KR20110104461A - Preparation method of antigen-immobilized immuno- fluorescence slide and the immuno-fluoroscence slide made by the method - Google Patents

Abstract

According to the present invention, a method of preparing a protein chip by immobilizing a C-reactive protein on a slide, mixing an antibody specifically binding to a protein to be analyzed with streptavidin and labeling it with fluorescent nanoparticles, the antibody Mixed competitive immunization step, analysis with a fluorescence camera, in order to immobilize the C-reactive protein on the slide, preparing a slide modified with 3-aminopropyltrimethoxysilane, 3-aminopropyl Hydrating the modified slide with trimethoxysilane, activating the modified slide using a glutaraldehyde solution, adding the C-reactive protein in a 30-70 mM phosphate buffer solution (pH 6.5-7.8) to 0.01-0.5 Dissolving to a mg / ㎖ concentration to prepare an antigen solution for immobilization, Petridi containing the slide on the spotting guide A method for producing an antigen-immobilized immunofluorescent slide for immobilizing antigen by spotting the prepared antigen solution at 1-100 μl dropping point and reacting the slide prepared by the above step for 1-6 hours. An immunofluorescent slide prepared is disclosed.

Description

FIELD OF THE INVENTION A method for producing an antigen-immobilized immunofluorescent slide and an immunofluorescent slide produced by the same, the present invention relates to an immunofluorescent slide prepared by the present invention and to an immunofluorescent slide prepared by the same.

The present invention provides a method for preparing a protein chip by immobilizing a C-reactive protein on a slide, mixing an antibody that specifically binds to a protein to be analyzed with striptevidin and labeling it with fluorescent nanoparticles. The present invention relates to a method for producing an antigen-immobilized immunofluorescence slide comprising an immunoreaction step and an analysis with a fluorescence camera, and an immunofluorescence slide produced thereby.

Biochip is an immobilization of various types of probes on the surface of a solid-phase support of a unit area.By using such a biochip, a small amount of sample can be used to diagnose disease, high throughput screening (HTS), and enzyme activity measurement. Experiments can be easily performed on a large scale.

Most of the methods of fixing a probe to a slide or slide are made by fixing the probe on a glass slide pretreated with a coating material. To this end, various surface chemicals have been proposed, and a technique such as a self-assembled monolayer is typically applied (Korean Patent Publication No. 2003-0038932).

In response, a three-dimensional immobilization method has been developed (Gill and Ballesteros, Trends in) in an attempt to increase the amount of probe immobilized and to maintain the activity of the probe. Biotechnology 18: 282, 2000). For example, a three-dimensional immobilization method using Packard Bioscience's Hydrogel ^® coating slide, Biocept's polyethylene glycol-based hydrogel, LG Chem's Solgel, and the like can be mentioned.

The slide for the immunosensor is selected from the group consisting of glass, silicone, hydrogel, metal, ceramic, porous membrane.

Currently, antibodies or antigen probe-plates on which an antibody or antigen is immobilized on a substrate based on the specific reactivity of the corresponding antigen or antibody are widely used for detection. Three representative kits for this use are rapid test kits, routine test kits and biochip kits.

These are used to detect target antigens or target antibodies corresponding to immobilized antibody probes or antigen probes.

In some cases, several reactors are combined into one probe-plate, each of which comprises a test strip that detects an antigen or an antibody, respectively.

On the other hand, with the recent entry into force of the Health Functional Food Act, the door to manufacture food materials and processed foods based on natural products and be recognized as health functional foods has been opened in Korea. However, to this end, it is necessary to present data that scientifically demonstrate the functional functionality of health functional foods, such as cardiovascular disease prevention, anti-aging, cancer prevention, obesity prevention, immune regulation, and gastrointestinal disease prevention. Therefore, the importance of food functional evaluation is increasing, and as a result, the possibility of commercialization of functional evaluation field is very high.

Functional assessment of foods in vitro around six functional types above (in in vitro ) functional evaluation, in vivo using experimental animals such as rats ( in in vivo ) functional evaluation and clinical trials. From the perspective of the food industry, the necessity of the development of in vivo functional retrieval technology using laboratory animals for efficient and rapid evaluation of functional ingredients and functional foods is increasing. have.

To be recognized as a functional food, the desired functionality, such as cardiovascular functionality, has to be scientifically proved, and the importance of assessing the functionality of the food cannot be overemphasized (Kim et al. Detection of reactive proteins, Journal of Biotechnology and Bioengineering , Vol. 22, p. 443, 2007). In vivo functional evaluation using laboratory animals as a preliminary step prior to clinical trials has traditionally been to measure changes in physical factors such as body weight, blood pressure, and organ morphology. However, these methods not only result in uneven results, but also require a variety of evaluation protocols, skilled experts, and expensive analytical instruments. A uniform assessment of food functionality in vivo is to measure the rise and fall of specific biomarker proteins associated with specific diseases or metabolic symptoms in laboratory animals such as rats. In this case, the animal containing the food to be recognized as a functional food is administered to the experimental animal.

Korean Patent No. 10-921237 discloses a new method for evaluating in vivo functionality that can quickly detect cardiovascular functionality of food materials and functional foods, and is a major biomarker for coronary artery disease and hypertension. It is known as a pentameric protein with a molecular mass of about 118 kilodaltons (kDa), which is synthesized by induced stimulation by interleukin-6 and interleukin-1β in mammalian liver. C-reactive protein ( Biosensors and Bioelectronics , Vol. 19, p. 1193, 2004; Clinical Chemistry , Vol. 47, p. 403, 2001; New England Journal of Medicine , Vol. 340, p. 448, 1999; Biochemical Journal , Vol. 327, p. 425, 1997) have been described for a high sensitivity detection method using a quartz crystal microbalance immunosensor.

