Biochemical blocking layer for liquid crystal assay

专利摘要:
The friction matrix structure for use in the liquid crystal analysis device comprises: a biomolecule recognition material deposited on one surface of a support containing a biochemical blocking compound and a biochemical blocking layer chemically fixed to one surface of the support forming the biochemical blocking layer. Include. The biomolecule recognition material includes a recognition site capable of selectively recognizing a target species to be detected by the liquid crystal analysis device. In addition, one surface of the support containing the biochemical blocking layer is rubbed to have a characteristic of inducing uniform fixation of the liquid crystal when the liquid crystal comes into contact with one surface of the support containing the biochemical blocking layer. Methods of making a friction substrate structure for use in liquid crystal analysis devices, optical cells for use in liquid crystal analysis, kits for use in liquid crystal analysis, and methods for detecting the presence of target species using liquid crystal analysis devices are also described.
公开号:KR20020074235A
申请号:KR1020027010047
申请日:2001-02-15
公开日:2002-09-28
发明作者:애보트니콜라스엘；김승렬
申请人:위스콘신 얼럼나이 리서어치 화운데이션；
IPC主号:

专利说明:

Biochemical blocking layer for liquid crystal analysis {BIOCHEMICAL BLOCKING LAYER FOR LIQUID CRYSTAL ASSAY}
[2] Methods for detecting the presence of biological materials and chemical compounds in samples are areas that have continued to evolve in the field of analytical chemistry and biochemistry. Various methods have been developed that allow for the detection of various target species in samples taken from the same source as the environmental or living organism. Detection of the target species can often be undertaken with the prescribed treatment and is often necessary in clinical situations prior to diagnosing the disease.
[3] While many conventional assays apply very well to detect the presence of a target species, many conventional assays are expensive and often difficult to use routinely in the field, requiring instrument use and highly trained professionals. Accordingly, there is a need for analytical devices and systems that are easier to use and capable of evaluating samples from remote locations.
[4] Recently, an analysis apparatus using liquid crystals has been published. For example, liquid crystal analyzers using mixed self-assembled monolayers (SAMs) containing octanethiol and biotin supported on anisotropic gold films deposited obliquely on glass have recently been reported. Gupta, V. K .; Skaife, J. J .; Dubrovsky, T. B., Abbott N. L. Science, 279, (1998), pp. 2077-2079. In addition, PCT publication WO 99/63329, published December 9, 1999, describes an analytical device using a SAM attached to a substrate and a liquid crystal layer fixed by the SAM.
[5] Although the described liquid crystal-based analyzer using an anisotropic gold film is suitable for use in determining whether it is present in a target paper sample, the manufacture of the anisotropic gold film by gradient deposition is difficult. For example, the production of gradient deposited gold films requires complex washing steps and high vacuum deposition. Thus, there is a need for a substrate structure that is easy to manufacture and that inhibits non-specific adsorption by proteins that can result in false positive test results.
[6] Summary of the Invention
[7] The present invention provides a friction substrate structure for use in a liquid crystal analyzer, an optical cell manufactured using the friction substrate structure, a method for producing a friction substrate structure, a kit for use in liquid crystal analysis, and detection of a target species using the liquid crystal analyzer. Provide a method.
[8] The friction matrix structure for use in the liquid crystal analyzer according to the present invention is characterized in that biomolecule recognition deposited on one surface of a support containing a biochemical blocking compound and a biochemical blocking layer chemically fixed to one surface of the support forming the biochemical blocking layer. Contains substances. The biomolecule recognition material includes a recognition site capable of selectively recognizing a target species to be detected by the liquid crystal analyzer. One side of the support containing the biochemical blocking layer and the deposited biomolecule recognition material is characterized by inducing uniform settling of the liquid crystal when the liquid crystal is in contact with one side of the support containing the biochemical blocking layer and the deposited biomolecule recognition material. To be rubbed. In another preferred friction substrate structure, the liquid crystal is in contact with one side of the support containing the biochemical blocking layer, and when the biomolecule recognition material is deposited on the friction surface containing the biochemical blocking layer, the support of the support containing the biochemical blocking layer One surface is rubbed to have a characteristic of inducing uniform fixation of the liquid crystal.
[9] Another friction substrate structure for use in the liquid crystal analyzer according to the present invention includes: a biochemical blocking layer having a biochemical; Bifunctional spacer compounds having a first end and a second end; A surface modification compound having a first end and a second end; And a support having at least one surface containing a biochemical blocking layer. At least one of the biochemicals is covalently bonded to the first end of the bifunctional spacer compound via a first chemical reaction between the reactor of the biochemical prior to the first chemical reaction and the reactor at the first end of the bifunctional spacer compound before the first chemical reaction . The surface modification compound is added to the second end of the bifunctional spacer compound through a second chemical reaction between the reactor at the first end of the surface modification compound before the second chemical reaction and the reactor at the second end of the bifunctional spacer compound before the second chemical reaction. Covalently binds. In addition, the surface modification compound is covalently bonded to one surface of the support containing the biochemical blocking layer through a third chemical reaction between the reactor on the surface before the third chemical reaction and the reactor on the second end of the surface modification compound before the third chemical reaction. . Finally, one surface of the support containing the biochemical blocking layer is rubbed to have a characteristic of inducing uniform fixation of the liquid crystal when the liquid crystal comes into contact with one surface of the support containing the biochemical blocking layer.
[10] Preferred friction matrix structures as described above also include biomolecule recognition materials deposited on one side of the support containing the biochemical blocking layer. The biomolecule recognition material has a recognition site capable of selectively recognizing a target species to be detected by the liquid crystal analyzer.
[11] In a preferred friction matrix structure, the bifunctional spacer compound is an organic compound having the formula below before the first and second chemical reactions:
[12] Chemical formula
[13]
[14] In the above formula,
[15] n is an integer having a value ranging from 1 to 20, more preferably 2 to 10 and even more preferably 5 to 8.
[16] Most preferably, the bifunctional activating compound is disuccinimidyl suverate.
[17] In another preferred friction substrate structure, the reactor at the second end of the surface modification compound before the third chemical reaction is a halogen-silicon bond or an alkoxy-silicon bond, while in another preferred friction substrate structure, the surface modification before the second and third chemical reactions The compound is a silicon atom; An alkoxy group bonded to the silicon atom via an oxygen-silicon bond; And an aminoalkyl group which bonds to a silicon atom via a carbon-silicon bond. In a more preferred friction substrate structure, the surface modification compound before the second and third chemical reactions is aminoalkyltrialkoxysilane, more preferably aminopropyltriethoxysilane.
[18] In another preferred friction substrate structure, the biochemical of the biochemical blocking layer is serum albumin, more preferably bovine serum albumin.
[19] In other preferred friction substrate structures the biomolecule recognition material is an immunoglobulin or part of an immunoglobulin, while in other preferred friction substrate structures, the biomolecule recognition material is a peptide or carbohydrate or a sequence of peptides or carbohydrates, or a sequence of DNA or RNA. In other preferred friction matrix structures, the biomolecule recognition material can recognize peptide, carbohydrate, DNA, RNA or fragments thereof or binding domains that bind to proteins, viruses, bacteria or micropathogens.
[20] At least two regions of the support surface containing the biochemical blocking layer are rubbed under different pressures for different periods of time so that at least two regions of the support surface containing the biochemical blocking layer have different susceptibility to the target species. do.
[21] Methods of making a friction substrate structure suitable for use in liquid crystal analyzers include reacting a biochemical blocking compound having at least one reactor with an activated and modified surface of a support. The activated and modified surface of the support has at least one functional group capable of reacting with the reactor of the biochemical blocking compound so that a covalent bond is formed between the biochemical and the support to produce a support having a surface containing the biochemical-blocking compound. . The method also includes the step of rubbing the surface of the support containing the biochemical-blocking compound to produce a friction surface characterized by inducing uniform settling of the liquid crystal when the liquid crystal contacts the friction surface.
[22] A preferred method of preparing a friction matrix structure suitable for use in a liquid crystal analyzer also includes reacting a surface modifying compound having a first end and a second end with a support to produce a surface modified support and between the surface modifying compound and the first end. Forming a covalent bond. The preferred method also reacts the surface of the support that is activated and modified by the reaction between the second end of the surface modifier and the first end of the bifunctional activator by reacting a bifunctional active agent having a first end and a second end with a surface modified support. Forming a covalent bond to produce.
[23] The optical cell for use in the liquid crystal analyzer has a biochemical blocking layer of friction matrix structure facing each other, including a spacing material located between the two friction matrix structures and the biochemical blocking layer of the two friction matrix structures. It is to be separated by a cavity which can be filled with liquid crystal.
[24] Liquid crystal analyzer according to the present invention comprises a friction substrate structure; A surface for uniformly fixing the liquid crystal; And a spacing material positioned between one surface of the biochemical blocking layer of the friction matrix structure and the surface for uniformly fixing the liquid crystal. The surface of the friction matrix structure includes both a biochemical blocking layer and a biomolecule recognition material. In a preferred liquid crystal analyzer, the surface of which the liquid crystal is uniformly fixed includes: another friction substrate structure having a biochemical blocking layer and a biomolecule recognition material; A friction matrix structure containing no biomolecule recognition material; Glass slides treated with octadecyltrichlorosilane; Friction uncoated glass slides; Glass slides with shear-settled Teflon; Or a glass slide having a gradient deposited gold film.
[25] Kits for use in liquid crystal analysis include friction substrate structures; A surface for uniformly fixing the liquid crystal; A spacing material, preferably a film, positioned between the friction matrix structure and the surface for uniformly fixing the liquid crystal; And liquid crystal compounds. In a preferred kit for use in liquid crystal analysis, the surface on which the liquid crystal is uniformly fixed is another frictional substrate structure. In another preferred kit, the friction matrix structure, the surface to uniformly fix the liquid crystal and the spacing material are preassembled into cells with the spacing material positioned therebetween. In such kits, samples that may contain the target species will be flushed through the cell for a predetermined time. The liquid crystal will then be placed in the cell and flushed through the cell, and thus can be used to determine if the kit is present in the target paper sample.
[26] Methods of detecting the presence of a target species using a liquid crystal analyzer include incubating the friction substrate structure with the sample to be tested for the presence of the target species; A spacing material, preferably a film, is placed between the incubated friction substrate structure and the surface for uniformly fixing the liquid crystal such that one side of the biochemical blocking layer of the friction matrix structure faces the surface for uniformly fixing the liquid crystal; Drawing the liquid crystal into the region between the incubated friction substrate structure and the surface which uniformly anchors the liquid crystal; Determining whether the liquid crystal is uniformly fixed on the friction substrate structure.
[27] An apparatus for detecting the presence of one or more target species in a sample is provided. The apparatus includes a support having a friction surface having a biochemical blocking layer. The device also includes a first target species detection region in a first portion of the support having a biochemical blocking layer, the first target species detection region having a first biomolecule recognition material capable of binding to the first target species. The device further comprises at least one other target species detection region in at least one other portion of the support having a biochemical blocking layer, wherein the at least one other target species detection region is capable of binding to at least one other target species. At least one other biomolecule recognition substance. The first target species detection region uniformly anchors the liquid crystal in the absence of the target species, and the at least one other target species detection region uniformly anchors the liquid crystal in the absence of at least one other target species. Uniform settling of the liquid crystal in the first target species detection region collapses when the first target species detection region is exposed to the first target species, and uniform settling of the liquid crystal in at least one other target species detection region results in at least one other target species It disintegrates when the detection region is exposed to at least one other target species.
[28] Particularly preferred devices for detecting the presence of a target species in a sample include surface rubbing and the first biomolecule recognition material and the at least one other biomolecule recognition material, respectively, the first target species detection region and the at least one other target species detection region. It includes what is present.
[29] The invention further provides a kit for use in detecting the presence of a target species in a sample, the kit comprising at least one friction substrate structure and a liquid crystal compound. Methods are also provided for detecting the presence of a target species in a sample using this type of kit. The method comprises contacting a portion of the friction substrate of the kit with a quantity of sample; Placing the liquid crystal of the kit on a portion of the friction matrix structure in contact with the sample; Determining whether the uniform fixation of the liquid crystal has collapsed.
[30] Further objects, features and advantages of the present invention will become apparent from the following detailed description taken in conjunction with the accompanying drawings.
[1] The present invention relates generally to the field of analysis of biological materials and chemicals, more particularly to blocking layers for use in liquid crystal analysis.
[31] In the drawing:
[32] 1 is a schematic showing the various steps used to chemically bind bovine serum albumin (BSA) to a glass plate. First, clean, dry glass slides are silylated with 3-aminopropyltriethoxysilane. Second, one side of a bifunctional crosslinker such as disuccinimidyl suverate (DSS) is reacted with a silylated glass slide to provide an activated surface for the reaction of bovine serum albumin with amino groups. Finally, the BSA is reacted with the free side and the reactive side of the attached DSS to provide a stable amide bond that immobilizes the BSA to the glass as shown in the figure.
[33] FIG. 2 is a diagram illustrating an apparatus used to rub a glass slide and a silicon wafer made using the procedure shown in FIG. 1. Various components of the apparatus include a glass slide 1; Electric friction machine 2 (modified strip chart recorder); Velvet polyester fabric friction material (3); Aluminum block weight 4; A fixed stopper 5; Double-sided tape 6; And chart paper as the movement guide 7.
[34] 3 is a bar graph showing the ellipsometric thickness of the BSA layer on a glass slide before (white) and after (black) friction using the apparatus shown in FIG. 2. The BSA layer is physically adsorbed onto clean untreated glass slides and OTS-treated glass slides. The BSA is chemically immobilized on the modified glass slide by reaction with APES followed by reaction with DSS as shown in FIG. 1. The friction film of BSA is made by applying a pressure of 10 ³ Pa for 1 minute at a speed of 5 mm / sec. The bar graph shows that when the glass slide with the chemically fixed BSA is rubbed, the BSA loses significantly less BSA layer compared to the BSA layer on the other slide.
[35] FIG. 4 shows friction with interference polarization and chemically immobilized BSA prepared according to FIG. ) And non-friction ( ) A graph showing the partial transmittance of light between 5CB fixed on a glass slide. The partial transmittance is shown as a function of the angle between the sample and the polarizer. The partial transmittance is the ratio of the intensity of light transmitted through the optical cell containing the liquid crystal and the intensity of light between the interference polarization to the highest intensity of light transmitted through the hollow cell under parallel-polarization.
