KR101872091B1 - Device for manufacturing vertically encoded hydrogel microparticles for multiplexed detection of target biological molecules, method for preparing the same, and method for preparing the microparticles using the device - Google Patents

Abstract

The present invention relates to an apparatus for producing multilayer-encoded hydrogel particles for target biomolecule multiplex detection, a method for producing the same, and a method for producing the hydrated gel using the same, and more particularly, to a biomolecule A first channel through which the specifically bound material and the polymer precursor fluid communicate; A second channel vertically stacked above or below the second channel and communicating with a fluorescent dye for fluorescence analysis of a polymer precursor and a substance specifically binding to the biomolecule; And pressure means for communicating with the first channel and the second channel and for controlling the flow of fluid in the first channel and the second channel, the apparatus comprising: an apparatus for producing a multilayered coded hydrogel particle for target biomolecule multiplex detection, And a method for producing the hydrogel particles using the same.
According to the present invention, it is possible to increase the production amount of microparticles, improve quality by lowering the degree of defects of microparticles without being influenced by external stimuli, and to encode a coded portion using a fluorescent dye instead of a geometric pattern, An apparatus for producing multilayer-encoded hydrogel particles for target biomolecule multiplex detection capable of remarkably lowering the rate of occurrence of errors at a time and capable of easily increasing an encoding region and a code region through control of wavelengths of laminated water and fluorescent dyes, And a method for producing the hydrogel particles using the same.

Description

TECHNICAL FIELD [0001] The present invention relates to a device for manufacturing multilayer-encoded hydrogel particles for target biomolecule multiplex detection, a method for producing the same, and a method for manufacturing the hydrogel particles using the same. method for preparing the microparticles using the device}

The present invention relates to an apparatus for producing multilayer-encoded hydrogel particles for target biomolecule multiplex detection, a method for producing the same, and a method for producing the hydrogel particles using the same.

Techniques using barcoded hydrogel microparticles are capable of providing a high sensitivity and processing speed and are capable of detecting a variety of biomolecules such as RNA or DNA from biological samples. It is in the limelight. In particular, bar-coded hydrogel particles have the ability to detect biomolecules with very high detection sensitivity at the level of theatomol, the ability to detect multiple molecules through thousands of code implementations, .

Generally, the bar-coded hydrogel microparticles are produced by a technique called Stop Flow Lithography (SFL). The fluid lithography process is a technique for synthesizing particles by irradiating ultraviolet rays onto a microfluid. So that they can flow without being mixed with each other in one microchannel. Therefore, it is possible to prevent the flow of various kinds of fluids in one microchannel.

In the case of the standard SFL process, the precursor fluid flowing into the microchannel includes a monomer having an acrylate group and a polymerization initiator, and then irradiates ultraviolet light to initiate a radical polymerization reaction to synthesize microparticles. When ultraviolet rays are irradiated using a mask, it is possible to synthesize microparticles having the shape of the mask. The prepared 3D hydrogels networks have a very good capturing ability for nucleic acid molecules as compared to microarrays or other surface immobilization techniques (Non-Patent Documents 1 to 3 and Patent Document 1).

On the other hand, despite the foregoing advantages, to date, techniques for producing bar-coded microparticles have largely used parallel flows systems, which require very precise alignment of the mask during grain synthesis It has requirements. Therefore, when the alignment of the mask is slightly changed due to an external stimulus such as vibration during the microparticle production process, the systems according to the above-described prior art can not be used because the morphology and region configuration of the microparticles are changed, There is a problem that it causes defects.

In addition, the systems according to the above-described prior art have a problem in that the micro-particle synthesis is performed one-dimensionally because the mask must be aligned on the parallel flow, and as a result, the production speed is not as high as about 16,000 per hour. Furthermore, the conventional bar-coded microparticles have a problem that the decoding process is very complicated and difficult due to the possibility of defects that may occur in the alignment process.

Patent Document 1: U.S. Published Patent Application No. 2012-0316082A1

Non-Patent Document 1: D. Dendukuri, S. S. Gu, D. C. Pregibon, T. A. Hatton and P. Doyle, Lab Chip, 2007, 7, 818-828. Non-Patent Document 2: D. C. Appleyard, S. C. Chapin, R. L. Srinivas and P. S. Doyle, Nat. Protoc., 2011, 6, 1761-1774. Non-Patent Document 3: E. Sokolovskaya, L. Barner, S. Braese and J. Lahann, Macromol. Rapid Commun., 2014, 35, 780-786.

Accordingly, it is an object of the present invention to solve the above-mentioned problems of the prior art and to improve quality by lowering the degree of defects of microparticles without being influenced by external stimuli while increasing the production amount of microparticles, It is intended to provide an apparatus for producing a multilayered coded hydrogel particle for detecting a target biomolecule multiplex, a method for producing the same, and a method for producing the hydrogel particle using the apparatus, .

In order to solve the above problems,

A first channel through which a substance specifically binding to a biomolecule to be detected and a polymer precursor fluid communicate with the sample to be analyzed;

A second channel vertically stacked above or below the second channel and communicating with a fluorescent dye for fluorescence analysis of a polymer precursor and a substance specifically binding to the biomolecule; And

And a pressure means for communicating with the first channel and the second channel and for controlling the flow of the fluid in the first channel and the second channel, and a device for manufacturing the multilayered coded hydrogel particles for target biomolecule multiplex detection do.

According to an embodiment of the present invention, the substance specifically binding to the biomolecule may be an antibody, RNA, or DNA.