The method of one of the enzyme immunoassays of the conventional method of measuring a C- reactive protein (enzyme-linked immunosorbent assay, ELISA ) (American Journal of Cardiology , Vol. 1, p. 155, 2005; American Journal of Veterinary Research , Vol. 1, p. 62, 2005; Journal of Clinical Laboratory Analysis , Vol. 18, p. 280, 2004), and it has the advantage of being easy to use without being affected by the measurement of coloring matters, and the measurement sensitivity is superior to or similar to other sensor methods ( Biosensors). and Bioelectronics , Vol. 22, p. 973, 2007; Biosensors and Bioelectronics , Vol. 21, p. 1987, 2006; Biosensors and Bioelectronics , Vol. 21, p. 1631, 2006; Biosensors and Bioelectronics , Vol. 21, p. 1141, 2006; Analytical Biochemistry , Vol. 328, p. 210, 2004).

Recently, the use of nanomaterials such as metal colloids, carbon nanotubes, fluorescent silica nanoparticles, and semiconductor quantum dots for sensor measurement using biosensors has been increasing. Increasingly, a number of studies have been reported to improve the sensitivity, stability, and selectivity of sensor signals through its application.Analytical Chemistry, Vol. 79, p. 630-707, 2007;Biosensors and Bioelectronics, Vol. 21, p. 1900, 2006;Langmuir, Vol. 22, p. 4357, 2006;Nano Letters, Vol. 5, p. 113, 2005;Biosensors and Bioelectronics, Vol. 20, p. 2454, 2005;Proceedings of the National Academy of Sciences, Vol. 100, p. 4984, 2003;Biochemical and Biophysical Research Communications, Vol. 274, p. 817, 2000).

When assessing the in vivo cardiovascular functionality of foods by measuring C-reactive protein as a biomarker present in the blood of experimental animals by biosensors, increasing sensor sensitivity is still important and the need for simple measurement is constantly raised. Has been.

The present invention provides a protein, antibody and fluorescence to be analyzed by immobilizing a certain volume of antigen on a slide using a micro dispensing pipette without using complicated devices such as a biochip arrayer or a micro-array scanner for the fabrication and measurement of biochips. Immune reactions between conjugates of nanoparticles or immobilization of immobilized antigens and mixtures of fluorescent nanoprobes and specific target molecules were carried out at the antigen-coated sites on the immunofluorescent slides. The present invention aims to provide a slide for a single-use immunosensor with high sensitivity of a sensor that can easily measure fluorescence intensity or number of fluorescent particles by a fluorescence microscope, and as a result, know the concentration of a specific target molecule such as a biomarker.

In order to solve the above problems, the present invention is to fix a protein (antigen) to be analyzed on a slide, to label the antibody specifically binding to the antigen with a fluorescent material, and to analyze the protein to be collected from a sample. After mixing with the fluorescently labeled antibody, the mixed mixture is reacted with the antigenic site immobilized on the slide, the unreacted antibody and antigen are washed with distilled water, the slide is placed in an incubator and dried, followed by fluorescence A microscope is used to measure the fluorescence intensity or the number of fluorescent particles.

Although the present invention shows the use of an indirect-competitive assay format for analyzing a sample, those skilled in the art to which the present invention pertains have a direct-competitive assay format. It will be readily understood that this can be done by a sandwich assay format.

Slide for immunosensor used in the present invention is selected from the group consisting of glass, silicon, hydrogel, metal, ceramic, porous membrane and has a horizontal and vertical dimensions of 20 ~ 40X70 ~ 80mm.

In the present invention, C-reactive protein (hereinafter referred to as "CRP") was used as a sample to be analyzed, but a person having ordinary knowledge in the technical field to which the present invention belongs may use the method of other food functional biomarker ELD. Blood loss biomarkers such as LDL, fibrinogen, angiotensin II, and cardiac troponin, anti-aging, cancer prevention, obesity prevention, immunomodulation, gastrointestinal disorders It will be readily understood that biomarker proteins related to prophylaxis and the like can be used.

The C-reactive protein antibody may be any one selected from antibodies prepared using C-reactive proteins derived from mammals such as rats, mice, rabbits, monkeys, and humans.

The sample collected from the sample may be any one selected from tissue extracts such as standard solution in which C-reactive protein is dissolved in the reaction buffer solution, blood, serum, plasma, saliva, body fluid, and liver.

      The solution used to block the sites that did not react with the antigen on the slide surface where the antigen is immobilized includes 1-5% of Bovine serum albumin (BSA), Rat serum albumin (RSA), and Human serum albumin (HSA). Any one selected from MSA (Mouse serum almumin) and GSA (goat serum albumin) solutions can be used.

Hereinafter, the present invention will be described in detail for each step.

       Immobilization of the C-reactive protein as an antigen on the slide of the present invention can be carried out by selecting one of the following two methods.

First, an antigen is immobilized using 3-aminopropyltrimethoxysilane (hereinafter referred to as "APTMS"). To use this method, first, a slide modified with APTMS is prepared. For the reforming, the slides were immersed in Piranha solution (Piranha solution, H ₂ SO ₄ : H ₂ O ₂ = 2: 1-4: 1, volume ratio) for 5-15 minutes, then rinsed with distilled water and then distilled water After sonication for 3 ~ 10 minutes, hydrated with 85 ~ 95 ℃ hot water for 30 ~ 90 minutes, put slides on Kimwipers and dry at room temperature for 20 ~ 120 minutes. The prepared slides subjected to surface washing are placed in a Petri dish containing APTMS solution dissolved in acetone at a concentration of 5 to 15%, and reacted at room temperature for 30 to 90 minutes. Rinse sequentially with acetone, 30-70 mM phosphate buffer solution (pH 6.5-7.8), distilled water, 3-4 times in front of the slide, and soak for 1 to 60 minutes in a beaker containing distilled water. Take this out and dry it at room temperature for 20 ~ 120 minutes on Kimwipes, heat it for 3 ~ 7 hours at 30 ~ 150 ℃ convection oven, cool it and store it in desiccator until it is used.

