261] obtain an image of the sample through the transmitting wall;
[00262] determining a background signal, wherein the background signal corresponds to at least the signal from the unbound reagent; and
[00263] reduce an image background signal, wherein the background signal varies less than 75% along the transmissive wall.
[00264] 74. The method of clause 70, wherein the average diameter of the microchannels is 5-200 microns.
[00265] 75. The method of clause 70, wherein the sample flows through the porous element substantially unfiltered.
[00266] 76. The method of clause 70, wherein the sample is a blood sample.
[00267] 77. The method of clause 70, wherein the optically active reagent comprises a fluorescently labeled antibody or antibody fragment and provides the mixture for formation of a fluorescently labeled sample.
[00268] Although the foregoing invention has been described in some detail by way of illustration and examples for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of the present disclosure that certain changes and modifications can be made. without departing from the spirit or scope of the appended claims.
[00269] Therefore, the foregoing merely illustrates the principles of the invention. It will be appreciated that those skilled in the art are able to devise different arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language cited herein are intended primarily to assist the reader in understanding the principles of the invention, this being without limitation to these examples and conditions specifically enumerated. Furthermore, all statements herein enumerating principles, aspects and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Furthermore, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, that is, any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is realized by the appended claims.

权利要求:
Claims (19)
[0001]
1. Microfluidic device (100), characterized in that it comprises: an application site (10) of the sample; a flow channel (30) in fluid communication with the sample application site (10); and a porous component (20) positioned between the sample application site (10) and the flow channel (30), wherein the porous component (20) comprises: a non-filtering porous matrix comprising pores; and a free assay reagent positioned within the porous matrix pores, wherein the porous matrix is configured to: be non-filtering with respect to the sample for which the device is configured to assay; and providing uniform mixing of the assay reagent with a sample flowing therethrough.
[0002]
2. Microfluidic device (100), according to claim 1, characterized in that the porous matrix comprises pores with diameters between 1 mm and 200 mm.
[0003]
3. Microfluidic device (100), according to claim 1 or 2, characterized in that the porous matrix comprises a pore volume between 1 ml and 25 ml.
[0004]
4. Microfluidic device (100), according to any one of the preceding claims, characterized in that the pore volume is between 25% and 75% of the porous matrix volume.
[0005]
5. Microfluidic device (100), according to any one of the preceding claims, characterized in that the porous matrix is a frit.
[0006]
6. Microfluidic device (100) according to any one of the preceding claims, characterized in that the porous component (20) additionally comprises a plug.
[0007]
7. Microfluidic device (100) according to claim 6, characterized in that the buffer comprises bovine serum albumin (BSA), trehalose, polyvinylpyrrolidone (PVP) or 2-(N-morpholino) ethanesulfonic acid or a combination of the same.
[0008]
8. Microfluidic device (100), according to any one of the preceding claims, characterized in that the reagent comprises a specific analyte-binding element.
[0009]
9. Microfluidic device (100) according to claim 8, characterized by the fact that the specific analyte-binding element is coupled to a detectable marker.
[0010]
10. Microfluidic device (100), according to any one of the preceding claims, characterized in that the device is configured to be portable.
[0011]
11. Microfluidic device (100) according to claim 1, characterized in that the porous matrix comprises a porous organic polymer.
[0012]
12. Microfluidic device (100) according to claim 1, characterized in that the device is configured to analyze a sample of whole blood.
[0013]
13. Microfluidic device (100), according to claim 1, characterized in that the device is configured to analyze proteins attached to the surface of a cell.
[0014]
14. Microfluidic device (100) according to claim 1, characterized in that the porous component (20) fills a region that extends from the sample application site (10) to the flow channel (30).
[0015]
15. Microfluidic device (100), according to claim 1, characterized by the fact that the porous matrix is configured to be non-filtering in relation to the cells of a cell sample.
[0016]
16. Microfluidic device (100) according to claim 5, characterized in that the frit comprises a sintered granular solid.
[0017]
17. Method, characterized in that it comprises: contacting a sample to an application site (10) of the sample of a microfluidic device (100) as defined in any one of claims 1 to 12 and 16; illuminating the sample in the flow channel (30) with a light source; and detect light from the sample.
[0018]
18. System, characterized by the fact that it comprises: a light source; an optical detector for detecting one or more wavelengths of light; and a microfluidic device (100) as defined in any one of claims 1 to 12 and 16.
[0019]
19. Kit characterized in that it comprises: a microfluidic device (100) as defined in any one of claims 1 to 12 and 16; and a container that houses the device.

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AU2014346787A1|2016-05-19|

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ES2856191T3|2021-09-27|

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AU2014346787B2|2017-04-27|

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WO2015069789A1|2015-05-14|

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法律状态:
2019-10-15| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-06-08| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-08-03| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 05/11/2014, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201361900590P| true| 2013-11-06|2013-11-06|

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US61/900,590|2013-11-06|

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PCT/US2014/064159|WO2015069789A1|2013-11-06|2014-11-05|Microfluidic devices, and methods of making and using the same|

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