WO2016133321A1

WO2016133321A1 - Microparticle for bioassay and method for manufacturing same

Info

Publication number: WO2016133321A1
Application number: PCT/KR2016/001493
Authority: WO
Inventors: 김상경; 정승원
Original assignee: 한국과학기술연구원
Priority date: 2015-02-16
Filing date: 2016-02-15
Publication date: 2016-08-25
Also published as: KR20160100689A; KR101758145B1

Abstract

The present invention relates to a microparticle for a bioassay and a method for manufacturing the same, using mask patterning, spotting and imprinting processes, wherein the bioassay has a probe area and an encoder area, which are not separated and overlappingly formed in the same substrate. According to the present invention, real-time PCR can be applied, and accurate quantification and recognition with respect to genetic information analysis is thus possible.

Description

Micro particles for bio assay and method for preparing the same

The present specification relates to microparticles for a bioassay and a method of manufacturing the same.

Molecular diagnostic systems are the next generation of convergence technologies across science and technology, including electronics, physics, chemistry, biology, materials engineering, IT, and NT, and are emerging as innovative technologies for the diagnosis and treatment of diseases.

In particular, the biochip, which is one of the representative fields of molecular diagnostic systems, attaches high density biomolecule probes such as DNA and proteins onto substrates such as glass, silicon, and polymers, and thus targets in the probes and samples. It detects the hybridization with a substance and is usefully used to analyze a very small amount of sample at a high speed.

As a result, gene information such as gene expression and response patterns, gene defects, and protein distribution can be analyzed to ultimately be used for diagnosis and treatment of human diseases, as well as for new drug development and reagent diagnosis.

Such biochips may be divided into DNA chips or protein chips according to the type of probe, and may be divided into microarray chips attached to a solid substrate and microfluidic chips attached to a microfluidic channel according to the attachment form of the probe.

In particular, by using the biochip to quantify DNA, RNA, protein and the like at the same time, reducing the analysis time, obtaining a variety of information at a low cost, that is, research on multiplexing analysis technology is active. In addition, there is a growing interest in cost reduction through analysis using a trace amount sample.

In general, when detecting hybridization between the probe and a target substance in a sample, in order to detect whether the hybridization is selective for the detection molecule, a fluorescent substance or radioactive material for obtaining a signal that can be directly measured by the researchers can be detected. Labeling materials such as substances and magnetic particles are used.

However, since such labeling materials are generally expensive, researchers' interest in reducing costs through trace analysis has focused on multiple analysis techniques that can obtain various information while using trace samples.

One of such multiple analysis techniques is by positional microarrays, and forms genetic probes or code regions specific to X and Y coordinates to analyze genetic information, which is an advantage for ultra high density analysis. have.

In addition, as a multi-analysis technique, by a suspension array (suspension array), a color coding method by a variety of dyes (fluorescent material) each expressing different emission colors (fluorescent material) a large amount of a large amount of The advantage is that the sample can be processed.

However, the planar microarray technique and the color coding scheme have poor reproducibility and are not easy to perform real-time PCR analysis, and thus, it is difficult to accurately recognize and quantify accurate genetic information. In addition, the detection information on the genetic information is inaccurate due to the interference and perturbation between the detection information according to the detection signal measuring method according to the labeling material, the direction and sensitivity of the detector, and the like. Is lacking.

In addition, multi-analysis technology provides a technique for particle-based diagnosis, and the use of small particles can increase the response surface area to increase responsiveness. There is an advantage that can proceed with various assays.

With this particle-based diagnostic technology, Korean Patent Application Publication No. 10-2009-0091117, "Multifunctional Encoded Particles for High Performance Analysis," flows fluorescently labeled monomers along microfluidic channels, and other adjacent ones. After flowing the probe-lead off monomers along the microfluidic channel, they are polymerized and exposed to the ultraviolet light transmitted through the photomask to form particles having the cord region and the probe region.