The slides modified with APTMS are hydrated by soaking in distilled water for 10-20 minutes. Take out the immersed slide, put it in a petri dish containing glutaraldehyde 1-4% solution, and react for 30-90 minutes to activate the protein to bind to the slide surface. Wash the front and back of activated slide 3-4 times with 30-70mM phosphate buffer (pH 6.5-7.8). Dip the slide for 1 to 60 minutes in a beaker of distilled water. Take it out and place the slide on Kimwipes for 20 to 120 minutes at room temperature.

Glutaraldehyde used in the above is prepared by adding 1 to 4 ml of glutaraldehyde in a concentration of 30-70% to 46 to 49 ml of distilled water and applying it to 30 to 70 ml.

      In order to immobilize the protein to be analyzed, for example, C-reactive protein, first, the C-reactive protein was dissolved in a concentration of 0.01-0.5 mg / ml in 30-70 mM phosphate buffer solution (pH 6.5-7.8) to prepare an antigen solution for immobilization. do. Place Petri dishes containing glutaraldehyde-activated slides on a grid spotting guide, 1-100 µl of the antigen solution prepared previously, and spot the lid on the slide corresponding to the dropping point on the spotting guide. Cover and react for 1 to 6 hours to immobilize the antigen, and then wash the front and back of the slide 3 ~ 4 times with 30-70mM phosphate buffer solution (pH 6.5-7.8) and soak for 1 minute in a beaker containing distilled water. Small plastic petri dishes containing 1-5% BSA solution dissolved in 30-70 mM phosphate buffer solution (pH 6.5-7.8) to block the sites that did not react with the antigen on the surface of the glutaraldehyde-activated slide that had immobilized the antigen. Put the slide on the lid and react for 1 ~ 5 hours.After the reaction, wash the front and back of BSA-blocked slide with 30-70mM phosphate buffer solution (pH 6.5-7.8) 3 ~ 4 times and put it in the beaker with distilled water. Immerse the slide for 1-60 minutes. Remove it and place the slide on Kimwipes for 20-120 minutes at room temperature.

Second, the method of immobilization by MPTMS-GMBS (3-Mercaptopropyltrimethoxysilane-N-gamma-maleimidobutyryloxy succinimide ester) method is used. First, the surface is immersed in a mixed solution of concentrated hydrochloric acid (18-36%) and methanol (50-100%) (HCl: MtOH = 1: 1 ~ 1: 2, volume ratio) for 15 to 45 minutes for surface cleaning. Rinse the front and back three to four times with distilled water. The slides are washed with distilled water at 85 ~ 95 ℃, placed on Kimwipes and dried at room temperature for 20 ~ 120 minutes.

In order to silanize the slide thus prepared, 50 to 500 mL of MPTMS solution dissolved in an organic solvent solution selected from the group consisting of 1-3% toluene, dimethyl formamide, and acetone is prepared. Put the slide in the Petri dish containing the solution and react for 1 to 3 hours at room temperature. After washing the front and back of the slide three to four times with the same organic solvent used in the preparation of the MPTMS solution, place on a Kimwips and dried at room temperature for 20 to 120 minutes.

In order to activate the slide surface 1 ~ 3mM GMBS solution is prepared as follows. Dissolve 14-42 mg of GMBS sample in 100 μl of DMF (N, N-Dimethylformamide; Sigma-Aldrich, USA) and add 49.9 mL of ethanol. The silanized slide was put in a Petri dish containing GMBS solution and reacted at room temperature for 30 to 90 minutes. Wash the front and back of the slide three to four times with distilled water and 30-70 mM phosphate buffer solution (pH 6.5-7.8). The slides are then placed in Kimwipes and allowed to dry at room temperature for 20 to 120 minutes.

Immobilization of the CRP antigen is performed as follows. First, the immobilized antigen solution is prepared by dissolving C-reactive protein in a concentration of 0.01-0.5 mg / ml in a 30-70 mM phosphate buffer solution (pH 6.5-7.8). Place petri dishes containing GMBS activation slides on the spotting guide and spot 1-100 μl of the antigen solution prepared on the spotting guide on the slide corresponding to the dropping point on the spotting guide. After immobilization, rinse the front and back sides 3 ~ 4 times with 30-70mM phosphate buffer solution (pH 6.5-7.8) and immerse the slide in beaker containing distilled water for 1 ~ 60 minutes. To increase the accuracy of the assay, place the slide in a small plastic Petri dish containing 1-5% BSA solution and cover and react for 1-5 hours. Using the phosphate buffer solution as above, wash the front and back of the slide three to four times. After soaking for 1 to 60 minutes in a beaker containing distilled water, place the slide on the Kimwipe and dry at room temperature for 20 to 120 minutes.

       A schematic representation of the two methods is shown as FIG. 1.

In analyzing a specific protein using the slide for immunosensor of the present invention, an antibody that specifically binds to an antigen immobilized on the slide is labeled. Labeling the antibody uses a highly sensitive and reproducible fluorescence staining method, and for this purpose, fluorescent silica nanoparticles (hereinafter referred to as "FSNP") are used in the present invention.