[36] FIG. 5 is a bar graph showing the elliptical polarization thickness increase of friction (black) and non-friction (white) silicon wafers with BSA chemically fixed on the surface after immersion in various solutions. An increase in the thickness of the ellipsometry of the BSA-fixed substrate was 10 mg / ml of BSA; 0.2 mg / ml fibrinogen; 100 nM anti-BSA; And immersed in 100 nM PBS-buffered solution of anti-FITC for 2 hours. The figure shows that BSA-fixed substrates result in a significant increase in thickness when immersed in anti-BSA PBS-buffered solution.
[37] FIG. 6 is a graph showing the partial transmission of light between interference polarization and 5CB settled in a friction film of BSA after immersion in protein solution as a function of the friction direction of the cell and the angle between polarizers. For reference, the partial transmission of the friction film of the fixed BSA is measured without immersion in any further solution ( ). For non-specific adsorption by proteins, the fixed film of BSA was immobilized at 10 mg / ml of BSA for 2 hours ( ) And 0.2 mg / ml fibrinogen ( Incubated in PBS-buffered solution). For specific binding by the antibody, a friction film of immobilized BSA was applied at 100 nM of anti-BSA ( ) And PBS-buffered solution of anti-FITC (Δ) at 100 nM.
[38] FIG. 7 shows non-friction containing chemically immobilized biotin-BSA prepared according to FIG. 1 using interference polarization and biotin-BSA rather than BSA (FIG. ) And friction ( ) A graph showing the partial transmittance of light between 5CB fixed on a glass slide. The partial transmittance is shown as a function of the angle between the sample and the polarizer. The partial transmittance is the ratio of the intensity of light transmitted through the optical cell containing the liquid crystal and the intensity of light between the interference polarization to the highest intensity of light transmitted through the hollow cell under parallel-polarization.
[39] FIG. 8 is a graph showing the normalized light output of 5CB anchored to a friction film of biotin-BSA as a function of anti-biotin IgG concentration. Friction speeds, lengths, and pressures were approximately 2.1 mm / sec, 127 mm, and 1,000 Pa ( ); 2.1 mm / sec, 127 mm and 250 Pa ( ); And 2.1 mm / sec, 51 mm, and 250 Pa (Δ).
[40] FIG. 9 is a graph showing the elliptical polarization thickness of a biotin-BSA film covalently immobilized on the surface of a silicon wafer (with native oxide) as a function of anti-biotin IgG concentration in solution. Friction speeds, lengths and pressures were approximately 2.1 mm / sec, 127 mm and 1,000 Pa ( ); 2.1 mm / sec, 127 mm and 250 Pa ( ); And 2.1 mm / sec, 51 mm and 250 Pa (Δ).
[41] FIG. 10 is a graph showing the standardized light output of 5 CB anchored to a friction film of Biotin-BSA as a function of the amount of anti-biotin IgG bound to the Biotin-BSA film. Friction speeds, lengths and pressures were approximately 2.1 mm / sec, 127 mm and 1,000 Pa ( ); 2.1 mm / sec, 127 mm and 250 Pa ( ); And 2.1 mm / sec, 51 mm and 250 Pa (Δ).
[42] 11 shows the normalized light output of 5CB film on the friction film of biotin-BSA as a function of friction pressure ( ) And the corresponding increase in thickness ( ) Is a graph. The friction speed and length are approximately 2.1 mm / sec and 127 mm, respectively. The friction film is immersed in a PBS solution of 20 nM anti-biotin IgG with stirring for 90 minutes.
[43] 12 shows the normalized light output of 5CB film on a friction film of biotin-BSA as a function of friction length ( ) And the corresponding increase in thickness ( ) Is a graph. Friction speed and pressure are approximately 2.1 mm / sec and 1,000 Pa. The friction film is immersed in a PBS solution of 20 nM anti-biotin IgG with stirring for 90 minutes.
[44] The following abbreviations are used throughout this application:
[45] APES: 3-aminopropyltriethoxysilane
[46] BSA: Bovine Serum Albumin
[47] DMSO: Dimethyl Sulfoxide
[48] DSS: Disuccinimidyl Subversate
[49] OTS: Octadecyltrichlorosilane
[50] PBS: phosphate-buffered saline
[51] 5CB: 4-cyano-4'-pentylbiphenyl
[52] All ranges listed herein include all combinations and recombinations encompassed within the limits of that range. Thus, the range of "5-92%" includes the range of "5-84%", "16-75%", and the like. Ranges of "less than 1000 Pa" will include "less than 400 Pa", "less than 250 Pa", and the like.
[53] In general, the present invention includes a friction substrate structure for use in a liquid crystal analyzer; A method of producing a friction substrate structure; An optical cell made from a friction substrate structure; A kit containing a friction substrate structure; And a method for detecting the presence of a target species using a liquid crystal analyzer.
[54] Friction substrates are useful for specifically binding biological target molecules and in order to induce liquid crystal reorientation, they must have special characteristics. Reorientation of the liquid crystal is necessary because this allows the analytical device assembled from the friction substrate structure to be used to determine if the target species is present in a given sample. Some of the features that a suitable friction substrate should have are: the ability to block non-specific adsorption; The ability to orient the liquid crystal uniformly; And possession of anisotropic structures in which specific binding of the target species can be partially or completely eliminated. The latter characteristic leads to non-uniform settling of the liquid crystal indicating that it is present in the target paper sample.
[55] Tribological biochemical blocking layers such as tribological BSA inhibit non-specific adsorption of other species such as proteins. In addition, friction substrates containing such friction blocking layers provide a uniform alignment of liquid crystals that can collapse when combined with biomolecule recognition materials on the target paper surface.
[56] Friction matrix structures for use in liquid crystal analyzers generally comprise a biochemical blocking compound immobilized on at least one side of the support. Immobilization of the biochemical blocking compound to the support forms a biochemical blocking layer on the support.
[57] As will be apparent to those skilled in the art, a wide variety of materials can be used as the support in the friction substrate structure according to the present invention. Preferred supports include polymers containing hydroxyl groups for reacting with surface modification compounds or materials and silica-containing materials. Examples of polymer supports include, but are not limited to, polystyrenes, polycarbonates, and polymethyl methacrylates that have been plasma treated to have hydroxyl or carboxylic acid functional groups present. Other materials suitable for use as a support include, but are not limited to, metal oxides such as indium oxide, tin oxide and magnesium oxide, which preferably react with sulfur-containing compounds containing reactive functional groups such as hydroxyl or carboxylic acid groups. And metals such as, but not limited to, gold, silver and platinum. Other materials that can be used as the support include cellulosic materials such as nitrocellulose, wood, paper and cardboard and sol-gel materials. Particularly preferred supports include glass, quartz and silica, and most preferred supports include glass slides and silica wafers. Preferably, this support is washed before use. For example, the glass slides are preferably treated with “piranha solution” (70% H ₂ SO ₄ /30% H ₂ O ₂ ) for 1 hour and then washed with deionized water before drying under a nitrogen stream. To be clean. The "piranha solution" reacts violently with organic compounds and requires careful handling and should not be stored in a closed container.
[58] Various materials such as, but not limited to, serum albumin, bipolar polymers, adsorbed lipid layers, dextran and other sugars, crosslinked lipids, polyethylene oxides, polyoxazolines, and hydrogels may be used in biochemical blocking layers. It may be suitable for use as a compound. Preferred substances for use as biochemical blocking compounds include serum albumin such as bovine serum albumin, human serum albumin, rodent serum albumin, dog serum albumin, cat serum albumin, pig serum albumin, horse serum albumin and rabbit serum albumin; It is not limited to this. Bovine serum albumin is a particularly preferred biochemical blocking compound for use in forming the biochemical blocking layer in the friction matrix structure according to the present invention.
[59] The friction matrix structure for use in the liquid crystal analyzer preferably comprises a biomolecule recognition material deposited on one side of the support containing the biochemical blocking layer. The biomolecule recognition material comprises a recognition site capable of recognizing and preferably binding to the target species to be detected by the liquid crystal analyzer when present in the target paper sample.
[60] The biochemical blocking compound can be placed on the support using physical adsorption without chemically immobilizing the biochemical blocking compound on the support. For example, a glass slide or silicon wafer support may be dipped in PBS-buffered BSA solution overnight and then dried. Such BSA-coated supports can be prepared using a variety of supports including, but not limited to, clean untreated glass slides and OTS-treated glass slides. More preferably, the biochemical blocking layer is chemically fixed to the surface of the support. This can be accomplished by treating the biochemical blocking layer physically adsorbed on the support with a crosslinker such as, but not limited to, glutaraldehyde. More preferably, surface modifiers are used with the bifunctional spacer compound or active agent to immobilize the biochemical blocking compound on the surface of the support.
[61] 1 is a reaction schematic showing the steps used in a method of chemically fixing a biochemical blocking layer to a surface of a support for use in a liquid crystal analyzer. As shown in FIG. 1, a surface having one end having a reactor capable of reacting a support generally with a functional group on the surface of the support and another end having a reactor capable of reacting with a reactor having one end of the bifunctional spacer compound Primary treatment with modifiers. In preferred surface modification compounds, reactors that can react with the functional groups of the support include, but are not limited to, functional groups such as halogen-silicon bonds or alkoxy-silicon bonds. These functional groups react with hydroxyl groups on the support such as silica wafers or glass to form covalent bonds that bind the silicon compound to the support surface. Preferred surface modification compounds also include those having a reactor capable of reacting with a reactor at one end of the bifunctional spacer compound. Such preferred reactors on surface modifying compounds include, but are not limited to, alkylamines. Thus, preferred surface modifiers include silicon atoms; At least one alkoxy group bonded to a silicon atom via an oxygen-silicon bond; And an aminoalkyl group which bonds to a silicon atom via a carbon-silicon bond. More preferred surface modification compounds include aminoalkyltrialkoxysilanes, such as those having aminoalkyl groups of 2 to 8 carbon atoms. Especially preferred such compounds are aminopropyltriethoxysilane (APES).
[62] Those skilled in the art will appreciate that alkoxy groups such as methoxy, propoxy, butoxy and phenoxy may be used instead of ethoxy groups. In addition, those skilled in the art will appreciate that sulfyldyl-terminated silanes such as aminoalkyldialkylchlorosilanes, 3-mercaptopropyltrimethoxysilane, and other silanes such as silanes having double bonds such as allyltrichlorosilane and allyltrialkoxysilane ( It will be appreciated that it may also be used as a surface modification compound. Those skilled in the art will appreciate that silanes having sulfidyl groups, such as 3-mercaptopropyltrimethoxysilane, may form disulfide bonds between both the hydroxyl and biochemical blocking compounds on the surface of the support and the sulfidyl group of the silane and the sulfidyl group of the protein. You will recognize that you will respond. Thus, a bifunctional spacer compound may not be necessary if such a surface modification compound is used. However, n-succinimidyl 3- (2-pyridythiothio) propionate (SPDP) or succinimidyloxycarbonyl-methyl- (2-pyridythiothio) toluene (SMPT), or succinimidyl, if necessary Heterodifunctional crosslinkers such as 4- (N-maleimido-methyl) cyclohexane-1-carboxylate (SMCC) or maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) may modify these sulfhydryl-containing surface modifications. Can be used with silicone compounds.
[63] The reaction between the surface modification compound and the support produces a support having a modified surface that can be activated by reaction with a bifunctional spacer compound. Since water in the reaction mixture may result in unwanted reactions with the surface modifying compound, the reaction between the surface modifying compound and the support is preferably carried out using anhydrous solvents and conditions, but those skilled in the art will appreciate that the presence of some water is acceptable. Will be recognized.
[64] In a method of chemically fixing a biochemical blocking layer to the surface of a support, a bifunctional spacer compound or a bifunctional activator reactor typically reacts with the modified surface to activate the surface to remove the activated and modified surface of the support. Form. Preferred bifunctional spacer compounds have two ends that may have similar or different functional groups. Preferred such bifunctional spacer compounds will have leaving groups at each of the two ends with one end reacting with a group such as the amine of the biochemical blocking compound and the other end reacting with a group such as the amine of the bound surface modification compound. Preferred difunctional spacer compounds or activators include structures having the formula:
[65] Chemical formula
[66]
[67] In the above formula,
[68] n is an integer having a value ranging from 1 to 20, more preferably 2 to 10 and even more preferably 5 to 8.
[69] Most preferably, the bifunctional spacer compound or activator is disuccinimidyl suverrate, where n has a value of 6.
[70] Those skilled in the art will recognize that a wide variety of bifunctional spacer compounds can be used in place of the above disuccinimidyl species and will prove to be effective in immobilizing biochemical blocking compounds on the surface of the support. Examples of homobifunctional spacer compounds that will react with the amine of the surface modifying compound and the amine of the biochemical compound of the biochemical blocking layer include: disuccinimidyl suverate; Bis (sulfosuccinimidyl) suverrate; Disuccinimidyl glutarate; Dimethyl adipimidate; Dimethyl suverimidate; Dimethyl pimelimate; Dimethyl 3,3-dithiobispropionimidate; Methyl N-succinimidyl adipate; And 1,5-difluoro-2,4-nitrobenzene, including but not limited to. Examples of homobifunctional spacer compounds that will react with the sulfidyl group of the surface modifying compound and the sulfidyl group of the biochemical compound in the biochemical blocking layer include: 1,11-bis-maleimidotetraethylene glycol; Bismaleimidohexane; 1,6-hexane-bis-vinylsulfone; 1,8-bis-maleimidotriethylene glycol; 1,4-bis-maleimidobutane; And bismaleimidoethane, but are not limited to these.
[71] In addition to the homobifunctional spacer compound shown above, it is also possible to use a heterobifunctional spacer compound in the present invention. Examples of bifunctional spacer compounds having one end capable of reacting with an amine and one end capable of reacting with sulfhydryl include: N- (κ-maleimidoundocanoyloxy) sulfosuccinimide esters; Succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxy- (6-amido-caproate); N- (κ-maleimidodecanoic acid); Succinimidyl 4- [-maleimidophenyl] butyrate; Succinimidyl-6 [(β-maleimidopropionamido) hexanoate]; Succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate; N-succinimidyl (4-iodoacetyl) aminobenzoate; N- [y-maleimidobutyryloxy] succinimide ester; m-maleimidobenzoyl-N-hydroxysuccinimide ester; N-ε-maleimidocaproic acid; N- [ε-maleimidocaproyloxy] succinimide ester; N-succinimidyl- [4-vinylsulfonyl] benzoate; N- [β-maleimidopropyloxy] -succinimide ester; Succinimidyl 3- [bromoacetamido] propionate; N-β-maleimidopropionic acid; N- [α-maleimidoacetoxy] succinimide ester; N-succinimidyl S-acetylthiopropionate; And N-succinimidyl iodoacetate. Bifunctional spacer compounds having one end capable of reacting with an amine and one end capable of reacting with a carboxyl group include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride. Examples of heterobifunctional spacer compounds having one end capable of reacting with sulfidyl groups and one end capable of reacting with hydroxyl groups include N- [-maleimidophenyl] isocyanates.
[72] The biochemical blocking compound preferably reacts with the activated and modified surface of the support produced by reaction with a bifunctional spacer compound. For example, one of the amines, such as the ε-amino group of the lysine residue, reacts with the unreacted terminus of the bifunctional spacer compound to form a covalent amide bond that immobilizes the biochemical blocking compound on the surface of the support. something to do.
[73] As noted above, the biomolecule recognition material is deposited on one side of the support containing the biochemical blocking layer. The biomolecule recognition material may be deposited before, during or after the biochemical blocking layer is fixed to the surface of the support. The biomolecule recognition material may be adsorbed on the surface of the support, but preferably it will also be chemically fixed to the surface of the support or otherwise attached by bonding with a biochemical blocking layer. Preferred biomolecule recognition substances more preferably include immunoglobulins or portions of immunoglobulins such as IgG that can recognize binding epitopes and binding domains that bind proteins, viruses, bacteria and other micropathogens. Other preferred biomolecule recognition materials include peptides or sequences of peptides, proteins, carbohydrates or sequences of carbohydrates, RNA and DNA. Other preferred biomolecule recognition materials can recognize and bind peptide sequences, proteins, carbohydrates and carbohydrate sequences, DNA, RNA or fragments of DNA or RNA. Preferably, the amine group of the biomolecule recognition material will react with the activated and modified surface of the support and then the biochemical blocking compound will be added and immobilized on the surface of the support. Small molecules can be used as biomolecule recognition material. For example, small molecules such as biotin can be bound to the friction surface of a biochemical blocking layer such as BSA to specifically bind the protein. Thus, friction substrates having such biomolecule recognition materials can be used to screen for small molecule-protein interactions that would be useful in drug development.