However, in the above technique, since the probe region and the code region are separated, it is not easy to distinguish between recognition and quantification, and a photomask must be used. Therefore, resolution is limited and resolution is limited, so that the formation of a precise code region is possible. There is a disadvantage that is not easy.

In addition, the above method forms particles intermittently retaining the probe region and the code region by the photomask along the microfluidic channel, so that the loss of material between particles generated by the photolithography process along the microfluidic channel is reduced. There is an uneconomic problem.

In addition, the particles produced by the above method is impossible to implement the real-time PCR, there is a disadvantage that cannot accurately quantify and recognize the genetic information.

The present invention is to solve the above problems, it is possible to manufacture the microparticles for bioassay using the mask patterning process and the spotting and imprinting process, in particular, since the probe region and the encoder region is not separated, real-time PCR application It is an object of the present invention to provide microparticles and methods for their preparation for this possible bioassay.

In order to achieve the above object, an embodiment of the present invention has a flat three-dimensional shape with a flat bottom surface, and a probe region for quantitatively detecting bio-assay information and an encoder pattern formed on the bottom surface. Provided is a microparticle for a bioassay, comprising an encoder region for recognizing a kind of bio-assay information, wherein the probe region and the encoder region are superimposed within a substrate.

In addition, an embodiment of the present invention is to form a photoresist layer on a substrate, a first step of manufacturing a first mold having an embossed encoder pattern made of a photoresist by a mask patterning process, and on the first mold Forming a imprint resin layer and forming a master mold formed of an imprint resin by an imprinting process to form a negative encoder pattern; spotting a solution for bioassay on the negative encoder pattern region formed in the master mold ( spotting to form droplets and curing the droplets formed on the master mold, and then separating them on the master mold to form microparticles having an encoder pattern formed thereon. It provides a method for producing micro particles for a bio assay comprising.

The manufacturing method according to an embodiment of the present invention uses a mask patterning process, a spotting and an imprinting process, to form microparticles for a bioassay in which a probe region and an encoder region are not separated in the same substrate and overlapped. Can provide. The microparticles can be applied to real-time PCR has the effect of accurate quantification and recognition for genetic information analysis.

In addition, the mask patterning process ensures the precision of the encoder pattern (5 µm or less), and enables encoding of more than 10 ⁹ , enabling detection of various information of ultra-fine samples, spotting only on the negative encoder pattern formed on the master molder. Since the droplets are formed by the process, waste of the probe material can be reduced, thereby reducing the cost.

Micro particles according to an embodiment of the present invention is formed in a flat bottom surface, the patterned encoder region is formed to always be horizontal to the bottom surface, it is possible to fix the encoder in a certain direction. As a result, the accuracy of the detection information can be guaranteed, and even with a very small amount of sample, high sensitivity can be detected, thereby improving the reproducibility of the experiment.

In addition, one embodiment of the present invention can detect whether or not hybridization with the target material from the contrast and the change in the concentration of the encoder pattern even with a normal CCD camera, regardless of the use of the labeling material, and easy to experiment, There is an effect that can reduce the cost for using the labeling material.

1 is a schematic diagram of a method for preparing microparticles for bioassay according to an embodiment of the present invention.

Figure 2 is a view showing a side photograph of the droplet formed on the master mold prepared according to an embodiment of the present invention.

Figure 3 is a view showing a photograph (a), a photograph (b), (c), (d) of the micro-particles for the master mold prepared according to an embodiment of the present invention.

Figure 4 is a view showing a picture of the microparticles according to one embodiment of the present invention.

FIG. 5 is a diagram illustrating a BF image (a) after PCR, and a fluorescence image (b) before and after PCR for microparticles according to an embodiment of the present invention.

6 is a view showing a schematic diagram ((a) cross section, (b) bottom view) of the microparticles constituting the composite structure prepared according to an embodiment of the present invention.

The present invention relates to microparticles for multiplexing bioassays that can reduce analysis time and obtain a variety of information at low cost by simultaneously quantifying and recognizing bioassays, particularly DNA, RNA, proteins and the like. The present invention aims to provide microparticles for a bioassay in which a probe region and an encoder region are not separated in the same substrate and overlapped using a mask patterning process and an imprinting process.