A method for producing fluorescent silica nanoparticles (FSNP) is outlined. FSNP, which is used to label antibodies that specifically bind to antigens, is a dye entrapment incorporating organofluorescence into the silica structure, and covalently binds the activated organofluorescence with an aminosilane compound and then silica. Core shell method for incorporation into the structure, Inverted core shell method for incorporating functional groups by reacting particles formed by incorporating organic fluorescent dyes into the silica structure with organosilane compounds What was manufactured by etc. can be used. The organic fluorescent dyes include dichlorotris (1,10-phenanthroline) ruthenium (II) monohydrate (dichlorotris (1,10-phenanthroline) ruthenium (II) hydrate), fluorescein, rhoda Rhodamine B, 5 (6) -carboxytetramethylrhodamine (5 (6) -Carboxytetramethylrhodamine) and the like can be used, and as an activated organic fluorescent dye, Fluorescein isothiocyanate, 5 ( 6) -carboxytetramethylhodamine N-succinimidyl ester (5 (6) -Carboxytetramethylrhodamine N-succinimidyl ester), tetramethylrhodamine 5-isothiocyanate, bis (2,2 ' -Bipyridine) -4'-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester hexafluorophosphate (Bis (2,2'-bipyridine) -4'-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester-bis (hexafluorophosphate), bis (2,2'-bipyridine) -4,4'-dicarboxybipyridine-ruthenium di (N-succinimi Diyl ester) bis (hexafluorophosphate) (Bis (2,2'-bipyridine) -4,4'-dicarboxybipyridine ruthenium di (N-Succinimidyl ester) bis (hexafluorophosphate)) and the like. The (3-aminopropyl) triethoxy silane ((3-Aminopropyl) triethoxysilane) and the like can be used, the organosilane compound carboxylate (Carboxylate), amine (Amine), amine / phosphonate (Amine / Compounds having phosphonate, poly (ethylene glycol), octadecyl, and carboxylate / octadecyl functional groups can be used.

The method of labeling antibodies using FSNP is described. First, it is necessary to modify the FSNP with streptavidin (hereinafter referred to as "SA") which specifically binds to biotin, which means that the SA-attached FSNP as described below is combined with a biotinylated antibody. To combine. Thiol-modified FSNPs are first prepared by reacting with 3-mercaptopropyltrimethoxy silane ((3-Mercaptopropyl) trimethoxy silane, hereinafter referred to as "MPTMS") to modify the FSNP to SA. Add 1 ~ 5mg of FSNP and 5 ~ 15ml of alcohol such as ethanol, methanol, propanol to 10 ~ 70ml cap tube, shake well, and sonicate for 10 ~ 30 minutes to completely dissolve FSNP in alcohol. . After that add MPTMS 30-70 ~ 200μl to the cap tube and react for 2-6 hours while stirring at a low speed of 30-200rpm at room temperature. The precipitate obtained by centrifuging the reaction mixture at 7000-17000 rpm for 20 to 40 minutes at 3 to 10 ° C. was washed 1 to 5 times using the same alcohol used in the reaction, and after each washing, a precipitate was obtained by centrifugation under the same conditions. . Remove the cap from the tube and allow it to dry at room temperature with a little aluminum foil. The precipitate is redissolved in 2-6 ml of 30-70 mM phosphate buffer solution (pH 6.5-7.8) to prepare a thiol-modified FSNP solution.

Maleimide-activated SA is added to thiol-modified FSNP to prepare SA-attached FSNP. More specifically, 0.1-0.5 mg of SA-maleimide and 3-7 ml of 30-70 mM phosphate buffer solution (pH 6.5-7.8) were added to a 10-70 ml cap tube to dissolve the SA-maleimide solution. . 0.5 to 2.0 ml of the thiol-modified FSNP solution prepared above was added thereto and the mixture was stirred at a low speed of 30 to 200 rpm for 1 to 3 hours at room temperature. The precipitate is taken by centrifugation at 7000-17000 rpm for 20-40 minutes at 3-10 ° C. The precipitate taken in the above step is washed 1 to 5 times with 30 ~ 70mM phosphate buffer solution (pH 6.5-7.8) and centrifuged under the same conditions after each wash to obtain a precipitate. The precipitate washed in this step is resuspended in 1-5 mL of 30-70 mM phosphate buffer solution (pH 6.5-7.8). This is wrapped in an aluminum foil in a cap tube and refrigerated to react with the biotinylated antibody described below.

Biotinylation of C-reactive protein antibodies can be carried out using either of the following two methods.

As a first method, a carbonic acid buffer solution is added to a C-reactive protein antibody to obtain a carbonic acid buffer solution containing a C-reactive protein antibody, and biotin dissolved in dimethyl formamide, an organic solvent. Reacting the biotinamidohexanoic acid N-hydroxysuccinimide ester (NHS-LC-biotin) solution with the carbonate buffer solution of the above step, the solution obtained in the step And dialysis in sodium chloride solution to remove unreacted biotinylation reagent.

More specifically, adding a carbonic acid buffer solution to the C-reactive protein antibody to obtain a carbonic acid buffer solution having a C-reactive protein antibody concentration of 1.0 ~ 3.0mg / ㎖, dissolved in 1 ~ 5mg / ㎖ concentration in dimethyl formamide Reacting 30-100 μl of NHS-LC-biotin solution with 25-75 μl of carbonic acid buffer solution of the above step under ice water for 1-5 hours, 75-175 μl of the solution obtained in the above step is 0.1-0.3 M Dialysis overnight in sodium chloride solution to remove unreacted biotinylation reagent.