According to another embodiment of the present invention, the polymer precursor may be an ethylene glycol monomer containing an acrylate group, a 2-methacryloyloxyethyl phosphoryl choline (MPC) monomer, or a mixture thereof.

According to another embodiment of the present invention, the fluorescent dye may be a quantum dot fluorescent dye, upconversion nanoparticles, a fluorescent bead, or a mixture thereof.

According to another embodiment of the present invention, the first channel or the second channel may be laminated a plurality of times at least twice.

According to another aspect of the present invention,

Fabricating first and second master wafers, each having a first channel and a second channel embossed patterned;

A polymer precursor solution is poured onto the first and second master wafers and thermally cured, and then the first and second master wafers are separated from the cured polymer to form first and second channels, Forming a mold and a second channel forming portion;

Preparing a stamp for forming a first channel in which the first channel is embossed by pouring an ultraviolet curable polymer solution into the first polymer mold and curing the ultraviolet curable polymer solution by irradiation of ultraviolet rays;

Coating and partially curing the polymer precursor solution on a glass slide;

Forming a first channel forming portion in which a first channel is formed on the surface by stamping the first channel forming stamp on the partially cured polymer surface; And

And combining the first channel forming portion and the second channel forming portion

The present invention provides a method for manufacturing a multilayered coded hydrogel particle for a target biomolecule multiplex detection.

According to an embodiment of the present invention, the master wafer may be an epoxy resin-based polymer resin.

According to another embodiment of the present invention, the polymer precursor solution may be a solution containing polydimethylsiloxane.

According to another embodiment of the present invention, the partial curing may be performed by coating the polymer on the glass slide and leaving it at 30 DEG C to 150 DEG C for 5 minutes to 20 minutes.

According to another embodiment of the present invention, after stamping the first channel forming stamp, the first channel forming portion and the second channel forming portion may be bonded at a temperature of 30 to 150 DEG C for 5 to 20 Additional partial curing may be performed for a few minutes.

According to another embodiment of the present invention, the first channel forming part and the second channel forming part may be combined and allowed to stand at 30 ° C to 150 ° C for 3 hours or more.

According to another aspect of the present invention,

Wherein the first channel and the second channel of the apparatus for producing lamellar-encoded hydrogel particles for target biomolecule multiplex detection are each subjected to pressure-

A first solution containing a substance specifically binding to a biomolecule to be detected and a polymer precursor in a sample to be analyzed, and

Fluid communication of a second solution comprising a polymer precursor and a fluorescent dye for fluorescence analysis of a substance that specifically binds to said biomolecule; And

Preparing a hydrogel particle comprising a probe region formed by curing the first solution and an encoding region formed by curing the second solution by irradiating ultraviolet rays periodically at a lower end of the manufacturing apparatus; A method for producing multilayered hydrogel particles for multiple detection is provided.

According to an embodiment of the present invention, the first solution and the second solution may further comprise a photoinitiator.

According to another embodiment of the present invention, the pressurization may be performed by applying a pressure condition of 2 kPa to 1000 kPa.

According to another embodiment of the present invention, the periodic ultraviolet irradiation may be performed by irradiating ultraviolet rays having a wavelength of 200 nm to 500 nm at a time interval of 0.05 second to 2 seconds.

According to another embodiment of the present invention, the shape of the hydrogel particles can be adjusted by irradiating the ultraviolet rays through a patterned mask.

According to the present invention, it is possible to increase the production amount of microparticles, improve quality by lowering the degree of defects of microparticles without being influenced by external stimuli, and to encode a coded portion using a fluorescent dye instead of a geometric pattern, An apparatus for producing multilayer-encoded hydrogel particles for target biomolecule multiplex detection capable of remarkably lowering the rate of occurrence of errors at a time and capable of easily increasing an encoding region and a code region through control of wavelengths of laminated water and fluorescent dyes, And a method for producing the hydrogel particles using the same.

FIGS. 1A and 1B are diagrams schematically showing a conventional method for producing a hydrogel particle in a parallel flow system and a method for producing a hydrogel particle in a lamination flow method according to the present invention, respectively.
FIG. 2 is a schematic block diagram of an apparatus for producing multilayer-encoded hydrogel particles for multiple target biomolecule detection according to the present invention.
FIG. 3 is a schematic view of hydrogel particles having hydrogel particles according to the present invention having various numbers of coding regions and probe regions. FIG.
FIG. 4 is a schematic view illustrating a process for producing a device for producing multilayered coded hydrogel particles for detecting target biomolecules in accordance with the present invention.
5 is a graph showing the fluorescence intensity of the hydrogel particles produced according to the present invention over time.
FIG. 6A is a fluorescence microscope photograph of the hydrogel particles prepared by introducing quantum dots of two different colors. FIG. 6B is a schematic diagram of a process of preparing a three-layered hydrogel particles according to the present invention, FIG. 6C is a fluorescence microscope photograph of the hydrogel particles prepared by controlling the thickness of the code layers by the hydrodynamic resistance model. FIG.
7a to 7c are diagrams showing the results of single detection of Alzheimer miRNAs using quadrangular hydrogel microparticles prepared according to the present invention.
FIGS. 8A and 8B are diagrams showing the results of multiple detection of Alzheimer's miRNAs using the square hydrogel microparticles prepared according to the present invention. FIG.

Hereinafter, the present invention will be described in more detail.