According to the present invention, it is possible to prepare microparticles for bioassay in a significantly simpler method than the prior art. In particular, since the probe region and the encoder region are not separated, real-time PCR can be applied, and thus there is an advantage in that accurate quantification and recognition for genetic information analysis is possible.

Hereinafter, with reference to the accompanying drawings will be described in detail for the present invention.

Figure 1 shows a schematic diagram of a method for producing microparticles for bioassay according to an embodiment of the present invention. Figure 2 shows a side photograph of the droplet formed on the master mold prepared according to an embodiment of the present invention. Figure 3 shows a photograph (a) for the master mold prepared in accordance with an embodiment of the present invention, a photograph (b) for the droplets, a photograph (c), (d) for the microparticles. 4 is a photograph of microparticles according to various embodiments of the present disclosure. FIG. 5 shows a BF image (a) after PCR, a fluorescence image (b) before and after PCR for microparticles according to an embodiment of the present invention. Figure 6 shows a schematic diagram ((a) cross-sectional view, (b) bottom view) of the microparticles constituting the composite structure prepared according to an embodiment of the present invention.

As shown in FIG. 1, in the method of manufacturing microparticles for bioassay according to an embodiment of the present invention, a photoresist layer is formed on a substrate, and an embossed encoder pattern made of the photoresist is formed by a mask patterning process. A first step of manufacturing the formed first mold; A second step of forming an imprint resin layer on the first mold and fabricating a master mold having imprint resin patterns formed of imprint resin by an imprinting process; A third step of spotting a solution for bioassay on a negative encoder pattern formed in the master mold to form droplets; And curing the droplets formed on the master mold, and then separating them on the master mold to form micro particles having an encoder pattern formed on a bottom surface thereof.

In the present specification, the microparticles are used in a device for detecting and analyzing a target nucleic acid in a bioassay, and the micro size of nano ( ^10-6 ), micro ( ^10-3 ), millimeter ( ^10-2 ), etc. The concept includes all of the particles. In one embodiment, the microparticle includes a probe region for quantitatively detecting information about a target nucleic acid or a fixed primer reacting with the target nucleic acid, ie, bioassay information, and an encoder region for recognizing a kind thereof.

In one embodiment, the present invention has a flat three-dimensional shape with a flat bottom surface, and the bio-assay is formed by a probe region for quantitatively detecting bio-assay information and an encoder pattern formed on the bottom surface. assay) may include a microparticle for a bioassay, comprising an encoder region for recognizing a kind of information, wherein the probe region and the encoder region overlap each other in a substrate.

The microparticles according to one embodiment may be hemispherical or semi-elliptic. In addition, as an embodiment, an opaque metal layer or an array of photoresist layers may be formed on the encoder pattern of the microparticles.

In an embodiment, the microparticle may further include a probe region or an encoder region of another kind, to form a complex structure.

In addition, the microparticles according to an embodiment may further include pores in the particles.

In the method of manufacturing microparticles for bioassay according to an embodiment of the present invention, a photoresist layer is formed on a substrate and a first mold having an embossed encoder pattern made of a photoresist is formed by a mask patterning process. It may include (step 1, (a)).

In one embodiment, the substrate may be any flat substrate such as metal, silicon, glass, and polymer, and the area of the substrate may be adjusted according to the size and number of encoder patterns to be formed.

According to an embodiment, the photoresist layer may be coated on the substrate to form an encoder pattern, and the thickness of the photoresist layer may be adjusted in consideration of the size of the recess of the encoder pattern.

In one embodiment, the photoresist layer may be any resin as long as it is a resin that can be used in a mask patterning process by photolithography. For example, a radiation curable epoxy acrylate, urethane acrylate, polyester acrylate, and a substituent having a refractive index adjusted may be used. More specific examples may use SU-6, SU-7, SU-8, and the like.