[74] As noted above, the biomolecule recognition material can be located on the surface of the friction matrix structure using several methods. For example, an active surface containing DSS may be first treated with immunoglobulins and then reacted with the biochemical compounds that make up the blocking layer. In another procedure, the active surface containing DSS can be reacted with a biochemical blocking compound such as BSA and then rubbed. This friction surface can then be treated with ligands terminated with DSS and biotin, peptides, polypeptides and amine groups such as DNA or RNA and fragment sequences thereof. This can then be fixed to the friction BSA surface. In another procedure, the active surface containing bound DSS can be partially reacted with BSA and then rubbed. The resulting structure can then be immersed in a solution containing immunoglobulins. In another procedure, the DSS-activated surface is reacted with the protein. The surface with the protein is then immersed in an immunoglobulin-containing solution and then in a solution containing a biochemical blocking compound such as BSA. In a particularly preferred procedure, an active surface containing an active agent such as, but not limited to, DSS is reacted with a biochemical blocking compound that binds to a biomolecule recognition substance such as, but not limited to, biotinylated BSA. This produces a substrate containing both biochemical blocking compounds and biomolecule recognition materials that can be rubbed to induce uniform settling of the liquid crystal, such as but not limited to 5CB. Friction substrates, such as those prepared from Biotin-BSA, can be used to prepare optical cells and kits for detecting the presence of anti-biotin IgG. Various biomolecule recognition materials and biochemical blocking compounds can be attached to the support and rubbed in the manner described above and then produce substrates that will exhibit non-uniform settling of the liquid crystal upon exposure to certain target species that bind to the biomolecule recognition material. It will be apparent to those skilled in the art.
[75] According to one preferred procedure, the biochemical blocking compound to which the biomolecule recognition substance is attached is delivered as a liquid droplet to a specific portion of the active surface. In this way, biochemical blocking compounds with specific biomolecule recognition materials are limited to specific localized areas of the surface. A second droplet of liquid containing a biochemical blocking agent having a functional group different from the first is placed in the second position of the target. This procedure is repeated until the surface bears an array of regions, each covered by a blocking agent and a different recognition material. The entire surface can then be rubbed. This procedure provides a surface suitable for use as a biochemical microarray and allows for detection of species diversity in the sample. Those skilled in the art will appreciate that modifications of the above procedure may also be used to produce multiarrays. In one such preferred procedure, rather than "spotting" liquid droplets on the surface, a fluid channel (e.g., made of micromolded polydimethylsiloxane) is used to deliver the liquid to a confined area of the surface. do. In general, methods known to those skilled in the art for delivering liquids to localized areas of the surface can be used to produce the desired microarray device for the detection of multiple target species.
[76] The microarrays shown above provide an apparatus for detecting the presence of one or more target species in a sample. The apparatus includes a support having a friction surface with a biochemical blocking layer. The device also includes a first target species detection region in a first portion of the support having a biochemical blocking layer, the first target species detection region having a first biomolecule recognition material capable of binding to the first target species. The device further includes at least one other target species detection region in at least one other portion of the support having a biochemical blocking layer, wherein the at least one other target species detection region is capable of binding to at least one other target species. At least one other biomolecule recognition substance. The first target species detection region uniformly fixes the liquid crystal in the absence of the target species, and the at least one other target species detection region also uniformly fixes the liquid crystal in the absence of at least one other target species. Uniform fixation of the liquid crystal in the first target species detection region collapses when the first target species detection region is exposed to the first target species, and uniform fixation of the liquid crystal in the at least one other target species detection region is also at least one other target species It disintegrates when the detection region is exposed to at least one other target species.
[77] Particularly preferred devices for determining the presence of a target species in a sample include surface rubbing and the first biomolecule recognition material and at least one other biomolecule recognition material, respectively, detecting the first target species detection region and at least one other target species. It includes what exists in the realm.
[78] The surface of the substrate containing the biochemical blocking layer is preferably rubbed in one direction (but not limited to). In some procedures, it is desirable to rub different regions of the biochemical blocking layer in different directions. This allows the liquid crystal to create a pattern when biochemicals bind to recognition moieties. This pattern can be used to provide information to the user. In some procedures it is also desirable to rub the biochemical blocking layer using different friction conditions in each area in small areas. This allows for a variety of surfaces to be susceptible to bound target biochemicals. In general, in the absence of a substance to which the surface binds to the surface, the substrate has a characteristic of inducing uniform settling of the liquid crystal and collapse of the settling of the liquid crystal when the liquid crystal comes into contact with one surface of the support containing the biochemical blocking layer. Rub the surface. Those skilled in the art will use tools and devices that vary the friction of the substrate structure and various friction materials including, but not limited to, velvet polyester fabric, silk, velvet, cotton, wool, foil, canvas, nylon and polyester. It will be appreciated that this may be done. Preferred friction materials are velvet polyester fabrics. Friction methods include: pushing a palm-sized cloth across the substrate surface; Attaching the fabric to a tool similar to mechanical sandpaper for use in sanding wood and then holding it against a substrate surface; And rotating the cloth attached to the cylindrical roller over the substrate and then lowering the rotating cylinder toward the substrate. Those skilled in the art will appreciate that there are a number of methods, including methods of rubbing the surface developed for use in liquid crystal-based computer displays, which can be used to rub the surface so that the surface uniformly fixes the liquid crystal. Generally, friction of the substrate involves pushing the fabric or other material across the substrate surface while contacting the fabric with the surface.
[79] The method of surface friction of a substrate containing a biochemical blocking layer uses a modified strip chart recorder as shown in FIG. As shown in FIG. 2, the slide 1 is typically placed on a modified strip chart recorder 2 so that one side of the slide 1 containing the biochemical blocking layer faces the friction material 3. The aluminum weight 4 is then placed on the slide to apply pressure on the slide 1 which is properly held with a fixed stopper 5. Double-sided tape 6 is commonly used to secure the friction material to the top of the chart paper 7 used as a moving guide. Friction is preferably achieved using an application pressure of about 250 to about 1,000 Pa. The movement of the friction material is typically from about 5 mm / sec to about 2.1 mm / sec, and the friction is usually performed for a time from about 1 minute to about 30 seconds. Those skilled in the art will appreciate that various pressures, times and speeds can be used to rub the substrate structure. However, as described below, it has been surprisingly and unexpectedly found that the susceptibility of biochemical detection optical cells made from friction substrates can vary considerably by varying the friction rate, friction length and friction pressure. Friction pressures and friction lengths in particular have been found to affect sensitivity. It has been found that the drop in pressure used to rub the substrate significantly increases the susceptibility of the friction substrate to fixation of the liquid crystal at a given concentration of species to be detected.
[80] The biochemical blocking layer inhibits non-specific adsorption of non-target species. Non-specific adsorption of the resulting non-target species does not change the orientation of the liquid crystals on the surface and thus interferes with the interpretation of the liquid crystal orientation, suggesting binding of the target species. For example, the friction substrate structure of a silicon wafer or glass slide containing a biochemical blocking layer formed from bovine serum albumin prevents non-specific adsorption of fibrinogen, lysozyme, anti-FITC and anti-streptavidin. This important feature of the biochemical blocking layer is important in the friction matrix structure for use in liquid crystal analyzers because the non-target adsorption of non-target species disrupts the uniform settling of the liquid crystals in contact with the surface. Because it will result. Particularly preferred biochemical blocking layers containing BSA that inhibit non-specific adsorption have multiple binding sites for biomolecule recognition material, easily react with the active surface of the support, and result in uniform settling of liquid crystals such as 5CB. To be rubbed.
[81]
[82] Various types of liquid crystals can be used with the friction substrate structure. Examples include both nematic and smectic liquid crystals. Other classes of liquid crystals that can be used according to the invention include: polymeric liquid crystals, breast liquid crystals, thermotropic liquid crystals, columnar liquid crystals, nematic discotic liquid crystals, calamitic nematic liquid crystals, ferroelectric liquid crystals, discoids Liquid crystals and cholesteric liquid crystals are included, but are not limited to these. Examples of some liquid crystals that can be used are shown in Table 1 above. Particularly preferred liquid crystals for use in the present invention include 4-cyano-4'pentylbiphenyl.
[83] The optical cell for use in the liquid crystal analyzer preferably comprises a spacing material, preferably a film, positioned between the two friction substrates as described above and the two friction substrates resulting in a cavity that can be filled with liquid crystals. . Another preferred optical cell according to the invention comprises a friction substrate structure as described above; A surface for uniformly fixing the liquid crystal; And a spacing material positioned between the biochemical blocking layer of the friction matrix structure and the surface for uniformly fixing the liquid crystal. Thus, both sides of the optical cell need not be friction substrates. The spacing material is preferably a film of defined thickness and more preferably a film that is stable in the presence of the liquid crystal material, is easy to handle and does not contaminate the liquid crystal. As will be apparent to those skilled in the art, various films may be suitable for use as spacing materials in the optical cells according to the present invention. However, preferred film spacing materials are preferably made of polymeric material such as Mylar ^R film or Saran ^R wrap. The film spacing material is typically located between the friction substrates such that each surface of the friction substrate containing the biochemical blocking layer faces another such surface of the other friction substrate. The spacing material may also consist of finite diameter microspheres or rods that are dispersed in the liquid crystal to separate both sides forming the optical cell.
[84] The liquid crystal analyzer according to the present invention comprises a friction substrate structure as described above; A surface for uniformly fixing the liquid crystal; And a spacing material positioned between the biochemical blocking layer of the friction matrix structure and the surface for uniformly fixing the liquid crystal. The surface of the friction matrix structure includes both a biochemical blocking layer and a biomolecule recognition material. One surface of the friction matrix structure containing the biochemical blocking layer and the surface for uniformly fixing the liquid crystal are separated by the spacing material facing each other and positioned therebetween. The liquid crystal is drawn to the region between the friction matrix structure and the surface which uniformly fixes the liquid crystal. In a preferred analytical device, it may not be necessary, but the surface for uniformly fixing the liquid crystal is also a friction matrix structure which may contain biomolecule recognition material. Other materials suitable for use as a surface for uniformly fixing the liquid crystal include a glass surface modified by reaction with octadecyltrichlorosilane and a glass surface having a gradient deposited gold film. Other suitable surfaces for uniformly anchoring the liquid crystal include friction glass slides and glass slides with shear-deposited Teflon. As long as the surface fixes the liquid crystal uniformly, the presence of the target species in the sample will disrupt the orientation of the liquid crystal in the friction matrix structure with the biomolecule recognition material and will thus be detected.
[85] Kits for use in liquid crystal analysis include friction substrate structures according to the present invention; A surface for uniformly fixing the liquid crystal; A spacing material such as a film between the friction matrix structure and the surface uniformly fixing the liquid crystal so that the analytical device as described above can be manufactured; And liquid crystals. The surface for uniformly fixing the liquid crystal may be a friction substrate or another surface for uniformly fixing the liquid crystal as described above. Such kits may comprise instructions for detection of the target species. Such instructions will typically include methods for incubating with a sample that may contain a target species to detect the friction substrate. This will also preferably include instructions describing how the presence of the target species is identified and may also include steps that can be used to measure the concentration of the target species in the sample. In addition, preferred kits may include friction substrates prepared using a variety of friction conditions that can be used to detect the presence of various concentrations of target species. In some preferred kits, the friction matrix structure, the surface that uniformly anchors the liquid crystal, which may be another friction matrix structure, and the spacing material are preassembled into the optical cell. In such kits, the sample to be tested for the target species, followed by liquid crystal, can be drawn or flowed through the preassembled cell. Thus, such kits may also include one or more syringes for use in detection of the target species.
[86] Another kit according to the present invention comprises at least one friction substrate and a liquid crystal. Such kits can also be used to detect the presence of a target species in a sample. The method comprises contacting a portion of the friction substrate of the kit with a quantity of sample; Placing the liquid crystal of the kit in the portion of the friction substrate structure in contact with the sample; Determining whether the uniform fixation of the liquid crystal has collapsed. If the uniform fixation of the liquid crystal has collapsed, it is present in the target paper sample. The determination of whether the uniform fixation of the liquid crystal has collapsed can be accomplished in various ways. One such method includes observing a friction substrate through a cross polarizer.
[87] The method for detecting the presence of a target species using the liquid crystal analyzer as described above includes several steps. First, the friction substrate structure is incubated with the sample to be tested for the presence of the target species. Typically, the incubation time will be about 2 hours, but this may vary depending on the particular target species and biomolecule recognition material capable of specifically recognizing and binding to the target species. Secondly, a spacing material such as a film is placed between the incubated friction substrate structure and the surface for uniformly fixing the liquid crystal so that the biochemical blocking layer of the friction substrate structure faces the surface for uniformly fixing the liquid crystal. Third, a liquid crystal such as 5CB is drawn into the region between the incubated friction substrate structure and the surface where the liquid crystal is uniformly fixed. Typically, the liquid crystal is isotropic during this step. It may be necessary to heat the liquid crystal before drawing it into the area between the incubated friction substrate structure and the surface which uniformly anchors the liquid crystal. Liquid crystals can also be drawn to the cell in a nematic state. Finally, the person performing the analysis determines whether the liquid crystal is uniformly fixed to the friction matrix structure. If the liquid crystal is uniformly anchored to the friction matrix structure, the sample will be found to contain no target species. On the other hand, if the liquid crystal is not uniformly anchored to the friction matrix structure, the sample will be found to contain the target species.