DISCLOSURE OF THE INVENTION The present invention has been proposed to overcome the limitations of a one-dimensional production method on a parallel stream, which is a conventional method of producing bar code (encoded) hydrogel particles, and it has been proposed to produce hydrated gel particles encoded by a two- To increase the efficiency and further improve the error rate of the produced encoded hydrogel particles.

1A and 1B schematically show a conventional method for producing a hydrogel particle in a parallel flow system and a method for producing a hydrogel particle in a multilayer flow method according to the present invention. Referring to FIG. 1A, The method according to the present invention can produce the desired hydrogel particles only by precisely aligning the mask at the fluid interface during the manufacturing process of the particles and thus the external stimulus such as vibration is applied during the production process, The shape and the area configuration of the particles to be produced also vary. Furthermore, in the conventional method, since the mask must be aligned on the parallel flow, the particle synthesis is performed one-dimensionally, and the production rate of the particles is also very low, about 16,000 per hour. In addition, the encoding region is manufactured using geometrical patterning, which causes considerable errors in decryption, resulting in a great difficulty in actual use.

Accordingly, in order to solve the problems of the prior art, the present invention provides an apparatus for producing a multilayer-encoded hydrogel particle for a novel target biomolecule multiplex detection as shown in FIG. 1B, a method for producing the same, And a method for producing the same.

Specifically, the apparatus for producing a hydrogel particle according to the present invention comprises:

A first channel through which a substance specifically binding to a biomolecule to be detected and a polymer precursor fluid communicate with the sample to be analyzed;

A second channel vertically stacked above or below the second channel and communicating with a fluorescent dye for fluorescence analysis of a polymer precursor and a substance specifically binding to the biomolecule; And

And pressurizing means in communication with the first channel and the second channel for regulating fluid flow within the first channel and the second channel.

2, a device according to the present invention includes a first channel 110, a second channel 120, and a pressurizing means 130, do.

In other words, the apparatus according to the present invention differs from the conventional parallel flow type hydrogel particle production apparatus in that a first region for producing a probe region containing a substance specifically binding to a biomolecule to be a detection target A channel 110 and a second channel 120 for producing an encoding region including a fluorescent dye for fluorescence analysis of a substance that specifically binds to a biomolecule are stacked in a direction perpendicular to each other. Thereby, there is no need for precise mask alignment as described above, and the production efficiency of the particles can be remarkably increased through the production of two-dimensional particles.

The apparatus according to the invention further comprises a pressure means (130) for regulating the flow of fluid in communication with the first channel (110) and the second channel (120) And a UV irradiation device for producing cured particles by irradiating light to the polymer precursor contained in the fluid while flowing through the channels 110 and 120. When the first and second channels 110 and 120 are formed on a substrate made of glass or the like, the light irradiating device may be disposed at the lower end of the substrate and may be disposed in the first and second channels 110 and 120 It becomes possible to irradiate the flowing fluid with light.

In the present invention, various substances may be selected depending on the type of the sample to be analyzed, and may be various substances such as antibodies, RNA, DNA, and the like. For example, in the following examples, single strand DNA was used as a substance binding to a biomolecule in order to detect various miRNAs known to be closely related to Alzheimer's disease. However, It will be appreciated by those skilled in the art that various other materials may be used depending on the substance to be detected in addition to the DNA.

Meanwhile, the fluid flowing in the first and second channels 110 and 120 is cured by ultraviolet irradiation to form hydrogel particles, and the fluid includes a polymer precursor for forming the hydrogel particles in the fluid . As the polymer precursor, various materials capable of forming hydrogel particles after curing by irradiation with light such as ultraviolet rays may be used, and examples thereof include, but are not limited to, ethylene glycol monomers containing an acrylate group, MPC (2-methacryloyloxyethylphosphoryl choline) monomer or a mixture thereof may be used.

In addition, as described above, in the present invention, the coding region included in the hydrogel particles includes a fluorescent dye instead of the geometric pattern used in the prior art, and this configuration minimizes the error rate that can occur in decryption. The fluorescent dyes may include, but are not limited to, various fluorescent dyes such as quantum dot fluorescent dyes, upconversion nanoparticles, fluorescent beads, or mixtures thereof.

2 illustrates a structure including a first channel 110 and a second channel 120 stacked on top of the first channel 110, And it is possible to produce a hydrogel having various laminated structures by stacking the first channel and the second channel each a plurality of times at least twice. For example, FIG. 3 illustrates hydrogels of various structures having one to three coding regions and one or two probe regions. At this time, in order to produce a hydrogel having a structure in which a coding region and a probe region are laminated a plurality of times, this can be accomplished by appropriately changing the design of the second channel.

The present invention also provides a method of manufacturing a manufacturing apparatus according to the present invention as described above,

Fabricating first and second master wafers, each having a first channel and a second channel embossed patterned;

A polymer precursor solution is poured onto the first and second master wafers and thermally cured, and then the first and second master wafers are separated from the cured polymer to form first and second channels, Fabricating a mold 410 and a second channel forming portion 420;

Preparing a first channel forming stamp 440 in which the first channel is patterned by pouring an ultraviolet curable polymer solution 430 onto the first polymer mold 410 and curing the ultraviolet curable polymer solution 430 by ultraviolet irradiation;

Coating and partially curing the polymer precursor solution (460) on the glass slide (450);

Forming a first channel forming portion (470) having a first channel formed on the surface by stamping the first channel forming stamp (440) on the partially cured polymer surface; And

And coupling the first channel forming part 470 and the second channel forming part 420 together.

FIG. 4 is a schematic process diagram of a manufacturing method of a manufacturing apparatus according to the present invention.