In one embodiment, the photoresist layer is formed on a substrate, and then selectively exposed using a mask on which an encoder pattern is drawn in advance, and then a portion of the photoresist that is not cured by the exposure process is removed through a developing process, and the cured Only the photoresist may form an embossed encoder pattern.

In the present specification, the embossed encoder pattern means a pattern corresponding to the final encoder pattern. Since the final encoder pattern is formed by the master mold to be described later, the one having the opposite image is referred to as an embossed encoder pattern.

According to the exemplary embodiments, the first mold having the embossed encoder pattern made of the photoresist manufactured by the mask patterning process is guaranteed with precision (5 μm or less) of the encoder pattern, and capable of encoding more than 10 ⁹ . In the process to be described later, since droplets are formed by the spotting process only on the intaglio encoder pattern formed on the master molder, waste of the probe material may be reduced, thereby reducing costs.

An embodiment of the present invention may include forming an imprint resin layer on the first mold, and manufacturing a master mold formed of an imprint resin by an imprinting process to form a negative encoder pattern (second step, ( b) step).

According to an embodiment, the imprint resin layer may be imprinted on the first mold and compressed by using a roller or the like so that the negative encoder pattern of the first mold is imprinted on the imprint resin layer.

According to an embodiment, after imprinting, the imprint resin layer may be photocured to fix the pattern made of the imprint resin. When peeling this off, a master mold including the imprint resin is formed to form an engraved encoder pattern (a pattern corresponding to the embossed encoder pattern of the first mold).

For this photocuring, as an example, a photocurable resin may be used for the imprint resin layer. The photocurable resin used in the imprint resin layer may be the same as the resin used in the photosensitive resin layer, or may be a pre-polymer in which a polydimethylsiloxane (PDMS) solution and a curing agent are mixed.

According to one or more exemplary embodiments, the method may further include: hydrophilizing a surface of a negative encoder pattern region in the master mold after the second step; Alternatively, the method may further include hydrophobic treatment of the surface of the region except the negative encoder pattern region in the master mold.

According to the exemplary embodiment, the hydrophilic treatment is performed on the negative encoder pattern region or the non-spotted negative encoder pattern region is formed by spotting a bioassay solution to be described later when the hydrophobic treatment is performed. It is possible to set it correctly. In this case, droplets can be easily formed without using a spotting device.

In an embodiment, the hydrophilic treatment may include an oxide layer including at least one of oxide-based materials such as SOG, TEOS, LTO, silicon oxide, titanium oxide, and aluminum oxide using a mask on the negative encoder pattern region. It may be. In an embodiment, the hydrophobic treatment may be implemented by any one of deposition of hydrophobic material, coating of hydrophobic material, silanizing treatment, and plasma treatment using a mask in a region other than the negative encoder pattern region. .

Next, an embodiment of the present invention may include spotting a solution for bioassay on a negative encoder pattern formed in the master mold to form droplets (step 3, (c )step).

According to the exemplary embodiment, droplets may be formed by spotting the bioassay solution only on a region where the negative encoder pattern is formed in the master mold, thereby reducing waste of the bioassay solution (ie, probe material).

In one embodiment, the bioassay solution may be used without limitation as long as it is formed of a transparent material capable of reading an encoder pattern from the outside. For example, the polymer may include one or more polymers of polyethylene glycol-diacrylate (PEG-DA), polyacrylamide (PA), and agarose.

In addition, in one embodiment, the bioassay solution may be a polymerase chain reaction (PCR) primer other than the polymer, a cyanine-based dye that provides quantitative information of a nucleic acid that is amplified by complementarily binding to a target nucleic acid, EtBr. , TaqMan ^TM It may further include a fluorescent marker, such as a probe, oligo-nucleotide probe (Molecular Beacon probe) and the like.

In one embodiment of the present invention, the bioassay solution may act as a matrix of the microparticles according to the present invention to change the type of the substrate itself or to form primers or probes included in the substrate. This makes it possible to detect information on the kind, size or amount of different kinds of target nucleic acids or immobilized primers reacting with them at the same time.

