KR20130021068A - Microparticle for immunoassay and method for immunoassy using the same - Google Patents

Abstract

PURPOSE: An immunoassay method using a microparticle is provided to perform simple quantitation and qualitation of a small amount of antibodies or antigens with high sensitivity. CONSTITUTION: An immunoassay method using a microparticle comprises: a step of filling a silane mixture to a replication mold with a micromold having a predetermined pattern; a step of adding C8-C16 alkane to the replication mold; a step of heating the replication mold at 30-80 deg. C, and curing microparticles; and a step of collecting and washing the cured microparticles. The silane mixture contains: a silane compound which is selected from the group consisting of 3-glycidoxypropyltrimethoxysilane(GPTMS), (3-glycidoxypropyl)-dimethyl-ethoxysilane(GPMES), (3-glycidoxypropyl) methyldiethoxysilane(GPMDES), beta-(3,4-Epoxycyclohexyl) ethyltriethoxysilane(EEES), beta-(3,4-epoxy-cyclohexyl) ethyltrimethoxysilane(EEMS), and (3-glycidoxypropyl) triethoxysilane(GPTES); acid or basic; and alcohol.

Description

Microparticles for Immunoassay and Immunoassay Using the Same

The present invention relates to microparticles for immunoassay and an immunoassay method using the same.

Immunoassay is a method for qualitatively and quantitatively detecting a small amount of a target antigen or antibody from a biomaterial containing many impurities using specific binding ability between the antigen and the antibody. Immunoassay is classified into radioimmunoassay (RIA), immunofluorescence (FIA), immunoluminescence assay, enzyme immunoassay (EIA or ELISA) according to the labeling method.

Immunofluorescence assay immobilizes a sample on a slide or the like, reacts with a fluorescence-labeled antigen (antibody), washes it, and measures fluorescence. ) Is a method of detecting. According to immunofluorescence analysis, antigens or antibodies can be detected by a relatively simple method, but have a low detection sensitivity and a narrow range of detectable concentrations. In order to solve the problem of low sensitivity, an indirect method of fluorescence labeling has been introduced in the secondary antibody, but the reaction is complicated because the reaction with the secondary antibody is additionally included.

Radioimmunoassays have been widely used in the past because they can quantitatively detect antigens or antibodies by a simple process, but they have recently been replaced by other analytical methods due to the use of radioisotopes as markers.

Enzyme immunoassay (ELISA) is the most widely used method to analyze antigen-antibody reactions with similar sensitivity to radioimmunoassay. The enzyme is used as a marker to determine the degree of discoloration of the substance resulting from the enzyme-substrate reaction. The degree of antigen-antibody reaction is analyzed. Analysis of antigen (antibody) by ELISA is usually millimeter-centimeter It is made using a micro well plate in which dozens of wells having a unit diameter are formed. Antibodies that specifically bind to specific antigens are adsorbed on the inner wall of the microwell, and more detailed analysis methods are as follows. 1) Inject the sample to be analyzed into the well of the microwell plate to which the antibody has been adsorbed. When the biomaterial is injected into the plate, other impurities do not react, and only specific antigens react with the antibody adsorbed on the surface. When the reaction is complete, a washing step is performed to remove other impurities not involved in the reaction. 2) After the washing is completed, the microwell is allowed to react with the antigen and is washed after reacting with a second antibody labeled with an enzyme. This is a previous step for visually confirming whether the antigen is bound to the antibody adsorbed to the microwell in step 1). If the antigen is bound, the second antibody is bound to the antigen. If no antigen is bound, the second antibody is removed in the wash step. 3) Quantitatively and quantitatively analyze antigens by adding and adding a substrate to the microwell, which reacts with an enzyme labeled on a second antibody, to develop color. Although the above description has been based on antigen detection, in the case of antibody detection, the basic analysis method is the same except that the antigen is adsorbed on the microwell plate instead of the antibody.

As described above, the immunoassay according to the related art has a limitation in that the surface area of the reaction is small because the antigen-antibody reaction can be performed only with the antibody (antigen) adsorbed on the bottom of the plate well. In addition, the simple process method is low or dangerous, and the high sensitivity method is composed of several steps, and the process is complicated, and the commercialized ELISA kit is expensive and inefficient in terms of economy. Therefore, in order to efficiently analyze trace amounts of substances with higher sensitivity than conventional methods, it is necessary to develop a system capable of analyzing antigen-antibody responses by a simpler process at a large reaction surface area.