Referring to FIG. 4, first and second master wafers (not shown in the drawings) in which the first channel and the second channel are embossed patterned are manufactured. In the present invention, the manufacture of the master wafer is carried out using conventional photolithography ≪ / RTI > Then, the polymer precursor solution is poured on the manufactured first and second master wafers and thermally cured, and then the master wafer and the cured polymer are separated from each other. This time, the first channel and the second channel are separated from each other by the first The polymeric template 410 and the second channel forming part 420 are manufactured. As the polymer precursor solution that can be used at this time, a solution containing, for example, polydimethylsiloxane as a heat-curable polymer can be used.

Particularly, in the present invention, the apparatus according to the present invention is not manufactured by directly joining the first polymer mold 410 and the second channel forming unit 420 manufactured by the above- The first channel forming stamp 440, which is an embossed stamp, is fabricated using the first polymer mold 410, and then the second channel forming stamp 440 is stamped on the surface of the partially cured polymer precursor, (470).

At this time, the master wafer used in the above process may be an epoxy resin-based polymer resin, for example, a polymer resin material such as SU-8.

On the other hand, the partial curing can be performed by coating the polymer on the glass slide and leaving it at 30 ° C to 150 ° C for 5 minutes to 20 minutes. The reason why the polymer is partially cured before stamping is that the seal or stamping In order to facilitate the process. This is because the polymer before the partial curing is similar to the liquid, so that it is difficult to perform the stamping at the desired point, and even if the stamping is performed, the channel slips and the channel is incorrectly imprinted. When the partial curing temperature is less than 30 ° C, the polymer is not partially cured sufficiently, and thus the first channel is not completely formed when the stamp is removed. When the partial curing temperature is higher than 150 ° C, There is a problem in that the polymer is completely hardened and the stamping is impossible because the polymer is not partially hardened when the leaving time is less than 5 minutes and the first channel is not completely formed when the stamp is removed And if it exceeds 20 minutes, there is a problem that the polymer is completely cured and the bonding of the first channel and the second channel is not smooth, which is not preferable.

Further, the partial curing process may be performed not only before the seal using the first channel forming stamp 440 but also after stamping the first channel forming stamp, and then the first channel forming portion and the second channel forming portion may be combined This additional partial curing process may also be carried out at 30 ° C to 150 ° C for 5 minutes to 20 minutes.

Then, as a final step, the device 480 according to the present invention can be manufactured by joining the first channel forming part 470 and the second channel forming part 420 manufactured by the above process, , The first channel forming part and the second channel forming part may be combined and allowed to stand at 30 ° C to 150 ° C for 3 hours or more. If the temperature after the bonding is less than 30 ° C, there is a problem that the bonded first channel forming portion 470 and the second channel forming portion 420 are not completely bonded. If the temperature is higher than 150 ° C, There is a problem that the physical properties of the polymer are affected, and if the holding time is less than 3 hours, the bonding between the combined channel forming portions becomes incomplete.

Furthermore, the present invention provides a method for producing hydrogel particles using the apparatus for producing multilayered coded hydrogel particles for target biomolecule multiplex detection according to the present invention manufactured by the above process,

The first channel and the second channel of the device according to the present invention, respectively under pressure,

A first solution containing a substance specifically binding to a biomolecule to be detected and a polymer precursor in a sample to be analyzed, and

Fluid communication of a second solution comprising a polymer precursor and a fluorescent dye for fluorescence analysis of a substance that specifically binds to said biomolecule; And

And preparing a hydrogel particle including a probe region formed by curing the first solution and an encoding region formed by curing the second solution by irradiating ultraviolet rays periodically at a lower end of the manufacturing apparatus.

As described above, the first solution forms a probe region of the hydrogel particles after curing, and the second solution forms an encoding region of the hydrogel particles after curing. Therefore, the solution may further contain a component for curing the polymer precursor, which may be a photoinitiator component that causes photo-curing by ultraviolet light irradiation. As a photoinitiator used for such a purpose in the field to which the present invention belongs, various materials exist. As a conventional art, it is possible to produce the hydrogel particles according to the present invention by appropriately selecting such photoinitiators.

On the other hand, the first solution and the second solution are pressurized by separate pressurizing means to form a fluid flow, wherein the pressurization can be performed by applying a pressure of 2 kPa to 1000 kPa. When the pressure is less than 2 kPa, the hydrogels produced in the channel can not be sufficiently pushed toward the outlet, so that a continuous production process is difficult. When the pressure exceeds 1000 kPa, the fluid does not stop well inside the channel, The uniformity of the hydrogel is lowered.

In addition, periodic ultraviolet irradiation is performed for photocuring of the polymer precursor contained in the first solution and the second solution. The periodic ultraviolet irradiation is performed at a time interval of 0.05 second to 2 seconds with a wavelength of 200 nm to 500 nm And then irradiating ultraviolet rays. However, the ultraviolet ray irradiation conditions are not limited thereto, and the ultraviolet ray irradiation conditions can be variously changed in consideration of the shape, composition and use of the hydrogel particles to be produced.

Lastly, the shape of the hydrogel particles produced according to the present invention may be controlled by irradiating ultraviolet rays through the patterned mask during the ultraviolet ray irradiation. For example, depending on the shape of the mask, various forms such as a cylinder, a square column, Of hydrogel particles can be produced.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following Examples are intended to assist the understanding of the present invention and are not intended to limit the scope of the present invention.