As an embodiment of the present invention, since the droplet is spotted on the master mold, the bottom surface may have a flat three-dimensional shape. Specifically, it may be hemispherical or semi-elliptic.

In one embodiment, the spotting may use a spotting device capable of fine control with a solenoid valve or the like, and may control the size of the droplet by controlling the spotting time and flow rate.

Next, an embodiment of the present invention may include curing the droplets formed on the master mold, and then separating them on the master mold to form micro particles having an encoder pattern formed on a bottom surface thereof. (Step 4).

As an example, a sacrificial layer may be separately formed therebetween to separate the cured droplets on the master mold. When the droplets cured in the peel-off method are separated from the master mold, micro particles having a final encoder pattern corresponding to the negative encoder pattern are formed on the bottom surface.

Since the encoder pattern according to an embodiment is generated by a mask patterning process, precision (5 μm or less) of the encoder pattern is guaranteed, and more than 10 ⁹ encodings are possible. There is an advantage that it is possible to detect a variety of information about the ultra-fine sample.

Since the micro particles according to an embodiment of the present invention are dropped into a droplet form by spotting and hardened, the bottom surface may have a three-dimensional shape with a flat shape. For example, the microparticles may be hemispherical or semi-elliptic. On the flat bottom surface, the encoder pattern by the master mold may be imprinted.

According to an embodiment, the microparticles may maintain a state in which the center of gravity is always horizontal. That is, the bottom surface on which the encoder pattern is formed may be arranged to be in contact with the ground or vice versa, so that the encoder pattern can always be recognized horizontally with respect to the ground.

Therefore, in one embodiment of the present invention, since the detector for the labeling substance is always used in a direction perpendicular to the microparticles, it is possible to ensure the accuracy of the detection information and to detect the high sensitivity even when using a very small amount of sample. To improve the reproducibility of the experiment.

In addition, according to an embodiment of the present invention, the microparticles of the present invention may include a probe region for quantitatively detecting bioassay information and an encoder region for recognizing the type of bioassay information by an encoder pattern formed on the bottom surface. Since it is not separated but formed in a superimposed manner, there is an advantage in that quantification and recognition can be simultaneously detected by recognition of an encoder pattern.

That is, according to an embodiment of the present invention, it is possible to manufacture microparticles for bioassay in a simpler and more economical manner than in the prior art by using a mask patterning process and a spotting and imprinting process. In particular, the microparticles according to the embodiment of the present invention are not separated from the probe region and the encoder region, and thus, real-time PCR can be applied, so that the reaction in the exponential phase can be confirmed in real time with a monitor. The amount of can be accurately quantified.

An embodiment of the present invention may further include forming an array of an opaque metal layer or a photoresist layer on the encoder pattern of the microparticles of the fourth step. In this case, since detection of bioassay information in various manners is possible, not only the detection information by fluorescence can be obtained, but also the detection information by a general bright field can be easily obtained.

Specifically, as an embodiment, the photoresist layer may include an epoxy-based negative photoresist, for example, SU-8. As an example, the metal layer may include at least one of a conductive metal and a conductive polymer. The metal layer may include a magnetic material. The magnetic material may include, for example, one or more of iron, nickel, cobalt, dynabeads, and bacterial magnetic particles.

In addition, as an embodiment, the roughness processing may be further performed on the encoder pattern of the microparticles of the fourth step, so that light scattering occurs more easily during the detection of fluorescence, so that more accurate information may be detected. have.

One embodiment of the present invention may further comprise the step of further forming pores in the microparticles of the fourth step. If more pores are formed, not only the surface of the microparticles is used for amplification and detection, but also the inside can be used to maximize the reactivity.

In one embodiment, the method for forming pores in the microparticles may include mixing a porogen with a solution for the bioassay and removing the pore copolymer after curing the droplets.

For example, the pore copolymer may be polyethylene glycol (PEG), and the pore may include a fluorescent marker that provides quantitative information of the nucleic acid to be amplified.