[88] In addition to the methods described above, the kits and analyzers used in accordance with the present invention may also pass or hold directly through a preassembled cell in which the sample to be tested comprises a surface that uniformly anchors the friction matrix structure, the spacing material and the liquid crystal It may be designed to be. Once a significant time has elapsed, the sample is removed and then liquid crystal is added to determine if it is present in the target species of the sample.
[89] In addition to the methods described above, the kits and analyzers used in accordance with the present invention may be designed so that the liquid crystal is positioned directly on the surface of the incubated friction substrate structure and the air aligns the orientation of the liquid crystal on one side of the liquid crystal on the friction substrate surface. have. That is, the liquid crystal is simply located on the surface. For example, it is well known that the orientation of 5CB is homeotropic at the liquid crystal air interface. Thus, the free surface of the liquid crystal may be substituted for the second surface that uniformly fixes the liquid crystal. Kits of this type are particularly useful for microarrays of pattern recognition groups.
[90] The following materials and methodologies are used in the examples described in more detail below.
[91] material
[92] The microscope glass slides used in the experiments are very advanced and silicon 100 wafers obtained and polished from Fisher Scientific, Pittsburgh, Pa., Are obtained from Silicon Sense, Nashua, New Hampshire, USA. The glass slides and silicon wafers are cleaned by treatment with a "piranha solution" (70% H ₂ SO ₄ /30% H ₂ O ₂ ) before use. The "piranha solution" reacts violently with the glass material and must be handled with great care and not stored in a closed container. After sweeping with "piranha solution" at 80 ° C. for 1 hour, the slides and silicon wafers are rinsed abundantly with deionized water and dried under a stream of nitrogen. Prior to use, the clean substrate is stored in a heated oven at 120 ° C. for at least 3 hours.
[93] Various chemicals were used in this experiment. Octadecyltrichlorosilane (OTS) and 3-aminopropyltriethoxysilane (APES) are both purchased from Gelest (Tulitown, Pa.). The silylation solution of the microscope glass slide using OTS was prepared using anhydrous toluene (Aldrich, Milwaukee, WI) as a solvent, while the silylation solution of the microscope glass slide using APES was 10 mM sodium acetate-acetic acid. Prepared using a buffer (pH 5.0) solution. Disuccinimidyl suverrate (DSS) is obtained from Pierce (Rockford, Ill.). DSS solutions are prepared using anhydrous methanol and dimethyl sulfoxide (DMSO) available from Aldrich (Milwaukee, WI). Bovine serum albumin (BSA, no IgG, lyophilized powder), anti-BSA (developed in rabbits), anti-streptavidin (developed in rabbits), anti-FITC (monoclonal, clone FL-D6, mouse ascites fluid) ), Fibrinogen (fractions I, III from human plasma), lysozyme (EC 3.2.1.17, III: from egg whites), and anti-biotin IgG (polyclones, developed in goats) are Sigma (St. Louis, MO) Material) and used as received. Biotin treated bovine serum albumin (Biotin-BSA, Biotin Mole / BSA Mole = 8) is obtained from Pierce (Rockford, Ill.). All proteins used in this study are dissolved in phosphate-buffered saline (PBS) buffer at pH 7.2. All aqueous solutions are prepared using Milli-Q ^plus brand deionized water (18.2 Pa.cm), available from Millipore, Bedford, Massachusetts. Buffer solutions are prepared using assay reagents. 4-cyano-4'-pentylbiphenyl (5CB), a nematic liquid crystal produced by BDH, is purchased from EM industries (Hawthorne, NY).
[94] Preparation of Substrate with Physically Adsorbed BSA Layer
[95] Hydrophobic and hydrophilic substrates were prepared for the study of physical adsorption of BSA onto these surfaces. Clean glass slides and silicon wafers were used as hydrophilic substrates. Hydrophobic substrates are prepared by treating glass slides and silicon wafers overnight with OTS solution (3% OTS in anhydrous toluene). To eliminate the potential for hydrolysis, silylation with OTS is carried out in a nitrogen underglove box (Model CC-40 from Vacuum Atmospheres Co., Hawthorne, CA). The substrate silylated with OTS is washed with toluene and dried at 120 ° C. for at least 3 hours before further use. BSA is physically adsorbed to hydrophilic and hydrophobic substrates by immersing it in 1 mg / ml BSA solution overnight in PBS buffer, pH 7.2.
[96] Preparation of Substrates with Chemically Fixed BSA Layers
[97] Substrates with chemically immobilized BSA layers are prepared using the experimental procedure schematically depicted in FIG. 1. The clean glass slides are aminopropylated by reacting with 10% APES in sodium acetate-acetic acid buffer (10 mM, pH 5.0) at 80 ° C. for 3 hours. The aminopropylated substrate is washed with deionized water and then dried at 120 ° C. for at least 3 hours and then activated with succinimide ester crosslinker (DSS) to facilitate coupling of the BSA to the surface by amide bond formation. . The aminopropylated substrate is immersed in anhydrous methanol and then 50 mM DSS stock solution in anhydrous DMSO is added in sufficient quantity to produce a 1 mM DSS solution. The mixture is stirred for 1 hour, washed with methanol and then immediately coupled with the amine groups of BSA. BSA coupling is accomplished by immersing DSS-activated glass slides in 1 mg / ml BSA solution in PBS buffer, pH 7.2 overnight.
[98] Preparation of Substrates with BSA Friction Films
[99] A friction BSA film was prepared by sliding friction material across a BSA-fixed glass slide using a friction strain strip chart recorder (Model No. SR-255 A / B from Heath Company) as shown in FIG. 2. do. Velvet polyester fabric (90% polyester / 10% spandex), available from Logantex Inc., New York City, NY, was used as friction material in this study. The friction material is attached to the top of the movement guide (chart paper) using a double sided tape to place the BSA-fixed glass slide over the friction material. Since the glass slide can be fixed in place, friction is achieved by the movement of the friction material that is guided by the chart paper. The friction time is 1 minute at a rate of 5 mm / sec with a chart recorder. The pressure applied is about 10 ³ Pa, which is achieved by loading the glass slide with weights (about 200 g of aluminum blocks having dimensions of about 1 inch by 3 inches).
[100] Preparation of Substrate with Chemically Fixed Biotin-BSA Layer
[101] Substrates with chemically immobilized biotin-BSA layers are prepared using the experimental procedure schematically depicted in FIG. 1 and using biotin treated BSA instead of BSA. The clean glass slides are aminopropylated by reacting with 5% APES in sodium acetate-acetic acid buffer (10 mM, pH 5.0) at 80 ° C. for 3 hours. The aminopropylated substrate was rinsed three times with sodium acetate-acetic acid buffer at 80 ° C. for 10 minutes in a sonication bath, washed with deionized water and then dried at 120 ° C. for at least 3 hours, followed by succinimide ester crosslinker Activation with (DSS) to promote coupling of biotin-BSA to the surface by amide bond formation. The aminopropylated substrate is immersed in anhydrous methanol and then 50 mM DSS stock solution in anhydrous DMSA is added in sufficient amount to produce a 1 mM DSS solution. The substrate is immersed in the stirred mixture for 1 hour, washed with methanol and deionized water and then immediately coupled with the amine group of biotin-BSA. Biotin-BSA coupling is achieved by immersing DSS-activated glass slides in 1 mg / ml biotin-BSA solution in PBS buffer, pH 7.2.
[102] Preparation of Substrates with Biotin-BSA Friction Films
[103] A friction film of Biotin-BSA crosses a Biotin-BSA coated substrate prepared as described above by sliding a velvet fabric (90% polyester / 10% spandex) available from Logantex Inc. (New York, NY, USA). Are manufactured. Friction is achieved using a friction strain strip chart recorder (model SR-255 A / B Heath Company) as shown in FIG. 2. The fabric is attached to the top of the transfer guide (chart paper) using a double sided tape, with the (biotin-BSA) -fixed glass slide facing the fabric. Since the glass slide may be fixed in place, friction is achieved by moving the friction material that is guided by the chart paper. The friction speed and length are adjusted by varying the feed rate and friction time of the chart recorder respectively. The rubbing pressure is controlled by placing different blocks of aluminum on the substrate before rubbing. As standard conditions, a friction speed of approximately 2.1 mm / sec (5 inches / minute), a friction length of approximately 127 mm (friction time of 1 minute) and a friction pressure of approximately 1,000 Pa (approximately 200 grams of mass and 2.54 cm × 7.62) aluminum blocks with dimensions in cm) are used.
[104] General Preparation of Friction BSA Films with Biomolecule Recognition Materials
[105] Except that the substrate having the chemically immobilized BSA layer and the chemically immobilized biomolecule recognition material is modified with the biomolecule recognition material immunoglobulin or fragment thereof and reacted with the DSS before treating the activated surface with BSA. It is prepared using the experimental procedure schematically shown in 1. The clean glass slides are aminopropylated by reacting with 10% APES in sodium acetate-acetic acid buffer (10 mM, pH 5.0) at 80 ° C. for 3 hours. The aminopropylated substrate was then washed with deionized water and dried at 120 ° C. for at least 3 hours and then activated with succinimide ester crosslinker (DSS) to surface the immunoglobulin or immunoglobulin fragments and BSA by amide bond formation. To facilitate coupling. The aminopropylated substrate is immersed in anhydrous methanol and then 50 mM DSS stock solution in anhydrous DMSO is added in sufficient quantity to produce a 1 mM DSS solution. The mixture is stirred for 1 hour, washed with methanol and then immediately coupled with the amine groups of the immunoglobulin or immunoglobulin fragments. Coupling of immunoglobulins or fragments thereof is accomplished by immersing DSS-activated glass slides in 100 ng / ml immunoglobulin or PBS buffer of this fragment overnight. The slide is then washed with deionized water and treated with BSA to produce the final substrate surface for friction. BSA coupling is achieved by immersing the glass slides in 1 mg / ml BSA solution in PBS buffer, pH 7.2 overnight. The surface of the substrate containing the immobilized BSA and immunoglobulin or fragments thereof is then rubbed according to the procedure outlined above.
[106] Friction substrate without BSA layer
[107] Friction glass slides without BSA layers and glass slides with shear-settled Teflon films were prepared for preliminary study of protein adsorption on friction films. A friction glass slide without a BSA layer is made by mechanically rubbing a glass slide containing non-BSA under the conditions described above for a slide containing BSA. Glass slides with shear-settled Teflon films are obtained by sliding flat Teflon blocks across the fused glass slides in an electric motor. A temperature of approximately 100 ° C. is used to shear-deposit Teflon on the glass slide because it provides a more complete and reproducible surface coverage than is achieved when lower temperatures are used. The pressure and speed applied are also controlled, 0.5 mm / s for about 10 ³ Pa and 15 seconds, respectively.
[108] Protein adsorption
[109] To study protein adsorption by various biochemicals, a friction film of chemically immobilized BSA is incubated with various protein solutions in PBS buffer (pH 7.2) for 2 hours. Such solutions include 100 nM polyclonal anti-BSA IgG solution for specific binding; 10 mg / ml BSA solution to study further adsorption of BSA; 100 nM anti-FITC IgG solution; 0.2 mg / ml fibrinogen solution; And 0.2 mg / ml lysozyme solution. Anti-FITC, fibrinogen and lysozyme solutions were prepared to investigate non-specific adsorption by chemically immobilized BSA substrates.
[110] Binding of Anti-Biotin IgG by Friction Film of Biotin-BSA
[111] The friction film of Biotin-BSA prepared as described above is incubated for 90 minutes in a PBS solution of anti-biotin IgG (pH 7.2) at different concentrations. During incubation, the IgG solution is stirred using a magnetic stir bar. After removal from the protein solution, the substrate is washed with deionized water and dried under a dry nitrogen stream.
[112] Optical cell
[113] An optical cell is paired with two glass slides and is spaced on one side of it using a film of Mylar ^R brand, approximately 10 μm thick, available from Dupont Films, Wilmington, Delaware, USA. By arranging the friction films to face each other, the friction direction of the films is parallel within the cell. The cells are held together using a "bulldog" clip located along the edge of the microscope glass slide. The cell is placed on a hot plate at 40 ° C. and heated with hot air for approximately 10 seconds. 5CB, which is a nematic liquid crystal, is heated in a glass syringe in an isotropic state (about 35 ° C). A droplet of 5CB is then placed at the edge of each cell in the hot plate. 5CB is then drawn by capillary action into an optical cell. Once the optical cell is filled with 5CB, the cell is removed from the hot plate and then cooled in air to room temperature. Upon cooling, the isotropic state of 5CB is converted to the nematic state.
[114] Polarizing microscope
[115] A polarization microscope (BX60, Olympus, Tokyo, Japan) was used to observe the optical texture formed by the light transmitted through the 5CB filled optical cell. All images were obtained using a 20 × objective lens with a visible area of 550 μm between the interference-polarized light except for the use of tribological biotin-BSA substrates. A 10 × objective lens with a 1.0 mm visible region between the interfering polarizations was used to obtain images of cells constructed from tribological biotin-BSA substrates. Images of optical aspects of liquid crystal optical cells made from tribological biotin-BSA substrates are taken with a digital camera connected to a polarizing microscope (C-2020 Z, obtained from Olympus America Inc., Melville, NY). Photographs of optical cells made using tribological biotin-BSA substrates are obtained using advanced mode (resolution of 1600 x 1200 pixels) with an aperture of f11 and a shutter speed of 1/160 sec. Analysis of the optical texture of cells made from tribological biotin-BSA substrates was carried out by Photoshop software (San Jose, Calif., USA) for calculating the average luminance (average pixel value in the range of 0-255) after image conversion to natural gray. Adobe Systems Incorporated). The azimuthal orientation of the liquid crystals for all optical cells is measured by the change in the interference color upon insertion of a quarter-wave plate (Normarski prism, 147.3 nm) into the optical path. All optical cells are placed on the microscope in the direction of friction parallel to the axis of low sensitivity of the quarter wave length plate corresponding to 45 ° rotation of the optical cell with respect to the axis of the polarizing plate. The axis with low photosensitivity is determined by observing the direction of interference movement. That is, the interference color shifts toward the larger delay in the Michel-Levy chart when the axis of the liquid crystal has a lower axis and the quarter wave length plate coincides.
[116] Optical cell transmittance
[117] The intensity of the light transmitted through each optical cell is recorded during the rotation of the sample between the interfering polarizations. The background intensity (I _Background ) of light transmitted through interference-polarized light and the highest intensity (I _Parallel ) of light transmitted through parallel polarization are recorded for the hollow optical cell (not filling 5CB). The recorded intensity values are corrected for the background intensity of the light transmitted through the interference-polarized light and normalized by the measured light intensity transmitted between the parallel polarizations (both hollow cells). That is, the corrected and normalized partial transmittance is given by the following equation:
[118]
[119] All intensity of transmitted light is measured by silicon photodiode (Silicone photodiode FDS100, Thorlabs, Inc., Newton, NJ).
[120] Elliptical Polarization Measurement Thickness
[121] For elliptical polarization measurements, silicon wafers are used as substrates instead of glass slides. Measurement sample substrates are prepared using the same procedure used to prepare glass slides for optical measurements. Elliptical polarization thickness is measured at three points of each sample using a Rudolph Auto EL ellipsometry (Flanders, New Jersey, USA) at a wavelength of 6320 Hz and an angle of incidence of 70 °. To interpret the elliptical polarization thickness of the binding protein, a simple two-layer model (an effective substrate of organic layer / SiO ₂ / Si) is used. To perform the calculation, a refractive index of 1.46 is used for the organic film formed on the silicon wafer.
[122] 5CB out-of-plane orientation
[123] Home-built optics are used to measure the out-of-plane orientation (inclined angle) of 5CB in the optical cell. The device includes a 10 mW He-Ne laser, a polarizer, a computer controlled step allowing the rotation of the sample, an analyzer and a photodiode. The optical cell is positioned between the interference-polarizing plates and illuminated at a normal incidence angle using a polarized He-Ne laser and then illuminated at an incidence angle rotated from -20 ° to + 20 ° relative to the normal incidence angle. A plot of the intensity of light transmitted through the cell against the angle of incidence is used to measure the tilt of the optical axis of the liquid crystal from the surface of the cell.
[124] Discussion of the Experiment Results
[125] Stability against friction of biochemical blocking layer