Two-layer structure Polydimethylsiloxane  ( PDMS Fabrication of Microfluidic Chip

A PDMS microfluidic chip with a two - layer structure was fabricated for the synthesis of rectangular hydrogel microparticles. PDMS (Corning, Sylgard 184) was mixed with the curing agent in a weight ratio of 10: 1, poured onto the SU-8 master, and then cured at 70 ° C for at least 4 hours. The SU-8 master includes a pattern of upper and lower positive-relief channels having a thickness of 50 [mu] m. Two inlets and one outlet through the replica of the upper PDMS channel were perforated with a biopsy perforator (diameter 1 mm). For lower PDMS channel replicas, Norland Optical Adhesive 81 (NOA81; Norland Products) was poured onto replica channels (negative-relief) and exposed to ultraviolet light (UV; 365 nm) for 4 minutes using a Vilber Lourmat, A NOA 81 stamp (positive-relief) was produced. The channel structure was printed on the PDMS layer using the NOA81 stamp, which was partially cured (12 minutes at 70 DEG C) on a glass slide. After further curing at 70 DEG C for 10 minutes, the NOA 81 stamp was removed. Finally, the upper PDMS channel was placed on the lower channel, and cured at 70 占 폚 for 3 hours while no air gap was formed between the two PDMS layers.

Fluid Mechanics Concentration Lithography  (Hydrodynamic Focusing Lithography, HFL Preparation of Quantum dot-coded quadrilateral hydrated gel microparticles

To perform the HFL, we developed a self-made circuit board and a UV light-emitting diode (LED) (Thorlabs) -pressure tuning system controlled by LabView (National Instrument) code. A pressure regulator (ITV0031-3BL, SMC pneumatics) was used to control the vertically stacked flow of the two-layer structure PDMS microfluidic chip. The regulator was positioned between a pressurized air source and a tying tube connected to the end of the 200 μL pipette (the inflow reservoir is shown in FIG. 2, Axygen). The square hydrogel microparticles were synthesized by UV LED through a film photomask (Hanall Technology) mounted on an inverted microscope (Axiovert 200, Zeiss).

During the HFL, the square particles were generated by the periodic emission of UV (200 ms) when the vertically stacked flow was stopped in an exactly synchronized manner. Each square microparticle consists of a cryptographic and probe region. The coding region contained 40% (v / v) PEG700DA (Sigma-Aldrich), 20% (v / v) PEG200 (Sigma-Aldrich) as a pore generating agent and 5% (v / v) Darocur 1173 Sigma-Aldrich) and also contains 27.5% (v / v) QDs (Nvigen), and 7.5% (v / v) deionized water. The wavelengths of quantum dots for color-coding are 615 nm (red), 535 nm (green), and 460 nm (blue).

As a result of characterization of the rectangular microparticles, the probe region was found to contain 40% (v / v) PEG700DA, 20% (v / v) PEG200, 5% (v / v) photoinitiator, 10% Beads (100 nm in diameter; Polyscience), and 25% (v / v) distilled water. For the detection of Alzheimer-miRNAs, a base free pre-polymer solution for the probe region was first prepared which contained 20% (v / v) PEG700DA, 40% (v / v) PEG200, 5% (v / v) And consisted of 35% (v / v) 3X Tris EDTA (TE) Buffer (Sigma-Aldrich). This base pre-polymer solution was then mixed with a volume ratio of 9: 1 (Integrated DNA technologies) with the acrylate-modified ssDNA oligomer dissolved in 1X TE buffer. At this time, in order to prevent possible contamination, nuclease free water (Sigma-Aldrich), autoclaved pipette tip and microtube were used.

The rectangular microparticles synthesized in the two-layer structure PDMS chip were collected in a microtube pre-filled with 400 μL of a mixture (1 × TET buffer) consisting of 1 × TE buffer and 0.05% (v / v) Tween-20 (Sigma-Aldrich) Respectively. The synthesized microparticles were mixed and centrifuged for about 1 minute. The microparticles were then washed by removing 340 μL of the supernatant buffer solution and resuspending in the same volume of fresh 1 × TET. This washing step was repeated 7 times. Finally, quadrangular hydrated gel microparticles were stored in 150 [mu] L of 1xTET at 4 [deg.] C prior to use.

Alzheimer's disease related miRNA  Multiple detection

miRNA detection was performed for a total volume of 50 μL. First, 25 μL 1xTET buffer was added to each microtube and 5 μL of the target miRNA was added. The performance of the miRNA assay according to the present invention was evaluated by varying the concentration of miRNAs. For multiple detection analysis and cross-reactivity testing, 200 ng of E. coli total RNA (Thermo scientific) was introduced to mimic the complex assay conditions. The mixture was heated in a heat agitator (Hangzhou Allsheng Instruments Co., Ltd) at 95 캜 for 5 minutes. After cooling to room temperature, three types of encoded rectangular microparticles (total 20 μL; ~ 100 particles / tube) were added to the microtube. Each assay volume contained 350 mM NaCl. The analytical microtube was incubated at 55 ° C for 90 minutes with stirring at 1,500 rpm in a heat agitator to hybridize the probes and the target miRNAs in the square micrographs.