It is an object of the present invention to provide microparticles capable of quantitatively / quantitatively detecting a small amount of antigen or antibody with high sensitivity by an economical and simple process.

Another object of the present invention is to provide a method for quantitatively / quantitatively detecting an antigen or an antibody using the microparticles.

The present invention for achieving the above object is (A) 3-glycidoxypropyltrimethoxysilane (GPTMS), (3-glycidoxypropyl)-dimethyl- ethoxysilane (GPMES) in a replication mold in which a micro mold having a predetermined shape and size is formed in a predetermined pattern , (3-glycidoxypropyl) methyldiethoxysilane (GPMDES), beta- (3,4-Epoxycyclohexyl) ethyltriethoxysilane (EEES), beta- (3,4-epoxycyclohexyl) ethyltrimethoxysilane (EEMS) and (3-glycidoxypropyl) triethoxysilane (GPTES) Filling at least one silane compound selected from the group consisting of a silane mixture comprising an acid or a base and an alcohol; (B) adding C8 ~ C16 alkanes to the replication mold filled with the silane mixture; (C) curing the microparticles by heating the replica mold to which C8 to C16 alkane is added at 30 to 80 ° C. in step (B); And (D) recovering and washing the cured micro particles; It relates to a microparticle for immunoassay characterized in that it is prepared to include.

Since the silane mixture, which is the alcohol solution, has hydrophilic properties, spherical particles are formed by interfacial tension as hydrophobic C8 to C16 alkanes are added. At this time, alkanes below C7 have a low boiling point, so they are easily evaporated in the following curing process, making it difficult to prepare spherical microparticles. Do. In the following examples, only the example using hexadecane, which is C16, is described, but all of the microparticles can be produced successfully by the use of alkanes of C8-C16.

In the silane mixture, alcohol is evaporated in the mixture by heating, and the silane compound is cured by the action of a catalyst to form spherical microparticles. At this time, since all the silane compounds include an epoxide group, the prepared microparticles are exposed to the surface of the epoxide group. Since the epoxide group is condensed with an amine group and all proteins have an amine group in the molecule, the antigen or antibody can be immobilized on the microparticles by a condensation reaction at a later immunoassay. Although no specific data is shown in the following examples, microparticles could be prepared similarly to GPTMS in the example using the silane compound, and protein A could be immobilized. At this time, the content of the silane compound in the silane mixture is preferably 5 ~ 30% by volume. If the content of the silane compound is too low, since there are few epoxide groups on the surface, the amount of the antigen or antibody to be immobilized is also reduced, thereby lowering the sensitivity in the future immune response. On the other hand, if the content of the silane compound is too high, the viscosity of the silane mixture increases, making it difficult to handle in the process of making particles using the micro mold, resulting in uneven particles.

The acid or base in the silane mixture serves as a catalyst in the polymerization reaction of the silane compound, and any polymer known to polymerize the silane compound may be used in the prior art. Of course, the appropriate acid or base can be selected and used. In the following examples, only hydrochloric acid is exemplified, but is not limited thereto, and an acid such as HCl, HNO 3, H 2 SO 4, HF, or a base such as ammonia, KOH, or NaOH may be used.

Step (C) is a step in which the microparticles are cured by polymerizing the silane compound and evaporating the alcohol. If the temperature is lower than 30 ° C., the polymerization reaction and the evaporation of the alcohol are too slow. Actually, the step (C) may proceed even at room temperature, but it takes more than one day to complete the reaction and is affected by other ambient conditions such as weather, so it is preferable to react at a temperature of 30 ° C. or more. The reaction is not impossible at a temperature of 80 ° C. or higher, but even if the reaction is completed at 80 ° C., since the reaction is completed within 1 hour, the reaction rate is fast enough, and if the temperature is too high, it causes inconvenience to the experimenter. ) Step is preferably heated to 30 ~ 80 ℃, more preferably 50 ~ 80 ℃.

In the silane mixture of step (B), it is possible to more firmly implement the microparticles produced by further comprising C1 ~ C4 tetraalkyl orthosilicate such as tetraethyl orthosilicate (TEOS), tetramethyl orthosilicate, tetrapropyl orthosilicate, tetrabutyl orthosilicate.