Another embodiment of the present invention may provide a method of forming another droplet on the droplet after curing the droplet formed on the master molder. In the present specification, as illustrated in FIG. 6, a droplet contained inside is referred to as a first droplet, and a droplet formed outside is referred to as a second droplet.

According to an embodiment of the present disclosure, after curing the first droplet formed on the master molder, a solution for bioassay different from the bioassay of step (c) is spotted on the first droplet and then placed therein. And forming a second droplet to include the first droplet (step (d)).

And, after curing the second droplet as an embodiment, separating the composite structure consisting of the first droplet and the second droplet on the master mold, to form a composite micro particles having an encoder pattern formed on the bottom surface (Step (e)).

The method may be useful when simultaneously detecting information on the type, size, or amount of two or more types of target nucleic acids or fixed primers reacting with one microparticle. In addition, the label material may be different between the first droplet and the second droplet, an encoder pattern may be formed only on the first droplet, or different encoder patterns may be formed on the first droplet and the second droplet, respectively. If necessary, the third droplet can be implemented.

The method of reading the encoder pattern of the microparticles manufactured as described above, that is, reading the information of the encoder is not limited, but a CCD camera or the like may be used as an embodiment. In addition, as an embodiment, the information of the encoder may be read individually for each microparticle, or the encoder information of each of the plurality of microparticles arranged in the array may be simultaneously read.

In addition, one embodiment of the present invention is a bio-test material, such as primers, target nucleic acid and the like in the solution and the microparticles according to an embodiment of the present invention after the bio assay, and arranged in an array (array) The present invention may provide a method of analyzing a bioassay result by reading an encoder pattern included in each micro particle.

Alternatively, after arranging bio experiments such as primers and target nucleic acids and microparticles according to an embodiment of the present invention, the microparticles arranged in the array are bioassayed, and the encoder pattern included in each microparticle. It can provide a method of analyzing the bioassay results by reading the.

Hereinafter, the present invention will be described in more detail with reference to an embodiment of the present invention. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

First, SU-8 resin (SU-8 3025, Microchem Co., Ltd.) was poured as a photoresist on a silicon substrate and coated under conditions of 500 rpm for 10 seconds and 200 rpm for 30 seconds.

Then, after 15 minutes soft bake on a hot plate at 95 ° C., selective 250 mJ UV exposure was performed using a mask on which an encoder pattern was drawn in advance. After exposure, bake for 5 minutes on a hot plate at 65 ° C for 1 minute and then remove the unexposed SU-8 using a SU-8 developer (Microchem). Work was performed. Then, baking was performed for 5 minutes on a hot plate at 200 ° C.

As a result, a first mold having a relief encoder pattern formed on a silicon substrate was fabricated.

An imprint resin layer was formed on the first mold on which the relief encoder pattern was formed. As the imprint resin, a pre-polymer in which a polydimethylsiloxane (PDMS) solution and a curing agent (trade name: SYLGARD® 184, manufactured by Dow Hitech Silicon) in a 10: 1 mixture was poured onto the first mold.

The gas was removed from the vacuum chamber for 60 minutes, then cured in an oven at 80 ° C. for 90 minutes, and separated from the first mold to prepare a master mold having a negative encoder pattern made of PDMS.

The droplets were formed by spotting a solution for bioassay on the negative encoder pattern formed on the master molder.

The solution for bioassay was 20% by volume PEG700DA (manufactured by Sigma Aldrich), 40% by volume polyethylene glycol 600 (PEG 600), 5% by volume Darocur 1173 (manufactured by Sigma Aldrich), 0.15% by volume as a photoinitiator. 35 volumes of 3X TE buffer containing tween-20 was used. Then, 5% by volume of EtBr (Interchelator binding to double-stranded nucleic acid and fluorescence with amplification) was further mixed with respect to the total volume of the bioassay solution.

The bioassay solution was spotted only on the negative encoder pattern region formed in the master molder by adjusting the open time of the spotting device to form droplets including the negative encoder pattern region therein.

The droplets are cured by irradiation with ultraviolet (UV) light for 6 minutes at 6 to 8 mW / cm ² , and then released on the master molder to release the microparticles having an encoder pattern formed on the bottom surface thereof. Formed.