[126] As mentioned above, biochemical blocking compounds such as BSA need not be, but are preferably covalently fixed to the support. To investigate the stability of the substrate structure with an unfixed biochemical blocking layer, the substrate structure is prepared using clean glass slides and hydrophobic slides modified by reaction with OTS. The BSA is then physically adsorbed onto the surface of the slide and then rubbed using the apparatus shown in FIG. 2. As shown in FIG. 3, measurement of elliptical polarization thickness indicates that at least 50% of the physically adsorbed BSA is lost upon friction, regardless of whether the support is a clean glass slide or an OTS treated slide. On the other hand, as shown in FIG. 3, slight changes in the thickness of the ellipsometry are observed when the substrate structure prepared according to the schematic diagram shown in FIG. 1 is rubbed. Thus, the fixation of the biochemical blocking layer using the method shown in FIG. 1 appears to overcome the reduction in thickness due to the friction of the biochemical blocking layer.
[127] Orientation of Liquid Crystals in a Fixed BSA Friction Film
[128] By examining the optical textures of various optical cells, the effects of friction on the fixation of liquid crystals drawn into the optical cells are evaluated. Specifically, the optical texture between 5 CB interference polarizers sandwiched between glass slide supports containing immobilized BSA prepared as shown in FIG. 1 is observed and recorded in photographs. Prior to friction, the optical texture of 5CB in contact with the BSA-fixed layer is not uniform. The friction of the BSA-fixed layer leads to 5CB uniform fixation when drawn into an optical cell made using a friction substrate structure. The azimuthal orientation of the liquid crystal in the frictional BSA layer is easily measured by the change of the interference color upon insertion of the quarter wave length plate into the optical path (see the detailed description in the experimental section). After inserting the 1/4 wave length plate, the interference color shifts the color towards the larger retardation, indicating that the alignment of the liquid crystals is parallel to the direction of friction.
[129] The out-of-plane orientation (inclined angle) of 5CB supported on the surface is also measured using the same cell used for the analysis. By fixing the cell to the crystal rotating device (see the detailed description in the Experimental paragraph), the inclination angle of the optical axis of 5CB from the plane containing the frictional BSA layer is measured to be 1.5 ± 0.5 °. Thus, these measurements show that the friction film of the fixed BSA induces a 5CB orientation that is 'planar' and 'parallel' with respect to the friction direction.
[130] Uniformity Analysis of Liquid Crystal Using Light Transmittance
[131] Light dissipation between the dark and bright images that occurs when the optical cell rotates is caused by the light transmitted through the optical cell between the interference-polarizing plate. The dark image observed when the direction of friction is parallel to the polarizer or analyzer indicates that the liquid crystal is uniformly fixed. The intensity of light transmitted through each optical cell is recorded as the sample rotates between the interference-polarizer plates. This technique is used to characterize the uniformity of liquid crystal fixation in a fixed BSA friction film (Fig. 4). ). As shown in the partial transmittance measurement (see the detailed description in the experimental paragraph), a large change in the intensity of the light transmitted through the interference-polarizer plate while rotating the cell with respect to the polarizer plate indicates uniform settling of the liquid crystal in the friction-fixed BSA substrate structure. On the other hand, a non-friction fixed BSA substrate showing non-uniform settling of the liquid crystal (in Figure 4 ), The intensity of the transmitted light is independent of the angle of rotation of the sample. This again confirms that a large change in the transmittance of light measured during rotation of an optical cell made of a frictionally fixed BSA substrate structure between the interference-polarizing plates of light results from uniform fixation of 5CB in the friction film of the fixed BSA.
[132] Effect of Anisotropy and Protein Adsorption of Friction Film
[133] As mentioned above, the fixed BSA friction film uniformly fixes the 5CB when the liquid crystal contacts the friction substrate structure. Experiments were performed to assess whether the binding of proteins removes the anisotropy in the alignment layer. For this purpose, a friction film having no BSA layer is produced. This is accomplished by rubbing the glass slides without any biochemical blocking compounds and by using glass slides with shear-deposited Teflon films. Glass slides with shear-deposited films are known to induce uniform settling of liquid crystals. Dennis, J. R .; Vogel, V. J. J. App. Phys. 83 (1998) p. 5195. Because BSA is easily adsorbed on most surfaces, it is predicted that BSA will completely cover the friction film by physical adsorption. In the friction glass slides and shear-deposited Teflon films, liquid crystals were uniformly aligned on the surface before being immersed in the BSA solution. However, after immersion in 0.1 mg / ml BSA solution, the 5CB nematic state supported by the friction glass slide and shear-deposited Teflon layer does not dissipate the light transmitted through the cell at a certain angle of the sample to the polarizer. Do not. In other words, the texture is completely non-uniform and there is no preferred orientation of the azimuthal arrangement. Thus, the change in shape caused by adsorption of BSA on the friction glass slide and shear-deposited Teflon film disrupts the anisotropy of the surface and results in non-uniform settling of the liquid crystal. Thus, adsorption of BSA on friction glass slides and shear-tipped Teflon films disrupts the anisotropy of the surface and results in non-uniform settling of the liquid crystals. Accordingly, it can be concluded that an alignment or biochemical blocking layer with selectivity in protein adsorption would be suitable as substrate structure for use in liquid crystal analysis devices. These results also show that since clean glass or Teflon films do not inhibit nonspecific protein adsorption, a biochemical blocking layer that inhibits nonspecific protein adsorption is required for the liquid crystal assay device.
[134] Nonspecific Adsorption of Proteins in BSA Layers
[135] As mentioned above, the biochemical blocking layer should effectively block the nonspecific adsorption of the protein if it is effective. After immersing the friction film in an aqueous solution containing 10 mg / ml BSA, the optical texture of the liquid crystal supported on the friction film of the fixed BSA was observed and recorded in a photograph. Compared with the liquid crystalline aspect in the friction film of BSA not immersed, the optical aspect of the liquid crystal is hardly changed by the immersion of the friction film of BSA in the BSA solution. This result contrasts with the optical behavior of the liquid crystals in the friction film of Teflon and the friction glass after immersion in an aqueous solution of BSA. The elliptical polarization thickness of the frictional and non-friction BSA film is measured after immersion in the BSA solution (FIG. 5). Examination of FIG. 5 shows that a covalently fixed film of BSA (non-friction) is immersed in and taken from an aqueous solution containing BSA and does not adsorb a measurable amount of BSA. In contrast, during friction, the covalently fixed layer of BSA adsorbs approximately 15 kPa of BSA. Thus, it is concluded that the nonspecific adsorption level of BSA is higher in the friction film of BSA compared to the non-friction BSA film. However, the nonspecific adsorption level of BSA in the friction film is insufficient to disrupt the uniform settling of the liquid crystal. As shown below, this result contrasts with the effect of specific binding of anti-BSA IgG on the friction film of BSA. In this case, specific binding of anti-BSA IgG is observed to induce non-uniform fixation of the liquid crystal in the friction film of BSA.
[136] The optical aspect of 5CB settled in a friction film of BSA taken by dipping in an aqueous solution containing fibrinogen and lysozyme was also investigated. Immersion of the BSA friction film into the lysozyme results in uniform fixation of the liquid crystal as observed and photographically recorded, but a number of defects (loop loci) appear in the optical texture of the liquid crystal supported on the film of the friction BSA immersed in fibrinogen. Although the drawback is evident in the optical aspect of the liquid crystal supported on the film of friction BSA immersed in fibrinogen, it should be noted that most liquid crystals are in a uniformly oriented state. As shown below, it is shown that the level of uniformity (measurement of partial transmission) is quantified and distinguished from the liquid crystalline aspect when specific binding of anti-BSA IgG to the friction film occurs. Oval polarization measurements of nonspecifically adsorbed BSA and fibrinogen, although the optical properties of the liquid crystals differed from each other after immersion in the aqueous solution of BSA and fibrinogen due to the small defect of the optical texture of the liquid crystal supported on the friction film after immersion in fibrinogen Thickness measurements show very similar levels of adsorption (FIG. 5). These results show that liquid crystals can be distinguished between adsorbed protein layers that are indistinguishable when characterized by ellipsometry. Nonspecific adsorption of fibrinogen (approximately 15 μs) was further measured on a film of immobilized BSA and found to be irrelevant whether the film was rubbing or not.
[137] The tilt angle of the liquid crystal is also measured after the nonspecific adsorption of BSA and fibrinogen to the friction film of BSA. The measured tilt angles were 3.8 ± 0.8 ° and 3.5 ± 0.5 ° for BSA and fibrinogen, respectively. As described above, the inclination of the liquid crystal was 1.5 ° ± 0.5 before immersing the friction film of BSA in an aqueous BSA or fibrinogen solution. This result explains that nonspecific adsorption causes a slight change in liquid crystal tilt (less than 2 degrees). Thus, it can be concluded that the friction film of the immobilized BSA inhibits nonspecific adsorption of the protein to a level that almost maintains a uniform planar orientation of 5 CB in a direction parallel to the friction direction of the BSA.
[138] Specific binding of proteins in tribological BSA layers
[139] In order to be suitable for use in the liquid crystal analysis device, the blocking layer must have an anisotropic structure which is removed by specific binding with the target species to be detected in the sample. The friction film of immobilized BSA is immersed in PBS-buffered 100 nM of various antibodies for 2 hours. After immersion in the anti-BSA solution, the texture of the optical cell containing the frictionally fixed BSA has a nearly non-uniform texture. Thus, the binding of anti-BSA by the BSA blocking layer in the friction matrix structure eliminates the anisotropy of the friction surface. In contrast, the immersion of the tribo fixed BSA substrate structure in antibody solutions such as anti-FITC and anti-streptavidin does not change the uniform texture similar to the results obtained when the friction substrate is in aqueous BSA solution. As shown in FIG. 5, the measurement of the ellipsometry thickness clearly shows the specific binding by anti-BSA and the variation of the non-uniform texture by the specific binding. Compared to immersion in BSA, fibrinogen and anti-FITC solutions, the specific binding resulting from immersion in anti-BSA solutions results in a significant increase in thickness (> 40 mm 3). This is also true when the optical cell from immersion in BSA and fibrinogen uses a substantially high concentration of protein. In addition, since the increase in thickness is independent of friction, it is inferred that the binding site of BSA is not damaged by friction for antigen-antibody reactions. Thus, it has been found that the friction film of BSA provides a surface that inhibits nonspecific adsorption and that the anisotropy is removed by specific binding.
[140] Permeability Analysis of Specific and Nonspecific Adsorption
[141] After soaking in the various solutions described above, permeation analysis of the frictionally fixed BSA substrate structure is performed for quantitative comparison between specific and nonspecific binding by the friction substrate structure. FIG. 6 shows that specific binding to anti-BSA eliminates cell rotational disappearance and cyclic permeability properties of the frictional BSA layer. Table 2 summarizes the partial intensities of the maximum (I _Max ) and minimum (I _Min ) values of each protein adsorption through immersion experiments and shows the disappearance difference between the highest and minimum permeability ([ I _Max -I _Min ] / I _Max ) is normalized. [I _Max -I _Min ] / I _Max of specific binding with anti-BSA was determined to be about 0.33. Thus, a significant decrease in specific binding occurs. This is especially true considering that the value of [I _Max -I _Min ] / I _Max of the friction film of the fixed BSA is about 0.94. Even for nonspecific adsorption, the normalized partial permeability value is at least 0.90 except for adsorption with fibrinogen. Although [I _Max- I _Min ] / I _Max of fibrinogen adsorption is relatively small compared to other nonspecific adsorptions (about 0.84), this value is closer to nonspecific adsorption than specific adsorption such as specific adsorption with anti-BSA. In addition, as shown in FIG. 6, the periodic nature of the light transmittance of the friction-fixed BSA substrate surface immersed in fibrinogen is clearly continued and clearly different from the light transmittance of specific binding resulting from immersion in anti-BSA solution. . Thus, with observations by optical texture and ellipsometry thickness, the results collected from the transmission measurements indicate that the friction-fixed BSA substrate structure can uniformly fix the liquid crystals, prevent nonspecific adsorption, and be removed by specific binding. It has an anisotropic structure.
[142] Partial transmittance ^a of liquid crystal cell by protein adsorption ^a Protein (concentration)Partial transmittance I _Max -I _Min ^d I _MaxI _Max ^b I _Min ^cReference ^e 0.63 ± 0.020.03 ± 0.010.94 ± 0.01 BSA (10 mg / ml)0.61 ± 0.020.05 ± 0.010.91 ± 0.02 Fibrinogen (0.2 mg / ml)0.58 ± 0.050.09 ± 0.020.84 ± 0.02 Lysozyme (0.2 mg / mL)0.49 ± 0.020.02 ± 0.010.94 ± 0.01 Anti-BSA (100 nM)0.34 ± 0.040.23 ± 0.020.33 ± 0.04 Anti-FITC (100 nM)0.58 ± 0.060.01 ± 0.010.96 ± 0.01 Anti-streptavidin (100 nM)0.57 ± 0.010.03 ± 0.010.94 ± 0.01^a Partial transmission is measured between interference-polarized light and 5CB settled in BSA friction film after immersion in protein solution for 2 hours. ^b The maximum value (I _Max ) of the partial transmittance is measured when the angle between the polarizing plate and the friction direction of the optical cell is 45 °, 135 °, 225 ° and 315 °. ^c Minimum value (I _Min ) is measured when these angles are 0 °, 90 °, 180 ° and 270 °. ^d [I _Max -I _Min ] / I _Max values are obtained from the paired partial transmittance at (0 °, 45 °), (90 °, 135 °), (180 °, 225 °) and (270 °, 315 °). Is calculated. ^{The e} reference represents the friction film of BSA immobilized prior to protein adsorption.
[143] Biotin-BSA Friction Film
[144] Changes in the optical texture of 5CB in optical cells made from frictional and non-frictional biotin-BSA substrates were observed and photographed as described above. When rotating between the interfering polarizers, a slight change (if any change) in the optical texture, indicating non-uniform fixation of 5 CB, is observed in the non-frictionated film of biotin-BSA. In contrast, the optical aspect of 5CB anchored between friction films was observed to change between dark and light by rotating the cell between the interference polarizers. This difference is illustrated graphically in FIG. 7. 4 and 7 show that optical cells made from frictional and non-frictional biotin-BSA substrates behave similarly to those made from frictional and non-frictional BSA substrates in terms of their ability to uniformly fix the liquid crystals. As in the case of an optical cell made from a BSA substrate, the liquid crystal appears dark when the nematic optical axis is aligned with the polarizer or analytical device of the optical cell made from the biotin treated BSA. If the friction direction (i.e., the optical axis in the nematic state of 5CB) is aligned parallel to the polarizer or analyzer, the polarization of the incident light does not change by transmission through the cell. Thus, the optical aspect of the liquid crystal is uniformly dark when viewed through an interference polarizer. However, rotation of the cell changes the polarization of the light to transmit incident light through the interfering polarizer, and the intensity of the transmitted light reaches its maximum at 45 ° rotation in the frictional direction with respect to the polarizer. 7 summarizes the average brightness trend of biotin-BSA films as a function of the rotation angle of the cell. As shown in FIG. 7, the sample showed no change when the cell rotates before friction, but noticeable periodic and large changes were observed in the friction sample. The highest brightness is observed at 45 °, 135 °, 225 ° and 315 ° and the minimum brightness is observed at 0 °, 90 °, 180 ° and 270 °.
[145] Optical Texture of Liquid Crystal by Anti-Biotin IgG Bound to Friction Film of Biotin-BSA
[146] Initially, standard friction conditions (1 minute friction at a friction rate of about 2.1 mm / sec and an applied pressure of about 1,000 Pa) were used to evaluate the chemically fixed biotin-BSA substrate. Under these conditions it was found that the optical texture of the optical cell prepared from the tribological biotin-BSA substrate was dependent on the concentration of anti-biotin IgG in the solution in which the substrate was immersed. The optical behavior of the liquid crystals becomes more complex and uneven as the concentration of anti-biotin IgG in the assay solution increases. At low concentrations, a uniform alignment is first observed by the appearance of a revolving line that causes light scattering. Although the number of loop loops increases with concentration, the rotation of the sample still results in a fairly large change in the intensity of light transmitted through the optical cell. As the concentration of anti-biotin IgG is further increased, the appearance of supported liquid crystals of very non-uniform texture is finally observed until the rotation of this sample causes little measurable change in the intensity of light transmitted through the cell. . The very non-uniformity of the liquid crystals indicates that the nematic state of 5CB is fixed without the desired azimuthal orientation in this film.