In addition, the silane mixture of step (B) may further include 2- [methoxy (polyethyleneoxy) propyl] trimethoxysilane (MPEGTMS) or mPEG-Silane (methoxyl silane PEG). PEG is known to play a role in preventing nonspecific binding in reaction with biomaterials (KSBB Journal, Vol. 24, No. 6, No. 119, 2009, 12) pp. 515-520), and MPEGTMS or mPEG-Silane are produced. By introducing a PEG group into the microparticles it serves to prevent the binding of unwanted proteins in the reaction with the antigen or antibody.

Another aspect of the invention relates to an immunoassay method using the microparticles. More specifically, the immunoassay method according to the antigen-antibody reaction of the present invention comprises the steps of: (A) reacting and washing by immersing the microparticles of claim 1 in a sample solution; (B) antigen-antibody reaction and washing by immersing the microparticles reacted in the step (A) in a solution of a labeled biomolecule that reacts specifically with the antigen or antibody to be detected; And (C) detecting the labeled biomolecules.

According to the immunoassay method of the present invention, the immunoassay can be performed with a very high sensitivity compared to the prior art, and thus it is possible to expand the scope of application of a relatively simple but low sensitivity method such as fluorescence immunoassay. In addition, in the enzyme immunoassay which can be analyzed with a relatively high sensitivity in the related art, a higher sensitivity can be analyzed and thus even a trace amount of the substance can be detected.

The sensitivity of the immunoassay with the same label is proportional to the surface area where the antigen or antibody is immobilized.

For conventional immunoassays, 96 well plates with a diameter of 8 mm and a depth of 8 mm are widely used. Even if the sample is reacted with each well of the well plate, the antigen or antibody is immobilized to the reaction volume of 400 mm ³ (well volume, 8 * π (8 mm / 2) ² ) because the antigen or antibody is immobilized only at the bottom of the well. The surface area at which the analytical reaction takes place is 50 mm ² (area, π (8 mm / 2) ² ).

On the other hand, when using the particles of about 70 ㎛ (0.07 mm) diameter prepared in the following example, by arranging the microparticles in a row in the well of the 8mm diameter can be filled with about 10,344. Considering the height of the well, microparticles can be stacked in at least 114 layers (8mm / 0.07mm) (which can be stacked higher because they are actually stacked between particles) and at least 1,179,216 microparticles in a reaction volume of 400 mm ³ Can be filled. Since the surface area of one micro particle is 0.015 mm ² (4π (0.07 mm / 2) ² ), the surface area of 1,179,216 micro particles is 17,688 mm ² (0.015 mm ² * 1,179,216 pieces), compared to the conventional method using a well plate. The reaction area is at least 350 times higher.

Since the well plate and the microparticles fluoresce by the same reaction, the fluorescence intensity of the reaction solution will be proportional to the reaction area if the reaction volume is the same. Therefore, the sensitivity of the immune response measurement of the microparticles according to an embodiment of the present invention will be at least 350 times higher than that of the immunoassay using a conventional well plate.

In the step (A), the microparticles bind to the compound having the amine group on the surface and the amine group in the sample to fix the protein in the sample. Subsequently, the samples which are not fixed to the microparticles are removed during the washing process. When (B) is specifically reacted with the antigen or antibody to be detected and immersed in the solution of the labeled biomolecule microparticles reacted in the step (A), the antigen or antibody immobilized on the microparticles and the biomolecule The biomolecule is anchored to the particles by the antigen-antibody reaction. Therefore, if there is an antigen or antibody to be detected in the sample, it will be immobilized to the microparticle in step (A), and it will bind to the labeled biomolecule in step (B), so that the labeled biomolecule is detected in step (C). Depending on whether or not immunoassay is possible. This immunoassay is not only a qualitative analysis but also a quantitative analysis by preparing and comparing a standard curve in advance.

At this time, if there is an epoxide group that has not reacted on the surface of the microparticles after the step (A), in the step (B) may be incorrect analysis results by non-specific binding to the biomolecule. To prevent this, between the steps (A) and (B), the microparticles reacted with the biomolecule of step (B) and a protein solution that does not react with the antigen or antibody to be detected in step (A) It is preferable to further comprise the step of immersing the reaction and washing. As the protein, non-reactive proteins such as bovine serum albumin or casein may be used.