As a second method, phosphate buffered saline is added to C-reactive protein antibody to obtain phosphate-buffered saline containing C-reactive protein antibody, and sulfosuccinimidyl-6- (bioti, a water-soluble biotinylation reagent dissolved in re-distilled water). Amido) hexanoate (Sulfosuccinimidyl-6- (biotinamido) hexanoate, sulfo-NHS-LC-biotin) solution is reacted with phosphate buffered saline of the above step, the solution obtained in the step of dialysis in phosphate buffered saline The method which consists of removing a reaction biotinylation reagent is mentioned.

More specifically, phosphate buffered saline is added to the C-reactive protein antibody to obtain phosphate-buffered saline having a concentration of 0.5-3.0 mg / ml of the C-reactive protein, and sulfo-NHS- dissolved in 2-20 mM in re-distilled water. 5 to 20 µl of LC-Biotin solution is reacted with 30 to 800 µl of phosphate buffered saline in the above step for 1 to 5 hours under ice water, and 55 to 820 µl of the solution obtained in the step is 0.05 to 0.25 M. Dialysis against phosphate buffered saline overnight to remove unreacted biotinylation reagent.

As a next step, an antibody to which a nanomaterial is attached is prepared. When the SA-modified FSNP prepared above and the biotinylated antibody are added and mixed, the biotin is specifically bound to SA, thereby producing an antibody labeled with a fluorescent material.

More specifically, the SA-modified FSNP solution prepared above was sonicated for 10-30 minutes, 200-600 μl of the biotinylated antibody solution 1-3 prepared at the concentration of 0.05-0.25 mg / ml prepared above. Add ㎖ and react for 1 to 3 hours at room temperature while stirring for 2 to 7 minutes at a low speed of 30 to 200 rpm at 30 minute intervals. Centrifuge at 7000-17000rpm for 20-40 minutes at 3 ~ 10 ℃, discard supernatant and take off precipitate. The precipitate taken in the above step is washed 1 ~ 5 times with 30 ~ 70mM phosphate buffer solution (pH 6.5-7.8), and after each washing, the precipitate is obtained by centrifugation under the same conditions as above. The precipitate washed in this step is resuspended in 0.4-1.2 ml of 30-70 mM phosphate buffer solution (pH 6.5-7.8).

A schematic method of labeling an antibody specifically binding to CRP of the present invention with a fluorescent substance is shown as FIG. 2.

In analyzing the specific protein using the slide for immunosensor of the present invention, the protein to be collected from the sample is mixed with the labeled antibody as described above.

More specifically, each concentration of C-reactive protein sample prepared by diluting stepwise using 30-70 mM phosphate buffer solution (pH 6.5-7.8) in an Eppendorf tube by specific binding of biotin and SA As the nano-material prepared as described above, an equal amount of FSNP-attached C-reactive protein antibody solution was added and mixed, followed by standing at room temperature for 5 to 300 minutes.

The next step is to react the mixture as described above with the antigenic site immobilized on the slide.

More specifically, the biotransducer immobilized C-reactive protein as an antigen by the glutaraldehyde process or the MPTMS-GMBS process and minimizing non-selective protein adsorption by a BSA (bovine serum albumin) blocking process. Put the Petri dish containing the slide as a transducer) on the spotting guide of the lattice and mix the above with the antigen-immobilized part on the slide as described above, and the protein to be analyzed obtained from the sample which was allowed to stand at room temperature for 5 to 300 minutes. After accurately spotting 1-100 μl of the labeled antibody mixture, react with the lid and react for 0.5 to 16 hours at room temperature. Indirectly between the antibody labeled with FSNP and the antigen immobilized on the slide and the protein to be analyzed A competition reaction is carried out. After the reaction, the unreacted antibody and antigen were washed three to four times before and after the slide using distilled water, and then immersed in the beaker containing distilled water for 1 to 60 minutes to remove.

Figure 3 illustrates how the antigens immobilized on the slides of the present invention and the fluorescently labeled antibodies bind by indirect competition. The 30-70 mM phosphate buffer solution (pH 6.5) is used instead of the C-reactive protein taken from the sample. -7.8) was mixed with the labeled antibody as described above, and then dropped into the site where the antigen on the slide was immobilized to change the number of fluorescent particles bound to the slide when antigen / antibody reaction was performed over time. .

Place the slide in an incubator and dry for 20-120 minutes.

Fluorescence microscopy is used to measure fluorescence intensity and number of fluorescent particles. If the CRP concentration in the sample is high, it reacts with the fluorescently labeled antibody so that the number of antigens immobilized on the slide and the fluorescently labeled antibody are less likely to bind. As described above, the antigens immobilized on the slides and the antigens in the sample show different fluorescence through competitive interaction with the fluorescently labeled antibody, so that the CRP can be easily measured in a short time.

According to the present invention, C-reactive protein, which is known as one of the major biomarkers for coronary artery disease, hypertension and inflammation, can be measured at nanomolar levels by coating a slide on a slide and reacting an antibody bound with fluorescent nanoprobes. Provided is a slide for an immunosensor that can easily measure the biomarker, an indicator of food functionality in vivo, with high sensitivity.

The present invention, in addition to the C-reactive protein in the future ultra-high sensitivity for biomarkers present in various concentration ranges in the blood of experimental animals such as low density lipoprotein (LDL), fibrinogen, angiotensin II (angiotensin II) Underlying techniques for evaluating food functionality required for high-speed simultaneous measurements can be established.