[147] The sensitivity of the optical texture to the binding of anti-biotin IgG in solution is investigated. In detail, the film of biotin-BSA is rubbed under different conditions to find certain rules (if any) in which friction conditions affect sensitivity. Regulation of sensitivity is important in the detection of IgG. If the detection sensitivity can be varied, the fluidity of the detection range can be provided in biological quantitative applications. First, the applied pressure decreases from 1,000 to 250 Pa without changing other friction parameters. The result of rubbing the biotin-BSA substrate to a light mass is that uniform settlement of 5CB is removed from low concentrations of anti-biotin IgG. For example, the biotin-BSA substrate rubbed at low pressure (about 250 Pa) shows a very non-uniform texture when exposed to anti-biotin IgG solution at a concentration of 20 nM. In contrast, a uniform alignment of 5CB is maintained when similar substrates rubbed under the same conditions except for a pressure of about 1,000 Pa were incubated in anti-biotin IgG solution at a concentration of 28 nM. Thus, the sensitivity of the optical cell made from the detection system and the friction substrate can be increased by reducing the frictional pressure. Changes in other frictional conditions have also been found to change sensitivity. For example, reducing the friction time from 60 seconds to 24 seconds using an applied friction pressure of 250 Pa further increases the sensitivity. These results demonstrate that the susceptibility of the friction film can be adjusted by simply changing the friction conditions. As a control experiment, a friction film of biotin-BSA is prepared using three friction conditions. Friction speeds, lengths and pressures in these experiments were approximately 2.1 mm / sec, 127 mm and 1,000 Pa; 2.1 mm / sec, 127 mm and 250 Pa; And 2.1 mm / sec, 51 mm and 250 Pa. Friction Biotin-BSA substrates prepared using these conditions are incubated in PBS buffer solution that does not contain any anti-biotin IgG. Friction makes the 5CB optical texture uniform (no distinction) and makes it difficult to discern optical cells made from friction substrates. Although the appearance of some circulating loops is observed in optical cells made from friction substrates with reduced frictional pressure and length, the optical texture is such that they can be distinguished from the optical cells with heterogeneity resulting from the specific binding of anti-biotin IgGs. Maintain sufficient uniform settling. This indicates that sensitivity can be increased without providing false positive test results.
[148] Quantitative Analysis of the Optical Aspects of 5CB Induced by Binding of Anti-Biotin IgGs
[149] Changes in the optical appearance of optical cells formed from the friction substrate of biotin-BSA upon exposure to a certain concentration of anti-BSA immunoglobulin are quantified by measuring the average brightness of the optical texture using the method described above. The corrected and standardized light output can be represented by the following equation:
[150] Equation 1
[151]
[152] From here,
[153] S is the highest luminance ratio between dark and bright images obtained by rotating the cell (L _Min / L _Max ),
[154] L _Min is the average brightness of the texture when the friction direction is parallel to the polarizer between the interfering polarizers,
[155] L _Max is collected when the friction direction is rotated 45 ° with respect to the polarizer,
[156] S _Max and S _Min are collected using a film of biotin-BSA before and after friction.
[157] The standardized light output using the luminance ratio (S) and the reference cell provides a quantitative measure of the degree of heterogeneity resulting from incubating in a solution with varying amounts of anti-biotin IgG. The degree of deviation found from point to point or sample to sample can also be minimized. 8 shows normalized light output collected from images of liquid crystals supported on a friction film of biotin-BSA after specific binding of anti-biotin IgG as a function of immunoglobulin concentration. 8 shows that the reduction in friction strength and length shifts the limit between uniform and non-uniform settling of the liquid crystal to lower concentrations of immunoglobulins. More detailed observation of non-uniform features using light output can be performed by measuring the amount of anti-biotin IgG bound to the friction film as described below.
[158] Anti-Biotin IgG Bound to Friction Film of Biotin-BSA
[159] To measure the amount of anti-biotin IgG specifically bound to the tribological biotin-BSA layer, the elliptical polarization measurement as described above was used to measure the increase in thickness due to IgG bound to the friction film of biotin-BSA immobilized on a silicon wafer. Measured using thickness measurement techniques (FIG. 9). FIG. 9 shows that the increase in thickness due to the binding of anti-biotin IgG is almost the same for each substrate despite different frictional conditions. This is true even if the standardized light output is significantly affected by the change in the friction conditions as mentioned above and illustrated in FIG. 8. As shown in FIG. 9, the thickness of the bound anti-biotin IgG slowly increases and reaches saturation at about 10 nm. Considering the size of IgG, generally measured at 4 nm × 10 nm × 14 nm, an increase in saturation thickness of about 10 nm indicates that the surface is almost completely covered by anti-biotin IgG in saturation.
[160] As described above and shown in FIG. 8, optical texture and standardized light output measurements provide concentrations of anti-biotin IgG in an amount that eliminates uniform settling of liquid crystals. In addition, as mentioned above and shown in FIG. 9, the measurement of the thickness of bound anti-biotin IgG provides a limiting amount to maintain or eliminate uniform fixation of 5CB by binding IgG. The review of FIGS. 8 and 9 thus shows the amount of bound anti-biotin IgG required to change the orientation properties of the liquid crystals anchored in the friction film. 10 illustrates that the amount of bound anti-biotin IgG due to specific binding to biotin on the surface of the biotin-BSA friction substrate leads to an increase in the non-uniform settling of the liquid crystals in the friction substrate. At standard friction conditions (2.1 mm / sec, 127 mm (friction time of 1 minute) and friction pressure of approximately 1,000 Pa (aluminum block with a mass of approximately 200 grams and dimensions of 2.54 cm × 7.62 cm)), Unexpected changes are observed at about 5 nm of binding anti-biotin IgG. The decrease in frictional strength shifts the threshold to lower levels of bound anti-biotin IgG. When the frictional pressure was reduced to 250 Pa, the limit thickness shifted to about 4 nm of bound anti-biotin IgG. The reduction of the frictional pressure from 1,000 Pa to about 250 Pa and the reduction of the friction time from 60 seconds to 24 seconds both shift the limit of bound anti-biotin IgG from about 5 nm to less than 2 nm. Therefore, sensitivity control in the light output of the liquid crystal by the binding protein can be achieved by simply changing the frictional conditions. Thus, friction substrates can be prepared for quantitative and qualitative applications at various concentrations of the target species.
[161] Susceptibility of light response due to changes in friction conditions
[162] The characteristics of liquid crystal alignment in terms of friction conditions are tested using a systematic approach. To do this, the friction speed, friction pressure and friction length (ie, friction time) are changed again. The aforementioned standard friction conditions are used for reference. At some point the work friction parameters are changed so that the effect on sensitivity and thickness can be observed independently. If the friction conditions change over the ranges shown in Figures 11 and 12, the friction produces a substrate that provides a very uniform texture upon exposure to 5CB (prior to incubation with the target analyte). In addition, similar to those shown in FIGS. 4 and 7, a large change is observed when the cell is rotated. In order to observe normalized light output by specific binding of anti-biotin IgG as a function of rubbing conditions, the rubbing substrate was subjected to anti-biotin IgG at a concentration of 20 nM showing the intermediate state of rubbing substrate light output under standard conditions. Incubate in solution. This makes it possible to change the optical image by changing the friction conditions to be specifically tested.
[163] FIG. 11 shows how the amount of bound anti-biotin IgG changes after incubation in a solution of anti-biotin IgG at a concentration of 20 nM as a function of pressure applied during friction, as indicated by increased thickness and normalized light output. 12 shows how the friction length affects the same parameters. If standard friction conditions were used, incubating the friction film of biotin-BSA to 20 nM anti-biotin IgG solution resulted in about 4 nm of binding IgG (Figure 9), slight regression loop appearance, and a standardized light output of about 0.2. (FIG. 8). As shown in FIGS. 11 and 12, the undetectable change in thickness after incubation in anti-biotin IgG solution occurs as a result of changing the pressure or friction length used during friction. However, the light output is significantly affected by the friction conditions used to make the friction substrate. Thus, the pressure applied during the friction process can be used to change the susceptibility of the optical cell produced from the friction substrate. 12 shows that the friction length has a significant effect on the optical behavior over a narrow range of friction lengths without affecting the thickness. Reduction of the friction length increases the non-uniformity of the optical texture, and a completely non-uniform feature is achieved at a friction length of 2.54 mm. Increasing the friction length causes the friction film to resist 5CB non-uniform settling. Thus, as the friction length increases to about 508 mm, the optical aspect of the friction film incubated with anti-biotin IgG shows a completely uniform texture and its standardized light output is near zero. This means that incubation in 20 nM anti-biotin IgG solution causes little change in the optical texture of the friction substrate. The effect of friction rate on increasing thickness and standardized power was also tested. However, little change in light output as a function of frictional speed was observed. These results demonstrate that friction pressure and friction length are very effective parameters that can be used to control and modify the susceptibility of optical cells fabricated using friction substrates.
[164] Based on the above results, image analysis of liquid crystal light output in the friction film of biotin-BSA can be used to quantitatively measure the amount of anti-biotin IgG bound to the surface of the frictional biotin-BSA substrate. In addition, such substrates can be used to determine the presence and amount of such substrates in a sample as described by anti-biotin IgG. In addition, the sensitivity of the light output can be easily adjusted by changing the frictional conditions used to produce the friction substrate. Thus, friction substrates are useful for imaging specific biomolecular interactions when used with liquid crystals.
[165] Preparation of Friction Substrates with Antibodies
[166] As mentioned above, various procedures can be used to prepare friction substrates containing antibodies as biomolecule recognition materials. A summary of six such procedures follows:
[167] Procedure 1
[168] 1. Covalently fix the antibody to the surface of the microscope glass slide using a DSS activated glass slide by immersing the activated glass in an aqueous solution of the antibody,
[169] 2. Mechanically rub the surface of the slide containing the immobilized antibody using a modified chart recorder,
[170] 3. Block the friction protein film by immersing it in 10 mg / ml BSA aqueous solution for 1 hour.
[171] Procedure 2
[172] 1. Covalently fix the antibody to the surface of the microscope glass slide using a DSS activated glass slide by immersing the activated glass in an aqueous solution of the antibody,
[173] 2. Block the substrate by immersing it in 10 mg / ml BSA aqueous solution for 1 hour,
[174] 3. Mechanically rub the immobilized antibody / BSA surface using a modified chart recorder,
[175] 4. Block the friction protein film by immersing it in 10 mg / ml BSA aqueous solution for 1 hour.
[176] Procedure 3
[177] 1.Covalently immobilize the protein on the DSS activated microscope glass by immersing the activated glass slide in aqueous protein solution,
[178] 2. The antibody specific for the immobilized protein is bound to the immobilized protein by immersing the substrate in an aqueous solution of the antibody,
[179] 3. Mechanically rubbing the surface of the substrate containing the immobilized antibody and protein using a modified chart recorder,
[180] 4. Block the friction protein surface on the substrate by immersing it in 10 mg / ml BSA aqueous solution for 1 hour.
[181] Procedure 4
[182] 1.Covalently immobilize the protein on the DSS activated microscope glass by immersing the activated microscope glass slide in an aqueous protein solution,
[183] 2. Mechanically rubbing the surface containing the immobilized protein using a modified chart recorder,
[184] 3. Immerse the slides in the aqueous antibody solution to bind the antibody specific for the immobilized protein to the immobilized protein, and
[185] 4. Block the friction protein film by immersing it in 10 mg / ml BSA aqueous solution for 1 hour.
[186] Procedure 5
[187] 1.Covalently fix the BSA to a DSS activated microscope glass slide by immersing the slide in an aqueous BSA solution,
[188] 2. Mechanically rub the surface of the fixed BSA using a modified chart recorder,
[189] 3. Covalently immobilize the antibody to immobilized BSA using DSS to reactivate the surface,
[190] 4. Block the surface of the friction substrate by immersing it in 10 mg / ml BSA aqueous solution for 1 hour.
[191] Procedure 6
[192] 1.Covalently fix the BSA to a DSS activated microscope glass slide by immersing the slide in an aqueous solution containing BSA,
[193] 2. covalently immobilize the antibody to the immobilized BSA surface using DSS to reactivate the surface,
[194] 3. Mechanically rub the surface containing the immobilized antibody and BSA using a modified chart recorder, and then
[195] 4. Block the friction substrate surface by immersing it in 10 mg / ml BSA aqueous solution for 1 hour.
[196] Nitrile triacetate / Ni ⁺² Preparation of Tribological Protein Substrates with
[197] Various procedures can be used to prepare a friction substrate having nitrilotriacetate (NTA) / Ni ⁺² . Such friction substrates are useful for detecting histidine fusion proteins.
[198] In the first procedure, the NTA-functionalized BSA is covalently fixed to the microscope glass slide using DSS by immersing the slide in a solution containing 10 mg / ml of NTS-functionalized BSA for 1 hour. The slide is then dried and mechanically rubbed using a modified chart recorder as described above. The NTS-BSA film is then immersed in an aqueous solution containing Ni ⁺² to form a complex.
[199] In the second procedure, the BSA is covalently primary fixed to the surface of the microscope glass slide by immersing the slide in 10 mg / ml BSA solution for 1 hour. The surface of the slide is then rubbed using a modified strip chart recorder. The friction surface is then activated with DSS and the activated surface is incubated for about 6 hours using NTA-ligand solution at a concentration of about 1 mM with NTA-ligand such as amino-terminal NTA available from Qiagen. The resulting substrate is then immersed in 10 mg / ml BSA solution for 1 hour to block the surface. The resulting substrate is then immersed for 3 hours in an aqueous solution containing Ni ⁺² at a concentration of about 10 mM.
[200] In a third procedure, the BSA is covalently primary fixed to the surface of the glass slide by immersing the slide in an aqueous solution containing about 10 mg / ml BSA for 1 hour. The surface of the BSA-coated substrate is then activated using DSS as described above. The activated BSA-coated substrate is then incubated in 1 mM NTA-ligand solution for about 6 hours. The substrate is then immersed in 10 mg / ml BSA aqueous solution to block the surface. After blocking the surface, the substrate is dipped in an aqueous solution with Ni ⁺² at a concentration of about 10 mM for about 3 hours. Finally, the surface of the resulting substrate is mechanically rubbed using the procedure described above.
[201] Those skilled in the art will soon recognize that the friction substrate of the present invention can be used to detect a variety of target species and that various biomolecule recognition materials can be used in the friction substrate. A partial non-limiting list of biomolecule recognition materials and target species for use in accordance with the present invention is as follows:
[202] Biomolecule Recognition Target Species
[203] Anti-Ras IgG Ras
[204] Histidine Fusion of RAF1 Activated Ras
[205] RAF1 Activated Ras
[206] GST Fusion of RAF1 Activated Ras
[207] Sialic acid influenza virus
[208] Anti-active p38, pAb, rabbit (pTGpY) p38
[209] Anti-pT183 MAPK pAb, rabbit MAPK
[210] Anti-active MAPK, pAb, rabbit, (pTEpY) activated MAPK
[211] Anti-ERK 1/2 pAb, rabbit ERK
[212] Anti-active JNK pAb, rabbit, (pTPpY) activated JNK
[213] Anti-Active CaM KII pAb, Rabbit, (pT286) Activated CaM KII
[214] Anti-pS473 Akt, pAb Akt
[215] Anti-phosphotyrosine pAb phosphotyrosine
[216] Donkey Anti-Rabbit Rabbit Rabbit IgG
[217] Mannos Concavalin A
[218] Anti-Hepatitis C IgG Hepatitis C Virus
[219] Anti-Hepatitis B IgG Hepatitis B Virus
[220] Anti-active p38 pAb activated p38
[221] Anti-CNP mAb CNP
[222] Anti-GBP pAb GBP
[223] Anti-human BDNF pAb BDNF
[224] Anti-human GDNF pAb GDNF
[225] Anti-human NT-3 pAb NT-3
[226] Anti-human NT-4 pAb NT-4
[227] Anti-human p75 pAb p75
[228] Anti-human tryptase mAb tryptase
[229] Anti-NGF mAb NGF
[230] Anti-Pan Trk pAb Trk
[231] Anti-Rabbit CNTF pAb CNTF
[232] Anti-TrkB In pAb TrkB
[233] Anti-TGF-b1 pAb TGF-b1
[234] Anti-VACht pAb VACht
[235] Anti-GFP green fluorescent protein
[236] NTA-Ni histitin fusion protein
[237] Glutathione GST Fusion Proteins
[238] Although the present invention is not limited to the embodiments detailed herein for illustrative purposes, it is understood that the present invention covers all such aspects as long as they are within the scope of the following claims.