The above-described analytical procedure describes only a method of directly reacting the sample with the microparticles, but the microparticles are reacted in advance with a second biomolecule that specifically reacts with the antigen or antibody to be detected before step (A) (A). More preferably, only the antigen or antibody to be detected in the step is reacted with the microparticles. The second biomolecule is to be distinguished from the biomolecule of step (B), and the biomolecule of step (B) is labeled with the specific reaction with the antigen or antibody to be detected and can be detected later. There is a difference and the biomolecule of step (B) may be the same as the second biomolecule, or may be another biomolecule. In this case, the reaction with the non-reactive protein may occur after the reaction of the second biomolecule, but before the reaction with the sample solution.

The label in step (B) may be a label by fluorescence, radioactivity or enzyme. In the following examples, the immunoassay by fluorescent labeling is exemplified, but it is obvious that the immunoassay by radioactivity or enzyme labeling can be performed by applying the same method as the prior art. In addition, in addition to the immunoassay by the direct method, indirectly reacting the biomolecule with the antigen-antibody in step (B) and then specifically reacting with the biomolecule, and reacting and analyzing the labeled second biomolecule. It may be.

In the following examples, the protein A solution was reacted with a sample solution, and the protein A was specifically reacted with the FITC-labeled IgG. This is only an example showing that the analysis can be performed successfully, but is not limited thereto.

As described above, according to the present invention, the microparticles for immunoassay can be prepared by a simple process, so that the immunoassay can be performed more economically.

In addition, according to the immunoassay method of the present invention using the microparticles, it is possible to quantitatively and qualitatively detect a small amount of antigen or antibody with high sensitivity.

1 is a micrograph of the microparticles prepared by one embodiment of the present invention.
Figure 2 is a schematic diagram showing a manufacturing process of the microparticles according to the present invention.
Figure 3 is a schematic diagram showing an immunoassay process according to an embodiment of the present invention.
4 and 5 are fluorescence micrographs showing the results of fluorescence immunoassay according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these embodiments are merely examples for explaining the content and scope of the technical idea of the present invention, and thus the technical scope of the present invention is not limited or changed. In addition, it will be apparent to those skilled in the art that various modifications and changes can be made within the scope of the present invention based on these examples.

Example 1 Preparation of Micro Particles

1) Fabrication of micro mold

As shown in FIG. 1, the micro mold was designed such that hundreds of circular micro wells having a width and length of 150 μm were arranged at regular intervals.

The negative photosensitive agent (SU-8, Microchem Co., USA) was evenly applied on the silicon wafer, and then spin-coated at 1,000 rpm to raise the 100 μm high photosensitive agent. Width and Length on Silicon Wafers UV was irradiated through a mask having a microwell shape of 150 μm to prepare a master mold having a shape opposite to that of the channel. Then, PDMS (Sylgard 184; Dow Corning, Midland, MI) was poured into the prepared master mold and then 4 hours at 65 ° C. Curing to produce a PDMS mold having a microchannel of the desired shape.

2) Preparation of Micro Particles

The schematic diagram of the manufacturing process of a microparticle is shown in FIG.

More specifically, 3-glycidoxypropyltrimethoxysilane (GPTMS), 2- [methoxy (polyethyleneoxy) propyl] trimethoxysilane (MPEGTMS), tetraethyl orthosilicate (TEOS), HCl (1N) and ethanol in the microwells of the micro molds prepared in 1). ) (99.9%) was mixed in a volume ratio of 3: 1: 3: 3: 16 in a sol state silane mixture using a micro pipette tip and excess mixture was removed.

Hexadecane was sprayed onto the mixture filled micro mold as shown in FIG. 2. At this time, the interaction between the hexadecane and the PDMS micro mold having hydrophobic properties causes the hexadecane to enter the micro mold wall, and the hydrophilic mixture has a spherical shape due to the interfacial tension with the hydrophobic hexadecane.

The micro mold was left in the oven at 65 ° C. for one day while sprinkled with hexadecane to obtain spherical micro particles. 1 is a micrograph showing spherical uniform microparticles produced in a microwell.

Example 2 Immunoassay Using Microparticles

1) Reaction of Microparticles with Protein A

The microparticles prepared in 2) of Example 1 were collected and washed with PBS buffer of pH 7. The washed microparticles were immersed in a solution of Protein A (Sigma-aldrich) diluted to 5 μg / ml concentration using PHS buffer, allowed to react at room temperature for 30 minutes, and centrifuged to remove the supernatant. After removing the supernatant, PBS buffer was added, the microparticles were suspended, and then washed four times by centrifugation to remove the supernatant.