1 is a schematic representation of two methods of immobilizing C-reactive protein to antigen.
2 is a schematic diagram showing a method of labeling an antibody that specifically binds CRP with a fluorescent substance.
3 is a view illustrating a method in which an antigen immobilized on a slide of the present invention and a fluorescently labeled antibody bind by indirect competition.
Figure 4 shows the time-dependent antigen by dropping 30-70 mM phosphate buffer solution (pH 6.5-7.8) instead of the C-reactive protein collected from the sample with the labeled antibody according to the present invention to the site where the antigen on the slide is immobilized. • A graph showing the change in the number of fluorescent particles bound to a slide when an antibody reaction was performed.
5, a, b, and c are diagrams showing that the inhibitory spots of contaminants such as impurities present on the surface of the slide before the treatment of the piranha solution are removed by the piranha treatment, and the inhibition of the fluorescence image even after silencing by APTMS Figure showing that no spots are observed. 5D is a diagram showing that no inhibitory spots are detected by taking a fluorescence image by treating a substrate treated with a glutaraldehyde 2.5% solution. 5E shows that no inhibition spots were found on the surface of the BSA blocking slide after antigen immobilization. Figure 5f is a diagram showing the fluorescence spot of the fluorescent silica nanoparticle-labeled antibody bound to the slide surface subjected to indirect competition by the method of the present invention.
6 shows an apparatus for fluorescence analysis.
FIG. 7 is a graph showing the calibration curve of antigen-immobilized immunofluorescence slides and FSNP labeled antibody based C-reactive protein.

Hereinafter, a specific method of the present invention will be described with reference to Examples. However, the scope of the present invention is not limited only to these examples.

The materials used in this experiment are as follows:

Recombinant histidine tagged rat CRP (pure state) expressed in mouse myeloma cell line (NSO) was obtained from R & D Systems, Inc. (Minneapolis, MN, United States) and used throughout the experiment. Its homopentameric structure consists of three non-covalent and two covalently bonded subunits. Monoclonal anti-rat CRP antibodies generated from hybridomas derived from fusion of mouse myeloma with purified B cells from mice immunized with purified, recombinant rat CRP derived from NSO-derived were also obtained from the above R & D Systems Inc. . Glass slides were obtained from Coring Inc. (Kennebunk, ME, United States). Aqueous biotylation reagent (sulfosuccinimidyl-6- (biothiamidido) hexanoate (sulfo-NHS-LC-biotin) was purchased from Pierce Biotechnology, Inc. (Rockford, IL, United States). Propyltrimethoxysilane (APTMS), glutaraldehyde and bovine serum albumin (BSA) were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, United States) All other compounds are warranted from various suppliers Double distilled water was used throughout the experiment.

Example  One: APTMS - GA  Antigen Immobilization on Treatment Slides

Fixing the antigen to the slide was prepared by the following procedure. In other words, the surface was immersed in a piranha solution (H ₂ SO ₄ : H ₂ O ₂ = 3: 1, volume ratio) for 10 minutes, rinsed with distilled water, sonicated in distilled water for 5 minutes and 90 ℃ After hydrated with hot water for 1 hour, the slide was placed on a Kimwipers and dried at room temperature for 30 minutes.

In order to perform silanization for introducing the amino group (-NH ₂ ), which is a reactor required for immobilization of the antigen, to the slide surface, the prepared slides subjected to surface washing are placed in a petri dish containing APTMS solution dissolved in acetone at 10% concentration. Reaction was carried out for 1 hour. Acetone, 50mM phosphate buffer solution (pH 7.4), distilled water was washed sequentially three times before and after the slide and immersed in a beaker containing distilled water for 1 minute. This was taken out, dried at room temperature for 30 minutes on a Kimwips, and then heat-treated in a convection oven at 120 ° C. for 5 hours to cool, and a fluorescent image was taken (FIG. 5 a, b, c). As shown in a, b, and c of FIG. 5, it can be seen that the inhibitory spots of contaminants such as impurities present on the surface of the slide before the Piranha treatment are removed through the Piranha treatment and fluorescence even after silanization by APTMS. It was found that no inhibition spot was observed on the image.

       Hydrated by soaking in distilled water for 15 minutes to activate the slide modified with APTMS. The immersed slide was taken out and placed in a petri dish containing 2.5% glutaraldehyde solution for 1 hour to activate the protein to bind to the slide surface. The slides were washed three times before and after the slides activated with a 50 mM phosphate buffer (pH 7.4), and the slides were immersed in a beaker containing distilled water for 1 minute. This was taken out and placed on a slide on Kimwipes, dried for 30 minutes at room temperature, and confirmed by taking a fluorescence image, no inhibition spot was found on the surface of the GA-activated slide (Fig. 5d).

       In order to immobilize the antigen (CRP, C-reactive protein) on a slide, the immobilized antigen solution was prepared by dissolving C-reactive protein at a concentration of 0.1 mg / ml in 50 mM phosphate buffer solution (pH 7.4). Place Petri dishes containing glutaraldehyde-activated slides on a grid spotting guide, and place 20 µl of the prepared antigen solution on the slide corresponding to the dropping point on the spotting guide. After the lid was capped and reacted for 1 hour to fix the antigen, the front and rear surfaces of the slide were washed three times with 50 mM phosphate buffer (pH 7.4) and immersed in a beaker containing distilled water for 1 minute. A small plastic petri containing 1% BSA (bovine serum albumin) solution dissolved in 50 mM phosphate buffer (pH 7.4) to block the sites that did not react with the antigen on the surface of the glutaraldehyde-activated slide that had immobilized the antigen. The slide was placed in a dish and the lid was covered for 1 hour. The slides were washed three times in the front and the back with 50mM phosphate buffer (pH 7.4), immersed in a beaker containing distilled water for 1 minute, placed on a Kimwipe, dried at room temperature for 30 minutes, and photographed for fluorescence imaging. No inhibition spots were also found on the surface of the BSA blocking slide after antigen immobilization (FIG. 5E).