权利要求:
Claims (56)
[1" claim-type="Currently amended] (a) a biochemical blocking layer comprising a biochemical;
(b) a bifunctional spacer compound comprising a first end and a second end;
(c) a surface modification compound comprising a first end and a second end; And
(d) a friction substrate structure for use in a liquid crystal analysis device comprising a support comprising at least one surface containing a biochemical blocking layer, wherein at least one of the biochemicals is at least one of a reactor and a first reactor of the biochemicals prior to the first chemical reaction; Covalently bonds with the first end of the bifunctional spacer compound via a first chemical reaction between the reactors of the first end of the bifunctional spacer compound prior to the chemical reaction; The surface modification compound is reacted with the second end of the bifunctional spacer compound through a second chemical reaction between the reactor at the first end of the surface modification compound before the second chemical reaction and the reactor at the second end of the bifunctional spacer compound before the second chemical reaction. Covalently bonded; The surface modification compound is covalently bonded to one surface of the support containing the biochemical blocking layer via a third chemical reaction between the reactor on the surface before the third chemical reaction and the reactor on the second end of the surface modification compound before the third chemical reaction; And a friction substrate structure in which one surface of the support containing the biochemical blocking layer is characterized by inducing uniform settling of the liquid crystal when the liquid crystal comes into contact with one surface of the support containing the biochemical blocking layer.
[2" claim-type="Currently amended] 10. The method of claim 1, further comprising a biomolecule recognition material deposited on one surface of the support containing the biochemical blocking layer, wherein the biomolecule recognition material is capable of selectively recognizing a target species to be detected by the liquid crystal analysis device. A friction substrate structure for use in a liquid crystal analysis device comprising a recognition site.
[3" claim-type="Currently amended] 3. The friction substrate structure of claim 2, wherein the biomolecule recognition material is chemically fixed to one side of the support containing the biochemical blocking layer.
[4" claim-type="Currently amended] 4. The friction matrix structure of claim 3, wherein the biomolecule recognition material is covalently bonded to at least some of the biochemicals of the biochemical blocking layer.
[5" claim-type="Currently amended] 4. The friction matrix structure of claim 3, wherein the biomolecule recognition material is covalently bonded to at least some of the bifunctional spacer compounds.
[6" claim-type="Currently amended] 4. The liquid crystal assay of claim 3 wherein the biochemical of the biochemical blocking layer comprises serum albumin and the biomolecule recognition material is an immunoglobulin, a portion of an immunoglobulin, a peptide, a polypeptide, a carbohydrate, a fragment of RNA or a fragment of DNA. Friction substrate structure for use in the device.
[7" claim-type="Currently amended] 4. The binding domain of claim 3, wherein the biochemical blocking layer comprises bovine serum albumin and the biomolecule recognition material binds to peptides, polypeptides, DNA, RNA, DNA fragments, RNA fragments or proteins, viruses, bacteria or micropathogens. A friction substrate structure for use in a liquid crystal analysis device capable of recognizing and combining.
[8" claim-type="Currently amended] The friction substrate structure of claim 1, wherein the reactor of biochemical is an amine.
[9" claim-type="Currently amended] The method of claim 1, wherein the bifunctional active agent is formulated before the first and second chemical reactions.
A friction substrate structure for use in a liquid crystal analysis device, comprising an organic compound wherein n is an integer having a value ranging from 1 to 20.
[10" claim-type="Currently amended] 10. The friction substrate structure of claim 9, wherein n is an integer having a value in the range of 5-8.
[11" claim-type="Currently amended] The friction matrix structure for use in a liquid crystal analysis device according to claim 1, wherein the bifunctional spacer compound is disuccinimidyl suverate prior to the first and second chemical reactions.
[12" claim-type="Currently amended] The friction substrate structure of claim 1, wherein the second terminal of the surface modification compound is selected from the group consisting of halogen-silicon bonds and alkoxy-silicon bonds.
[13" claim-type="Currently amended] The method of claim 1, wherein the surface modification compound prior to the second and third chemical reactions comprises a silicon atom; An alkoxy group bonded to the silicon atom via an oxygen-silicon bond; And a silicon compound comprising an aminoalkyl group bonded to a silicon atom via a carbon-silicon bond.
[14" claim-type="Currently amended] The friction substrate structure of claim 1, wherein the surface modification compound is an aminoalkyltrialkoxysilane prior to the second and third chemical reactions.
[15" claim-type="Currently amended] The friction substrate structure of claim 1, wherein the surface modifier is aminopropyltriethoxysilane before the second and third chemical reactions.
[16" claim-type="Currently amended] (a) a biochemical blocking compound chemically immobilized on one surface of a support to form a biochemical blocking layer; And
(b) a biomolecule recognition material deposited on one surface of the support containing the biochemical blocking layer, wherein the biomolecule recognition material comprises a recognition site capable of selectively recognizing a target species to be detected by the liquid crystal analysis device. In a friction substrate structure for use in a liquid crystal analysis device, one surface of the support containing the biochemical blocking layer is characterized by inducing uniform settling of the liquid crystal when the liquid crystal comes into contact with one surface of the support containing the biochemical blocking layer. Friction substrate structure to be rubbed so that.
[17" claim-type="Currently amended] 17. The friction matrix structure of claim 16, wherein the biomolecule recognition material is deposited on the friction surface of the support containing the biochemical blocking layer.
[18" claim-type="Currently amended] 17. The friction substrate structure of claim 16, wherein the biomolecule recognition material is deposited on one side of the support containing the biochemical blocking layer before one side of the support containing the biochemical blocking layer is rubbed.
[19" claim-type="Currently amended] 17. The friction matrix structure of claim 16, wherein the biochemical blocking compound is fixed to the support by crosslinking with a crosslinker.
[20" claim-type="Currently amended] 20. The friction substrate structure of claim 19, wherein the crosslinker is glutaraldehyde.
[21" claim-type="Currently amended] 17. The liquid crystal analysis device according to claim 16, wherein the biochemical blocking compound is fixed to the support by binding to the bifunctional spacer compound, wherein the bifunctional spacer compound binds to the surface modification compound and the surface modification compound binds to the functional group on the surface of the support. Friction substrate structure for use.
[22" claim-type="Currently amended] 17. The liquid crystal analysis of claim 16, wherein the biomolecule recognition material is immobilized to the support by binding to a bifunctional spacer compound, wherein the bifunctional spacer compound binds to the surface modification compound and the surface modification compound binds to a functional group on the surface of the support. Friction substrate structure for use in the device.
[23" claim-type="Currently amended] The friction substrate structure of claim 16, wherein the biomolecule recognition material is immobilized on the support by binding to a biochemical blocking compound.
[24" claim-type="Currently amended] The device of claim 16, wherein the biochemical blocking compound comprises serum albumin and the biomolecule recognition material is an immunoglobulin, a portion of an immunoglobulin, a peptide, a polypeptide, a carbohydrate, a fragment of RNA or a fragment of DNA. Friction substrate structure for
[25" claim-type="Currently amended] 17. The binding domain of claim 16, wherein the biochemical blocking compound comprises bovine serum albumin and the biomolecule recognition material binds to peptides, polypeptides, DNA, RNA, DNA fragments, RNA fragments or proteins, viruses, bacteria or micropathogens. A friction substrate structure for use in a liquid crystal analysis device capable of recognizing and combining.
[26" claim-type="Currently amended] 17. The method of claim 16, wherein at least two regions of the support surface containing the biochemical blocking layer are rubbed under different pressures for different times so that at least two regions of the support surface containing the biochemical blocking layer have different sensitivity to the target species. , Friction substrate structure for use in liquid crystal analysis device.
[27" claim-type="Currently amended] In the method for producing a friction substrate structure suitable for use in a liquid crystal analysis device,
(a) reacting a biochemical blocking compound comprising at least one reactor with an activated and modified support surface (the activated and modified surface of the support comprises at least one functional group capable of reacting with a reactor of the biochemical blocking compound) And a covalent bond is formed between the biochemical and the support to produce a support having a surface comprising a biochemical blocking compound),
(b) rubbing a surface comprising a biochemical blocking compound to produce a friction surface having a characteristic of inducing uniform settling of the liquid crystal when the liquid crystal contacts the friction surface.
[28" claim-type="Currently amended] 28. The method of claim 27, wherein the biochemical blocking compound is serum albumin.
[29" claim-type="Currently amended] 28. The method of claim 27, wherein the biochemical blocking compound is bovine serum albumin.
[30" claim-type="Currently amended] The method of claim 27,
(c) reacting a surface modification compound comprising a first end and a second end with a support, wherein a covalent bond is formed between the support producing the surface modified support and the first end of the surface modification compound;
(d) reacting a bifunctional activator having a first end and a second end with a surface modified support, wherein the covalent bond that produces the activated and modified support surface is a bifunctional active agent at the second end of the surface modifier. Formed by the reaction with the first end of the compound).
[31" claim-type="Currently amended] 31. The method of claim 30, wherein the surface modification compound is capable of reacting with hydroxyl groups on the surface of the support.
[32" claim-type="Currently amended] 31. The method of claim 30, wherein the bifunctional active agent is capable of reacting with the amines of the biochemical.
[33" claim-type="Currently amended] 31. The method of claim 30, wherein the first end of the surface modifier is selected from the group consisting of halogen-silicone bonds and alkoxy-silicone bonds.
[34" claim-type="Currently amended] 33. The method of claim 30, wherein the surface modifier comprises: a silicon atom; An alkoxy group bonded to a silicon atom via an oxygen-silicon bond; And a silicon compound comprising an aminoalkyl group bonded to a silicon atom via a carbon-silicon bond.
[35" claim-type="Currently amended] 31. The method of claim 30, wherein the surface modifier is an aminoalkyltrialkoxysilane.
[36" claim-type="Currently amended] 36. The method of claim 35, wherein the surface modifier is aminopropyltriethoxysilane.
[37" claim-type="Currently amended] 31. The bifunctional active agent of claim 30, wherein the bifunctional active agent A process for producing a friction substrate structure suitable for use in a liquid crystal analysis device, comprising an organic compound, wherein n is an integer having a value ranging from 1 to 20.
[38" claim-type="Currently amended] 38. The method of claim 37, wherein n is selected from the group consisting of integers having values in the range of 5-8.
[39" claim-type="Currently amended] 31. The method of claim 30, wherein the bifunctional activator comprises disuccinimidyl suverate.
[40" claim-type="Currently amended] 28. The method of claim 27, further comprising reacting the biomolecule recognition material with the activated and modified surface of the support comprising a reactive site and a recognition site capable of selectively recognizing and binding a target species to be detected by the assay device. Wherein the covalent bond that produces a support comprising a biomolecule recognition material having a recognition site capable of selectively recognizing and binding a target species to be detected by the assay device is activated and modified with a biomolecule recognition material A method for producing a friction substrate structure suitable for use in a liquid crystal analysis device, which is formed in the liver.
[41" claim-type="Currently amended] 41. The method of claim 40, wherein the biomolecule recognition material reacts with the activated and modified surface of the support before the surface comprising the biochemical blocking compound is rubbed.
[42" claim-type="Currently amended] 41. The method of claim 40, wherein the biomolecule recognition material reacts with the activated and modified surface of the support after the surface comprising the biochemical blocking compound is rubbed.
[43" claim-type="Currently amended] 41. The method of claim 40, wherein at least two regions of the surface comprising the biochemical blocking compound are rubbed for different times using different pressures.
[44" claim-type="Currently amended] 28. The method of claim 27, further comprising reacting the biomolecule recognition material with the activated and modified surface of the support comprising a reactive site and a recognition site capable of selectively recognizing and binding a target species to be detected by the assay device. Wherein the covalent bond that produces a support comprising a biomolecule recognition material having a recognition site capable of selectively recognizing and binding a target species to be detected by the assay device is comprised between the biomolecule recognition material and the biochemical blocking compound. A method for producing a friction substrate structure suitable for use in a liquid crystal analysis device formed.
[45" claim-type="Currently amended] The method of claim 27,
(c) reacting a first biomolecule recognition material comprising a first reactive site and a first recognition site with a first region of an activated and modified surface of the support, wherein the covalent bond is the first biomolecule recognition material And between the first region of the activated and modified support),
(d) reacting a second biomolecule recognition material comprising a second reactive site and a second recognition site with a second region of the activated and modified surface of the support, wherein the covalent bonds with the second biomolecule recognition material Formed between the second region of the activated and deformed support).
[46" claim-type="Currently amended] 46. The liquid crystal analysis device of claim 45, wherein the first and second biomolecule recognition materials react with the first and second regions of the activated and modified surface of the support before the surface comprising the biochemical blocking compound is rubbed. A method of making a friction substrate structure suitable for use.
[47" claim-type="Currently amended] (a) two friction substrate structures according to claim 2 or 16; And
(b) a spacing material located between the biochemical blocking layers of the two friction matrix structures, wherein the biochemical blocking layers of the friction matrix structures face each other but are separated into cavities that can be filled with liquid crystals; And an optical cell for use in a liquid crystal analyzer.
[48" claim-type="Currently amended] (a) a friction matrix structure according to claim 2 or 16;
(b) a surface for uniformly fixing the liquid crystal; And
(c) a liquid crystal analyzer comprising a spacing material positioned between the biochemical blocking layer of the friction matrix structure and the surface for uniformly fixing the liquid crystal.
[49" claim-type="Currently amended] (a) at least one friction substrate structure according to claim 2 or 16;
(b) a surface for uniformly fixing the liquid crystal;
(c) a spacing material located between the friction substrate and the surface for uniformly fixing the liquid crystal; And
(d) A kit for use in a liquid crystal analyzer comprising a liquid crystal compound.
[50" claim-type="Currently amended] 50. The kit according to claim 49, wherein the liquid crystal is 4-cyano-4'-pentylbiphenyl.
[51" claim-type="Currently amended] 50. The kit of claim 49, wherein at least one friction matrix structure, a surface for uniformly fixing the liquid crystal, and a spacing material are preassembled into an optical cell.
[52" claim-type="Currently amended] (a) incubating the friction matrix structure according to claim 2 or 16 with the sample to be tested for the presence of the target species;
(b) placing a spacing material between the surfaces of uniformly fixing the liquid crystals with the biochemical blocking layers of the frictional substrate structure incubated such that the biochemical blocking layers of the frictional substrate structures face the surfaces of uniformly fixing the liquid crystals;
(c) drawing the liquid crystal into the region between the incubated friction matrix structure and the surface to uniformly fix the liquid crystal;
(d) determining whether the liquid crystal is uniformly anchored to the friction substrate structure.
[53" claim-type="Currently amended] (a) a support having a friction surface comprising a biochemical blocking layer;
(b) a first target species detection region in a first portion of the support comprising a biochemical blocking layer, the first target species detection region comprising a first biomolecule recognition material capable of binding to the first target species; And
(c) at least one other target species detection region (at least one other target species detection region is capable of binding to at least one other target species) in at least one other portion of the support comprising a biochemical blocking layer A device for detecting the presence of one or more target species in a sample comprising a different biomolecule recognition material)
The first target species detection region uniformly anchors the liquid crystal in the absence of the target species and the at least one other target species detection region uniformly anchors the liquid crystal in the absence of at least one other target species, further detecting the first target species Uniform settling of the liquid crystal in the region collapses when the first target species detection region is exposed to the first target species and uniform settling of the liquid crystal in the at least one other target species detection region results in at least one A device that collapses when exposed to other target species.
[54" claim-type="Currently amended] 54. The apparatus of claim 53, wherein the surface is rubbed while the first biomolecule recognition material and the at least one other biomolecule recognition material are present in the first target species detection region and the at least one other target species detection region, respectively.
[55" claim-type="Currently amended] A kit for use in detecting the presence of a target species in a sample, the kit comprising at least one friction substrate structure according to claim 2 or 16 and a liquid crystal compound.
[56" claim-type="Currently amended] 56. A method of detecting the presence of a target species in a sample using the kit of claim 55,
(a) contacting the friction substrate structural portion of the kit of claim 55 with an amount of sample;
(b) placing the liquid crystal of the kit of claim 55 in the portion of the friction substrate structure in contact with the sample;
(c) determining whether the uniform fixation of the liquid crystal has collapsed.