After the hybridization, the microparticles were washed three times with 350 μL of 1 × TET containing 50 mM NaCl and 245 μL of ligation mix was added. The ligation mix contained 150 μL 10x NE buffer 2 (New England Biolabs), 1,350 μL 1xTET, 250 nM ATP (New England Biolabs), 40 nM Integrated DNA technologies, and 800 U / ml T4 DNA It is composed of Fermentas. Hybridization and ligation of universal adapters (total volume 295 [mu] L) were performed simultaneously at 21.5 [deg.] C for 45 minutes at a rate of 1,500 rpm in a heat agitator. After performing another washing step as described above, streptavidin-picoerythrine (SA-PE; Life Technologies) diluted 50-fold in 1xTET was added. The final incubation step was carried out at 21.5 [deg.] C for 45 minutes. Followed by a subsequent wash step with 500 μL PTET (consisting of 75% (v / v) 5 × TE, 25% (v / v) PEG 400 (Sigma- Aldrich), and 0.05% (v / v) Tween- , A suspension of 50 μL of square microparticles in PTET was used for image analysis. At this time, PTET was pre-sonicated for 5 minutes before use to remove the aggregated polymer.

Image analysis

RGB fluorescence images for hydrogel microparticles were obtained using a digital single-lens reflex camera (DSLR, EOS 6D, Canon). In order to obtain monochromatic fluorescence images for analyzing the target signal intensity from the probe region, a scientific complementary metal-oxide-semiconductor (sCMOS) camera (optiMOS, QImaging) The time was set to 100 ms. Both cameras were connected to an inverted microscope (Axio Observer.A1, Zeiss). In order to image the cipher area, LED lamp (SOLA SM II, Lumencor Light Engine) was filtered through cube set (XF02-2, Omega Optical) and another set (89101x, 89101m, 89100bs, Chroma Technology Co.). Monochromatic fluorescence images were rotated, edited and analyzed using a self-made Matlab (MathWorks) code in a semi-automatic manner. The fluorescence intensity of each edited probe region (18 x 20 μm) was averaged over at least nine square microparticles. To calculate the background signal, square microparticles were analyzed in the same manner as described above without hybridizing to the target.

Statistical analysis

The ratio intensity was used for statistical analysis of Prism (GraphPad). Statistical significance was analyzed using 2-way ANOVA and paired t-test, and p <0.05 was considered significant.

Review

One. Qdot  Encoded rectangle Hydrogels  Synthesis of microparticles

In order to synthesize the coded rectangular hydrogel microparticles, a PDMS microfluidic chip having a two-layer structure was prepared in the present invention. In contrast to the previously reported methods of fabricating two-layer structure PDMS microchannels (KW Bong, KT Bong, DC Pregibon and PS Doyle, Angew . Chem ., Int . Ed. , 2010, 49 , 87-90) The invention has developed a novel method of using NOA 81 as a stamping material to print a bottom channel on a partially cured PDMS layer.

Specifically, since NOA 81 has low hydrophobicity when compared to PDMS, it is easy to remove the NOA 81 stamp after the bottom PDMS layer is fully cured. In the present invention, stable microfluidic flow is caused in a microfluidic chip of a two-layer structure through a flow focusing technique. The tetragonal hydrogel microparticles encoded by quantum dots were synthesized via HFL. The HFL cycle was repeated consisting of stop (500 ms), exposure (200 ms), and hold (100 ms).

The provided two-dimensional array design made it possible to produce hydrogel microparticles at a faster production rate (3x) than the one-dimensional array synthesis method. Specifically, in a conventional method for producing multifunctional hydrated gel microparticles through a flow lithography (Stop Flow Lithography, SFL, one-dimensional array synthesis method), alignment between the photomask and the planar parallel flow is extremely important. Even minor changes to this arrangement will cause particle components to change or lose, ultimately changing the function of the hydrated microparticles.

By replacing the plane parallel orientation with a vertical flow system in the present invention, this mask alignment need is eliminated. This is because in the present invention, any region in the multilayer flow channel during the HFL process consists of a uniform proportion of each monomer. Therefore, according to the method of the present invention, the change in mask alignment does not affect the function maintenance of the hydrogel microparticles, and as a result, the efficiency of particle production is further enhanced.

The encoded quadratic hydrogels microparticles contain two distinct regions, the cipher and the probe region. The coding region functions to mark the particle, and the probe region contains the complementary sequence to the target miRNA (ssDNA oligomer). In the x-y plane, each rectangular microparticle has a square shape of 20 x 20 μm (width x length), and the height of the microparticles is about 40 μm along the z-axis. The width and length are controlled by the photomask pattern (array of squares). The height of the microparticles is determined by both the height of the PDMS microchannel and the thickness of the oxygen suppressing layer.

By optimizing the volume (i.e., width, length, and height) of the square hydrogel microparticles, the present invention allows the aspect ratio defined by height / width (or length) to be approximately 2 so that the xz or yz plane always lays down Respectively. In other words, the imaging plane of the microparticles is always oriented perpendicular to the direction of the excitation light. With this design, microparticles can be produced by axially exposing UV along the z-direction, and identification and analysis of the target along a transverse plane (x-z or y-z plane) is possible. Quantum dots were encoded as fluorescent markers on one end of quadrangular hydrogel microparticles. For example, in the case of Alzheimer's disease, these quantum dots are chosen to exhibit unique properties including symmetry, narrow emission spectrum and immunity to photobleaching. Furthermore, these quantum dots are biologically inert and have been used in many biological applications, such as for the delivery of siRNA, monitoring of living cells, and detection of DNA midpoint mutations.