Example  2: MTS - GMBS  Antigen Immobilization on Treatment Slides

Fixing the antigen to the slide was prepared by the following procedure. That is, the surface was immersed for 30 minutes in a solution of concentrated hydrochloric acid (35%) and methanol (70%) in a volume ratio of 1: 1 for surface cleaning, and then washed the slide three times with distilled water. After immersing in concentrated sulfuric acid for 30 minutes, the front and rear sides of the slides were washed three times with distilled water.

2 ml of MPTMS solution was added to 98 ml of toluene by adding 2 ml of MPTMS ((3-Mercaptopropyl) trimethoxy silane) to 98 ml of toluene for silanization to introduce thiol group (-SH), which is a reactor required for immobilization of antigen, to the slide surface. Prepared. The slide was placed in a Petri dish containing the above solution and reacted at room temperature for 2 hours. Thereafter, the slides were washed three times with toluene, and then the slides were placed in Kimwipes and dried at room temperature for 30 minutes.

To activate the slides modified with MPTMS, 28 mg of GMBS was dissolved in 100 μl of DMF (N, N-Dimethylformamide, chromosllvr ^® Plus, St. Louis, MO, United States, HPLC≥99.9%) followed by 49.9 mL of ethanol. 2 mM GMBS solution was prepared by adding thereto. The silanized slides were placed in a Petri dish containing 2 mM GMBS solution and allowed to react at room temperature for 1 hour. The front and rear surfaces of the slides were washed three times with distilled water and 50 mM phosphate buffer (pH 7.4) and dried at room temperature for 30 minutes in Kimwipes.

       In order to immobilize the antigen (CRP, C-reactive protein) on a slide, the immobilized antigen solution was prepared by dissolving C-reactive protein at a concentration of 0.1 mg / ml in 50 mM phosphate buffer solution (pH 7.4). Place petri dishes containing GMBS activation slides on the spotting guides, and add 20 µl of the antigen solution previously prepared to the slide site corresponding to the dropping point on the spotting guide, cover the lid, and react for 1 hour to immobilize the antigen. It was. The slides were washed three times in front of the slides with a 50 mM phosphate buffer solution (pH 7.4) and immersed in a beaker containing distilled water for 1 minute. To block antigen-unreacted sites on the surface of the GMBS-activated slides with immobilized antigen, place the slide in a small plastic Petri dish containing 1% BSA solution dissolved in 50 mM phosphate buffer solution (pH 7.4) and cover the lid for 1 hour. Reacted. The slides were washed three times, front and back, with 50 mM phosphate buffer (pH 7.4), immersed in a beaker containing distilled water for 1 minute, placed on a Kimwipe and dried at room temperature for 30 minutes.

       Fluorescence images were taken on slides subjected to surface cleaning, silanization, activation, antigen immobilization and BSA blocking, which are the stages of the antigen immobilization process on the MTS-GMBS treatment slide. , Volume ratio) No inhibition spot was detected on the slide surface except for the slide before treatment.

Example  3: SA in Modified FSNP Manufacture

2 mg of FSNP and 10 ml of ethanol were added to a 25 ml cap tube, followed by shaking for 15 minutes to completely dissolve FSNP in ethanol. Thereafter, 100 µl of MPTMS was added to the cap tube, and reacted for 4 hours while stirring at low speed at 100 rpm at room temperature. The precipitate obtained by centrifuging the reaction mixture at 13000 rpm for 30 minutes at 4 ° C. was washed three times with ethanol and centrifuged under the same conditions after each wash to obtain a precipitate. The cap of the tube was removed and slightly dried with aluminum foil. The precipitate was redissolved in 4 ml of 50 mM phosphate buffer (pH 7.4) to prepare a thiol-modified FSNP solution.

SA-maleimide solution was prepared by dissolving 0.25 mg of SA-maleimide and 5 ml of 50 mM phosphate buffer solution (pH 7.4) in a 25 ml cap tube. 1 ml of the thiol-modified FSNP solution was added thereto, followed by reaction at room temperature for 2 hours while stirring was continued at a low speed of 100 rpm. The precipitate was taken by centrifugation at 10000 rpm for 20 minutes at 4 ° C. The precipitate taken in the above step was washed three times with 50 mM phosphate buffer solution (pH 7.4), and after each washing, the precipitate was obtained by centrifugation under the same conditions as above. The precipitate washed in this step was resuspended in 2 ml of 50 mM phosphate buffer solution (pH 7.4). This was wrapped in aluminum foil in a cap tube and refrigerated to react with the biotinylated antibody described below.

Example  4: of antibody Biotinylation

1 mg vial of sulfosuccinimidyl-6- (biotinamido) hexanoate, sulfo-NHS-LC-biotin, a refrigerated water-soluble biotinylation reagent On the day of the experiment, the resultant was removed from the refrigerator to room temperature, and dissolved in 30-700 µl of monoclonal anti-rat CRP antibody by adding 30-700 µl of 0.1 M phosphate buffered saline (pH 7.2). 180 μl of distilled water was added to the 1 mg vial of sulfo-NHS-LC-biotin to prepare a 10 mM sulfo-NHS-LC-biotin solution, and 6.67 μl of the solution was added to the antibody solution to determine the molecular ratio of biotin and antibody 20. It adjusted to: 1. The vial containing the reaction mixture was gently shaken and mixed, put in an Eppendorf tube rack, and placed in iced water for 2 hours. The reaction mixture was placed in a Slide-A-lyzer kit (Pierce Biotechnology, Inc), and then dialyzed overnight with 0.1 M phosphate buffered saline (pH 7.2) to remove the unreacted reagent, and the contents of the dialysis membrane were recovered. Biotinylated antibodies were prepared by adjusting the total volume to 1 ml using saline.