类似技术:

---

公开号 | 公开日 | 专利标题

---

Ribaut et al.2016|Small biomolecule immunosensing with plasmonic optical fiber grating sensor

---

JP3787143B2|2006-06-21|Apparatus and method for detecting analysis target by optical interference

---

US9297800B2|2016-03-29|Biomaterial construct, its producing method, biomaterial support, target material purifying method, affinity chromatography container, separation chip, analyzing method and analyzing separator for target material, biomaterial complex, and its support, sensor chip, solid support with biomaterial fixed thereon

---

US5492840A|1996-02-20|Surface plasmon resonance sensor unit and its use in biosensor systems

---

CA2235183C|2009-08-11|Device and apparatus for the simultaneous detection of multiple analytes

---

Briand et al.2006|Building of an immunosensor: How can the composition and structure of the thiol attachment layer affect the immunosensor efficiency?

---

Tang et al.2006|Electrochemical immuno-bioanalysis for carcinoma antigen 125 based on thionine and gold nanoparticles-modified carbon paste interface

---

CA1303981C|1992-06-23|Polymer-coated optical structures

---

US9175421B2|2015-11-03|System and method for carrying out multiple binding reactions in an array format

---

DE3215484C2|1988-09-29|

---

US5077210A|1991-12-31|Immobilization of active agents on substrates with a silane and heterobifunctional crosslinking agent

---

Salamon et al.1999|Plasmon resonance spectroscopy: probing molecular interactions within membranes

---

US5427915A|1995-06-27|Multi-optical detection system

---

US5468606A|1995-11-21|Devices for detection of an analyte based upon light interference

---

Clare et al.2005|Orientations of nematic liquid crystals on surfaces presenting controlled densities of peptides: amplification of protein− peptide binding events

---

EP0589867B1|1996-04-10|Sensing surfaces capable of selective biomolecular interactions, to be used in biosensor systems

---

JP2710243B2|1998-02-10|Optical structure coated with polymer

---

US6627397B1|2003-09-30|Measuring chip for surface plasmon resonance biosensor and method for producing the same

---

US6594011B1|2003-07-15|Imaging apparatus and method

---

Skaife et al.2000|Quantitative interpretation of the optical textures of liquid crystals caused by specific binding of immunoglobulins to surface-bound antigens

---

EP1084394B1|2015-04-22|Optical amplification of molecular interactions using liquid crystals

---

JP4999249B2|2012-08-15|Multiple analyte assay method and assay apparatus

---

AU714839B2|2000-01-13|Three-dimensional colorimetric assay assemblies

---

US6861251B2|2005-03-01|Translucent solid matrix assay device for microarray analysis

---

US5756279A|1998-05-26|Analyte detection by means of an analyte-responsive polymer

同族专利:

---

公开号 | 公开日

---

WO2001061325A2|2001-08-23|

---

US20050260703A1|2005-11-24|

---

EP1255996B1|2010-05-26|

---

WO2001061325A3|2002-04-11|

---

AT469350T|2010-06-15|

---

JP2003523514A|2003-08-05|

---

KR100463979B1|2005-01-03|

---

US7651662B2|2010-01-26|

---

JP3512775B2|2004-03-31|

---

AU4719801A|2001-08-27|

---

US6692699B2|2004-02-17|

---

AU774673B2|2004-07-01|

---

DE60142220D1|2010-07-08|

---

CA2394966C|2007-01-09|

---

CA2394966A1|2001-08-23|

---

US20020055093A1|2002-05-09|

---

US20040161800A1|2004-08-19|

---

EP1255996A2|2002-11-13|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

法律状态:
2000-02-16|Priority to US18295300P

---

2000-02-16|Priority to US60/182,953

---

2001-02-15|Application filed by 위스콘신 얼럼나이 리서어치 화운데이션

---

2002-09-28|Publication of KR20020074235A

---

2005-01-03|Application granted

---

2005-01-03|Publication of KR100463979B1

优先权:

---

申请号 | 申请日 | 专利标题

---

US18295300P| true| 2000-02-16|2000-02-16|

---

US60/182,953|2000-02-16|