After washing, the protein A-coupled microparticles were immersed in a BSA (Sigma-aldrich) solution diluted in PBS buffer at 1% (w / w) and reacted with a rotator at room temperature for 30 minutes using the same method as described above. Washed again with PBS buffer.

2) Reaction with Fluorescently Labeled IgG

Protein A was detected using an anti-Mouse IgG-FITC antibody labeled with fluorescent FITC that specifically reacts with protein A.

More specifically, the anti-mouse IgG-FITC (Fluorescein isothiocyanate) obtained by diluting the protein A-implemented particles in PBS buffer at a concentration of 3 µg / ml in 1). It was immersed in the solution and reacted at room temperature for 30 minutes. After the reaction was completed, the microparticles were washed by the same method as in Example 1 using PBS buffer to remove the Anti-Mouse IgG-FITC remaining without reaction. 3 shows a schematic of the overall reaction.

The washed micro particles were observed using a fluorescence microscope (NIKON, TE2000, Japan) and the results are shown in FIG. 4. As can be seen in Figure 4, protein A could be detected from the FITC signal evenly appeared on the particle surface.

FIG. 5 is a fluorescence micrograph showing the results of immunoassay by microparticles prepared by another embodiment in which the ethanol content is 30% (v / v) in the mixture used to prepare microbeads. Except for a slight increase, it was confirmed that the FITC signal was evenly detected on the particle surface as in FIG. 4.

Claims (9)

(A) 3-glycidoxypropyltrimethoxysilane (GPTMS), (3-glycidoxypropyl) -dimethyl-ethoxysilane (GPMES), (3-glycidoxypropyl) methyldiethoxysilane (GPMDES) on a replication mold in which a micro mold having a predetermined shape and size is formed in a predetermined pattern. at least one silane compound selected from the group consisting of beta- (3,4-Epoxycyclohexyl) ethyltriethoxysilane (EEES), beta- (3,4-epoxy-cyclohexyl) ethyltrimethoxysilane (EEMS) and (3-glycidoxypropyl) triethoxysilane (GPTES) Filling a silane mixture comprising an acid or a base and an alcohol;
(B) adding C8 ~ C16 alkanes to the replication mold filled with the silane mixture;
(C) curing the microparticles by heating the replica mold to which C8 to C16 alkane is added at 30 to 80 ° C. in step (B); And
(D) recovering and washing the cured micro particles;
Immunoassay microparticles, characterized in that is prepared, including
The method of claim 1,
The content of the silane compound in the silane mixture of the step (B) is an immunoassay microparticles, characterized in that 5 to 30% by volume.
The method of claim 1,
The silane mixture of step (B) is an immunoassay microparticle, further comprising at least one compound selected from the group consisting of tetraethyl orthosilicate (TEOS), tetramethyl orthosilicate, tetrapropyl orthosilicate, tetrabutyl orthosilicate.
The method according to claim 1 or 3,
The silane mixture of step (B) is an immunoassay microparticles, further comprising 2- [methoxy (polyethyleneoxy) propyl] trimethoxysilane (MPEGTMS) or mPEG-Silane (methoxyl silane PEG).
In the immunoassay method by the antigen-antibody reaction,
(A) reacting and washing the microparticles of claim 1 by immersing it in a sample solution;
(B) antigen-antibody reaction and washing by immersing the microparticles reacted in the step (A) in a solution of a labeled biomolecule that reacts specifically with the antigen or antibody to be detected; And
(C) detecting the labeled biomolecules;
Immunoassay method characterized in that it comprises a.
The method of claim 5, wherein
Between the steps (A) and (B),
Reacting and washing the biomolecules of step (B) and the microparticles reacted in step (A) on a protein solution that does not react with the antigen or antibody to be detected;
Immunoassay method characterized in that it further comprises.
The method of claim 5, wherein
Before step (A),
(a) immersing and washing the microparticles in a second biomolecule solution that specifically reacts with the antigen or antibody to be detected;
Immunoassay method characterized in that it further comprises.
The method of claim 7, wherein
Between the steps (a) and (A),
Reacting and washing the biomolecules of step (B) and the microparticles reacted in step (A) on a protein solution that does not react with the antigen or antibody to be detected;
Immunoassay method characterized in that it further comprises.
The method according to any one of claims 5 to 8,
The label in step (B) is an immunoassay method characterized in that the label by fluorescence, radioactivity or enzyme.

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