To introduce the quantum dots into the photochemically rectangular microparticles, soluble methacylated quantum dots were used in the present invention. Although the PEGDA free-polymer solution is aqueous, the photoinitiator functions as the most hydrophobic component and causes aggregation of the quantum dots. In order to solve this problem, in the present invention, not only the composition of the pre-polymer solution was optimized, but also the quantum dots were sonicated for about 20 minutes immediately before introduction. As a result, it was confirmed that the optimally introduced quantum dots exhibited constant fluorescence intensity and shape stability for at least 6 weeks (Fig. 5). Assuming that the viscosity of the probe monomer is the same as the viscosity of the PEG 200, the time it takes for the quantum dots to diffuse more than half of the microchannel is about 595 seconds (for example, 3.7 nm in diameter). This time is much longer than the residence time of the monomer in the channel (1.2 seconds). Therefore, the diffusion of the quantum dots into the probe region is almost negligible.

2. Assignment of code number

In order to ensure diagnosis, it is inevitable to detect multiple disease related targets. For example, in the case of Alzheimer's, there are more than 20 miRNAs associated with these diseases. In the multiple detection analysis method according to the present invention, various code markers corresponding to different probes have been designed. In keeping with the increasing number of target miRNAs, three parameters have been considered in the present invention: 1) the wavelength of the quantum dots, 2) the number of code streams, and 3) the height of the code stream. In the case of the simplest code (one code stream), the present invention uses a new model through the concept of blocking pressure, which controls the ratio of the cords and probe areas to 1: 1. Figure 6a shows the introduction of quantum dots of two different colors (i. E., Red or green). For complex codes composed of more than one code stream, a hydrodynamic resistance model was used to adjust the thickness of the code and probe layers (KW Bong, J. Xu, J.-H. Kim, SC Chapin, MS Strano, KK Gleason and PS Doyle, Nat. Commun . , 2012, DOI: 10.1038 / ncomms1800). Rectangular microparticles with a 1: 1 ratio of cords and probe areas were synthesized. For additional coding regions, the total height of the two monomers was made identical to that of the probe region by simultaneously introducing two code monomers into the microfluidic channel (Fig. 6B). For this three-flow lamination, a two-layer PDMS channel was modified by adding an inflow point. The additional code stream was then transferred to the stacked monomers. Further, to increase the number of color codes, the ratio of green and blue quantum cords (i.e., 2: 1 and 1: 2) was adjusted by varying the inlet pressure (FIG. 6C). At this time, it is important to maintain the ratio between the code and probe areas at 1: 1 for semi-automatic image analysis. If we define 'n' as the number of quantum dot colors, 'm' as the number of code streams, and 's' as the combination of the height of the code stream, the expected number of codes is _n π _m · s.

3. Qdot  Encoded rectangle Hydrogels  Using microparticles miRNA  Detection: Single detection of Alzheimer's miRNA

First, the quantum dot-coded data is analyzed by a single detection analysis method similar to that reported previously (SC Chapin, DC Appleyard, DC Pregibon and PS Doyle, Angew . Chem ., Int . Ed. , 2011, 50 , 2289-2293) The analytical performance of square hydrogel microparticles was investigated. Briefly, a capture probe photochemically immobilized within PEGDA (i.e., a ssDNA oligomer functionalized with Acrydite ^TM at the 5 'end) has 1) a target-specific complementary sequence (miRNA probes; Figures 7a and 7b) and 2) a universal sequence for fluorescent reporting (adapter probes; FIGS. 7A and 7B). After hybridizing the miRNA targets with the hydrogel microparticles, an enzymatic binding was generated only between the miRNA targets hybridized by T4 DNA ligase and the biotinylated universal adapter (Fig. 7B). Subsequently, the fluorescence intensity from the SA-PE reporter fluorescence source varied with the content of miRNA targets (Fig. 7C).

The working duration of the analysis according to the invention was approximately 4 hours. Therefore, as compared with the analysis time reported previously spent on different miRNA detection strategy, analysis according to the invention Northern blotting (E. Koscianska, J. Starega-Roslan, K. Czubala and WJ Krzyzosiak, Sci. World J ., 2011, 11, will be much faster as compared to 102-117) and microarray methods (H. Wang, and B. RA Ach Curry, Rna, 2007, 13, 151-159).

In the present invention, two main analytical conditions are optimized, which are temperature and salt concentration. Each of these two factors influences sensitivity and selectivity in nucleic acid analysis. The optimized conditions were 55 ℃ and 350 mM NaCl concentration, respectively. In the present invention, there has been a recent report (L. Cheng, J. Doecke, R. Sharples, V. Villemagne, C., et al. Fowler, A. Rembach, R. Martins, C. Rowe, S. Macaulay and C. Masters, Mol. Psychiatry, 2014, 20, 1188-1196) were selected some kinds of miRNA 3 exo (ie, hsa-miR-18b -5p, hsa-miR-342-3p, hsa-miR-1306-5p). These three miRNAs are strongly associated with clinical classification. For detection of the three miRNAs, hsa-miR-18b-5p, hsa-miR-342-3p and hsa-miR-1306-5p were conjugated with blue, red and green quantum dots respectively, Lt; RTI ID = 0.0 &gt; of the particles.

20% (v / v) PEGDA (MW 700 Da) was used as the main composition of the probe region because this concentration not only enabled the diffusion of probe and target molecules, but also resulted in mechanical instability (e.g., Bending or folding of the particles). For each of the three AD-miRNA targets, 100 to 10,000 amol was applied independently within a total assay volume of 50 mu L. As a result, we have confirmed that ligation-based methods exhibit high efficiency without error values, which can occur in target amplification-based methods for direct detection of three Alzheimer's miRNAs.