Example  5: Nanomaterial  Preparation of Attached Antibodies

After sonicating the SA-modified FSNP solution for 15 minutes, 400 μl of this solution was added to 2 ml of the biotinylated antibody solution at a concentration of 0.1 mg / ml, and stirred for 5 minutes at a low speed of 100 rpm at 30 minute intervals for 2 hours at room temperature. I was. The supernatant was discarded by centrifugation at 10000 rpm for 20 minutes at 4 ° C, and a precipitate was taken. The precipitate taken in the above step was washed three times with 50 mM phosphate buffer solution (pH 7.4), and after each washing, the precipitate was obtained by centrifugation under the same conditions as above. The precipitate washed in this step was resuspended in 0.8 ml of 50 mM phosphate buffer (pH 7.4).

Example  6: Sample solution  Blend of labeled antibodies

Nanomaterial prepared as above by specific binding of biotin and SA to each concentration of C-reactive protein sample solution prepared by diluting stepwise using 50 mM phosphate buffer solution (pH 7.4) in Eppendorf tubes. As a mixture, an equal amount of C-reactive protein antibody solution with FSNP was added thereto, followed by mixing for 1 hour at room temperature.

Example  7: indirect competition

The Petri dishes containing the slides as biotransducers immobilizing the C-reactive protein as an antigen and minimizing non-selective protein adsorption by the BSA blocking process according to Examples 1 and 2 above were placed on a lattice spotting guide. Place it on the slide where the antigen on the slide is immobilized and mix as above and let stand for 1 hour at room temperature. Accurately add 20 µl of the mixture of the protein to be analyzed and the labeled antibody as described above, and close the lid. The reaction was carried out at room temperature for 2 hours to perform an indirect competition reaction between the antibody labeled with FSNP and the antigen immobilized on the slide and the protein to be analyzed. After the reaction, the unreacted antibody and antigen were washed three times before and after the slide using distilled water, and then, the slide was immersed in a beaker containing distilled water for 1 minute and removed.

Example  8: Fluorescence Image Analysis

The slide of Example 7 subjected to indirect competition was placed in an incubator and dried. The dried slides were analyzed by fluorescence microscopy to measure fluorescence intensity and number of fluorescence particles. FIG. 6 shows a device for fluorescence analysis, and the fluorescence spots of the FSNP-labeled antibody bound to the slide surface subjected to indirect competition were confirmed by taking fluorescence image photographs (FIG. 5 f). Figure 7 shows the calibration curve prepared by measuring the relative fluorescence from the concentration-dependent fluorescence image of the C-reactive protein using the FSNP-labeled antibody. As the CRP concentration in the sample increases, the degree of reaction with the labeling antibody is increased, the antigen immobilized on the slide The degree of fluorescence was low due to the decrease in the degree of binding to the labeled antibody and the linearity in the concentration range of very low C-reactive protein. The limit of detection for CRP measurement by immunofluorescent slide was 0.1 ~ 1.0 ng / ml. The sensitivity was very high.

Example  9: Immunofluorescence  Slide manufacturer

Disposable 3-aminopropyltrimethoxysilane or 3-mercaptopropyltrimethoxysilane-N-gamma using a transparent microscope slide having a horizontal and vertical dimension of 30 × 70 mm using the method of Examples 1-8. An immunofluorescent slide was prepared by modifying the slide surface with maleimidobutyryloxy succinimide ester and containing 1-100 μl of C-reactive protein at a concentration of 0.01-0.5 mg / ml.

Claims (3)

Manufacturing a protein chip by immobilizing the C-reactive protein on a slide, labeling an antibody that specifically binds the C-reactive protein with fluorescent nanoparticles, and labeling the C-reactive protein obtained from a sample with the fluorescent label In the step of mixing with the prepared antibody, the mixed mixture is reacted with the antigenic site fixed on the slide, and analyzed by measuring the fluorescence intensity or the number of fluorescent particles using a fluorescence microscope,
In order to immobilize the C-reactive protein on a slide,
Washing the slide by immersing the slide in a mixed solution of concentrated hydrochloric acid and methanol (HCl: MtOH = 1: 1 to 1: 2, volume ratio) for 15 to 45 minutes;
3-mercaptopropyltrimethoxysilane dissolved in one organic solvent solution selected from the group consisting of 1-3% toluene, dimethyl formamide, and acetone was prepared to prepare 3-mercaptopropyltrimethoxysilane solution. Silanizing the slide by washing the front and back sides of the slide three to four times with the same organic solvent used in the preparation;
Activating the slide surface using 1-3 mM N-gamma-maleimidobutyryloxy succinimide ester solution;
Preparing an immobilized antigen solution by dissolving C-reactive protein in a concentration of 0.01-0.5 mg / ml in a 30-70 mM phosphate buffer solution (pH 6.5-7.8);
Placing a petri dish including the slide on a spotting guide and spotting the prepared antigen solution at a dropping point of 1-100 µl; And
Method for producing an antigen-immobilized immunofluorescent slide, characterized in that the immobilized by reacting the slide prepared by the step 1-6 hours. The slide surface is modified with 3-aminopropyltrimethoxysilane or 3-mercaptopropyltrimethoxysilane-N-gamma maleimidobutyryloxy succinimide ester prepared by the method of claim 1, and 0.01-0.5 mg Immunofluorescence slide containing 1-100 μl of C-reactive protein at a concentration of / ml. A method for measuring a C-reactive protein in a sample using the immunofluorescent slide according to claim 2.

Images