Figure 2 shows a side photograph of the droplet formed on the master mold prepared according to an embodiment of the present invention, it can be seen that the shape of the droplet by spotting shows a flat hemispherical bottom surface.

Figure 3 (a) shows a photograph of a master mold manufactured according to an embodiment of the present invention. Figure 3 (b), (c) shows a photograph of the droplet formed by spotting the solution for bio assay on the master mold. Figure 3 (d) shows a photograph of the microparticles prepared by curing the droplets, separating the droplets from the master mold. 3 (a) to (c), it was confirmed that a high-precision encoder pattern was formed by a mask patterning process and an imprinting process.

Figure 4 shows a photograph of the microparticles formed with various encoder patterns, according to one embodiment of the present invention. According to one embodiment, the microparticles were formed in a hemispherical shape with a flat bottom surface, so that it was possible to read encoder patterns of all microparticles in a vertically upward direction of the microparticles.

Figure 5 (a) is a bright field (BF) image (a) after PCR for the microparticles according to an embodiment of the present invention. This was taken with a CCD camera, and it was confirmed that the encoder pattern could be read.

5 (b) and (c) show fluorescence images before and after PCR. As the PCR cycle increases, the color of fluorescence becomes brighter and the encoder pattern becomes clearer.

Claims

The bottom surface has a flat three-dimensional shape,

A probe region for quantitatively detecting bio-assay information; And

An encoder region for recognizing a type of bio-assay information by an encoder pattern formed on the bottom surface,

Wherein said probe region and said encoder region are superimposed within a substrate.
The microparticle of claim 1, wherein the microparticles are hemispherical or semi-elliptic.
The microparticle of claim 1, wherein an array of opaque metal layers or photoresist layers is formed on the encoder pattern of the microparticles.
The microparticle of claim 1, wherein the microparticle further includes a probe region or an encoder region of another kind, to form a complex structure.
The microparticle of claim 1, further comprising pores in the microparticle.
Forming a photoresist layer on the substrate and manufacturing a first mold having an embossed encoder pattern made of the photoresist by a mask patterning process;

A second step of forming an imprint resin layer on the first mold and fabricating a master mold having imprint resin patterns formed of imprint resin by an imprinting process;

Spotting a solution for bioassay on a negative encoder pattern region formed in the master mold to form droplets; And

Curing the droplets formed on the master mold and then separating them on the master mold to form micro particles having an encoder pattern formed on a bottom surface thereof;

Method for producing micro particles for a bio assay comprising a.
The method of claim 6, wherein after the second step,

Hydrophilizing the surface of the negative encoder pattern region in the master mold; or

Hydrophobic treatment of the surface of the master mold except for the negative encoder pattern region;

Method for producing micro particles for a bio assay further comprising.
The method of claim 6, wherein the droplet of the third step has a flat three-dimensional shape with a flat bottom surface.
7. The method of claim 6, wherein the droplet of the third step is hemispherical or semi-elliptical.
The method of claim 6, further comprising forming pores in the microparticles of the fourth step.
7. The method of claim 6, further comprising forming an array of opaque metal layers or photoresist layers on the encoder pattern of the microparticles of the fourth step.
Forming a photoresist layer on the substrate and manufacturing a first mold having an embossed encoder pattern made of the photoresist by a mask patterning process;

(B) forming an imprint resin layer on the first mold and fabricating a master mold having imprint resin patterns formed of imprint resin by an imprinting process;

(C) spotting a solution for bioassay on the negative encoder pattern region formed in the master mold to form a first droplet;

After curing the first liquid droplets formed on the master mold, a bioassay solution different from the bioassay solution of step (c) is spotted on the first droplets so that the first droplets are contained therein. (D) forming two droplets; And

After curing the second droplet, separating the composite structure consisting of the first droplet and the second droplet on the master mold to form a composite microparticle having an encoder pattern formed on the bottom surface (e) Method for preparing micro particles for sage.