Specifically, all three Alzheimer miRNAs showed linear dose-dependency over three log units (hsa-miR-18b-5p, hsa-miR-342-3p, and hsa-miR-1306-5p R ² = 0.9949, 0.9836, and 0.9987) (see FIG. Also, the degree of reaction variation between Alzheimer &apos; s miRNAs at each concentration was negligible (see FIG. 7C). This variation is due to the slightly different hybridization dynamics of the three miRNAs (for example, the hybridization rate depends on the GC content and melting point), which regulates the concentration of the miRNA probe in the experimental parameter PEGDA free-polymer solution This can be effectively reduced.

In the present invention, characterization of the performance of all assays was performed without adjusting the concentration of miRNA probes. Furthermore, we have found that by extrapolating the signal-to-noise ratio (SNR) line, the limit of detection (LoD) is calculated to give a sensitivity of atomol level (I.e., 21.8, 8.08, and 7.75 amol for hsa-miR-18b-5p, hsa-miR-342-3p, and hsa-miR-1306-5p, respectively). The data demonstrate that the quadrangular hydrogel microparticles according to the present invention exhibit excellent performance without finely adjusting the concentration of the probe in PEGDA and amplifying the signal.

4. Alzheimer's miRNAs  Multiple detection

To analyze the specificities and to simulate the complexity of the biological samples, detection of multiple Alzheimer miRNAs administered into a solution of E. coli RNA was performed in the present invention (see FIG. 8A). The reason for selecting the bacterial RNA is not only that it contains human plasma-rich non-human bovine RNA sequences, but also because bacterial RNA is often used to detect miRNAs associated with human diseases. Significantly, even when performing simultaneous detection of multiple miRNA targets, there was no significant difference in detection sensitivity, indicating that the detection sensitivity, expressed as the fluorescence intensity, in the two systems, single detection and multiple detection systems, Because there is no statistical difference in the detected values at the data points.

Table 1 below summarizes the detection limit values for the multiple detection and single detection assay methods. Referring to Table 1 below, it can be seen that the square hydrogel microparticles according to the present invention retained their specificity even in complex samples.

Alzheimer's miRNA Analysis method Detection limit (amol) hsa-miR-18b-5p Single detection 21.8 Multiple detection 25.9 hsa-miR-342-3p Single detection 8.08 Multiple detection 18.6 hsa-miR-1306-5p
Single detection 7.75 Multiple detection 10.8

Figure 8b also shows the background subtracted mean fluorescence intensity of the probe region for a total of 7 cases with 200 ng E. coli total RNA as well as the presence and absence of each Alzheimer &apos; s miRNA target (5 fmol). Referring to FIG. 8B, it can be seen that the present invention can detect Alzheimer's miRNAs without any fear of cross-reactivity even in the presence of abundant other nonspecific RNAs.

In summary, the present invention provides multilayered hydrated gel microparticles for multiple detection of a variety of target molecules such as Alzheimer &apos; s miRNAs. These encoded hydrogel microparticles contain vertically distinct portions (i. E., The cords and probe regions). In addition, since the microparticles are synthesized by the two-dimensional array method in the present invention, it is possible to produce microparticles with high productivity without the necessity of sophisticated mask alignment required in the prior art. In the present invention, the number of color codes is increased by using quantum dots of various wavelengths, increasing the number of code layers, or adjusting the thickness of the code layers. Furthermore, it has been successfully demonstrated that the encoded quadrilateral hydrogel microparticles can be used to multiplexly detect miRNAs associated with Alzheimer &apos; s disease with sensitivity and high selectivity at the atomol level. By detecting such miRNAs from the patient serum, The possibility of using particles for clinical diagnosis has been shown.

Claims (16)

delete delete delete delete delete Fabricating first and second master wafers, each having a first channel and a second channel embossed by a photolithography process;
A polymer precursor solution is poured onto the first and second master wafers and thermally cured, and then the first and second master wafers are separated from the cured polymer to form first and second channels, Forming a mold and a second channel forming portion;
Preparing a stamp for forming a first channel in which the first channel is embossed by pouring an ultraviolet curable polymer solution into the first polymer mold and curing the ultraviolet curable polymer solution by irradiation of ultraviolet rays;
Coating and partially curing the polymer precursor solution on a glass slide;
Forming a first channel forming portion in which a first channel is formed on the surface by stamping the first channel forming stamp on the partially cured polymer surface; And
And combining the first channel forming portion and the second channel forming portion
Wherein the method comprises the steps of: (a) The method according to claim 6,
Wherein the master wafer is an epoxy resin-based polymer resin. A method for manufacturing a multilayer-encoded hydrogel particle producing apparatus for multi-target biomolecule detection. The method according to claim 6,
Wherein the polymer precursor solution is a solution containing polydimethylsiloxane. 2. The method of claim 1, wherein the polymer precursor solution is a solution containing polydimethylsiloxane. The method according to claim 6,
Wherein the partial curing is carried out by coating the polymer on the glass slide and leaving the mixture at 30 ° C to 150 ° C for 5 minutes to 20 minutes. The method according to claim 6,
After the stamp for forming the first channel is stamped, additional partial curing is carried out at 30 ° C to 150 ° C for 5 minutes to 20 minutes before bonding the first channel forming portion and the second channel forming portion Wherein the method comprises the steps of: The method according to claim 6,
Wherein the first channel forming unit and the second channel forming unit are combined and then allowed to stand at 30 DEG C to 150 DEG C for 3 hours or more. delete delete delete delete delete

Images