KR101171738B1 - Fabrication of DNA-conjugated hydrogel microparticles via replica molding and DNA hybridization assay using the microparticles - Google Patents

Abstract

The present invention can be prepared in a uniform shape and size of the DNA-conjugated hydrogel microparticles using a small amount of samples, and is not limited to the spherical DNA-conjugated can be produced in various shapes as needed A method for preparing hydrogel microparticles is provided. More specifically, the method for producing a DNA-conjugated hydrogel microparticle of the present invention comprises the steps of: (A) preparing a replication mold in which a micro mold having a predetermined shape and size is formed in a predetermined pattern; (B) filling the replica mold with a composition comprising a polyacrylate monomer, a probe DNA modified at one end with an acrylate group to be copolymerizable with the monomer and a photoinitiator; And (C) photopolymerizing the composition by irradiating ultraviolet rays to the replica mold.

Description

Fabrication method of DNA-conjugated hydrogel microparticles by replication molding and nucleic acid hybridization analysis method using the same {Fabrication of DNA-conjugated hydrogel microparticles via replica molding and DNA hybridization assay using the microparticles}

The present invention can be prepared in a uniform shape and size of the DNA-conjugated hydrogel microparticles using a small amount of samples, and is not limited to the spherical DNA-conjugated can be produced in various shapes as needed A method for preparing hydrogel microparticles is provided. The present invention also relates to a method for hybridizing DNA using the DNA-conjugated hydrogel microparticles.

Hybridization method of nucleic acid is very useful for the detection of pathogens and biohazards, diagnosis of diseases. DNA chips in which microarrays of probe DNAs are formed on flat surfaces are widely used for hybridization analysis, but have disadvantages such as long incubation time, low probe titer, and expensive equipment and software. Complementing this disadvantage, a method for analyzing hybridization of DNA using hydrogel microparticles conjugated with probe DNA has been proposed. Hydrogels are hydrophilic polymers with three-dimensional conformation that can easily contain large amounts of water and genes, peptides, proteins and cells. They are highly valuable for biosensors due to their inherent biocompatibility and hydrophilic properties. In the conventional DNA chip, hybridization analysis is performed on a two-dimensional plane, whereas porous hydrogel microparticles conjugated to DNA allow hybridization analysis on a porous three-dimensional network. Hybridization can be analyzed quickly.

The microparticles according to the prior art are prepared by a method of emulsion polymerization in which a monomer droplet to form microparticles is mixed with a fluid which is not mixed with a monomer, and then a monomer droplet is formed by stirring and cured by ultraviolet irradiation or heating. It became. However, it is difficult to obtain monodisperse particles having a uniform shape and size of the particles by this method.

In order to obtain monodisperse microparticles having a uniform shape and size, the present inventors inject spherical particles together with a monomer and a monomer-free mixed phase solution into a microfluidic chip in Korean Patent No. 957200 and Korean Patent Publication No. 10-2010-16799. A method of preparing spherical microparticles by forming a monomer droplet of and irradiating with ultraviolet rays is provided. The monodisperse particles having uniform shape and size of the particles can be produced by the above method using the microfluidics technology, but there is a limitation that the monomer does not deviate greatly from the sphere because the monomer forms droplets in the continuous phase by surface tension. . In addition, the photopolymerization must occur in the course of the droplets formed in the microfluidic equipment, so complex equipment and high-brightness UV lamps are required and the experimental conditions must be finely controlled. Since the prepared microparticles are present in a mixed state with the monomers which have not reacted with the continuous phase solvent, the washing process for removing them is very important.

In order to solve this problem, methods for preparing microparticles by a batch process have been proposed. Recent studies using replica molding are based on printing techniques such as those proposed by Rolland et al. (J. Amer. Chem. Soc 2005, 127, 10096-10100), which are mainly used for pharmaceutical delivery systems. Used to make scale hydrogel particles. In this method, the monomer solution is dropped onto the non-wetting substrate, and the mold, which is also wettable, is placed on the solution and pressed and printed. Thereafter, the adhesive material is coated on the mold to recover the microparticles from the mold, and then the adhesive material is removed to obtain the microparticles. This printing technique generates a lot of defects when using diluted samples or samples that evaporate well, and there is a concern that the collection of manufactured microparticles is complicated and the manufactured microparticles are contaminated with adhesive materials. In addition, there is a problem in applying to the preparation of 10 ~ 200 ㎛ size DNA-conjugated hydrogel microparticles required in the present invention is relatively large compared to the case of the size of the particle size nm. Carlos J. Hernandez et al. (J. Phys. Chem. 2007, 111 (12), pp 4477-4480) spin-coated a monomer solution on a silicon wafer and then irradiated with UV using a photomask to produce only the desired shape. Photopolymerization reaction occurred to prepare microparticles of various two-dimensional structure. Nature materials 2006, 5, 365-369 and Science 2007, 315, 1393-1396 proposed a method for preparing microparticles of various shapes while using microfluidics. More specifically, in the microfluidic chip, highly reactive monomers flow into the channel to induce photopolymerization by irradiating UV from the bottom, while placing various types of photomas on the path to which ultraviolet rays are irradiated. By allowing this to penetrate, microparticles having the same shape as the photomask are produced. At this time, by using the PDMS surface capable of oxygen permeation at the bottom surface of the channel of the microfluidic chip, adhesion to the PDMS surface should be prevented by inhibiting photopolymerization near the PDMS surface during photopolymerization. This method has the advantage of not using a separate mobile phase, but the washing process is also important because the prepared microparticles are suspended in the monomer.

Conventional methods using such replica moldings are difficult to apply in bio-applications where expensive samples are required because only some of the monomer solutions are used for the preparation of the microparticles and the rest are discarded. Therefore, there is a need for a method capable of producing microparticles of various shapes by a simple method while minimizing the use of a sample.

Republic of Korea Patent No. 957200 Republic of Korea Patent Application Publication No. 10-2010-16799

 J. Amer. Chem. Soc 2005, 127, 10096-10100.  J. Phys. Chem. C, 2007, 111 (12), pp 4477-4480.  Nature materials 2006, 5, 365-369.  Science 2007, 315, 1393-1396.

The present invention for solving the problems of the prior art aims to provide a method for producing a DNA-conjugated hydrogel microparticles of various shapes in a uniform size and shape.

It is another object of the present invention to provide a method for economically preparing DNA-conjugated hydrogel microparticles without waste of a sample.

It is still another object of the present invention to provide a method for the hybridization analysis of nucleic acids in a two-dimensional mold or in a three-dimensional suspension using DNA-conjugated hydrogel microparticles.

The present invention for achieving the above object is (A) Manufacturing a replica mold in which a micro mold having a predetermined shape and size is formed in a predetermined pattern; (B) filling the replica mold with a composition comprising a polyacrylate monomer, a probe DNA modified at one end with an acrylate group to be copolymerizable with the monomer and a photoinitiator; And (C) irradiating the replica mold with ultraviolet light to photopolymerize the composition; It relates to a method for producing a DNA-conjugated hydrogel microparticles comprising a. 1 is a conceptual diagram showing a method for preparing a DNA-conjugated hydrogel microparticle according to the present invention.

Probe DNA in the present invention means a gene fragment having a sequence complementary to the target DNA to be hybridized with the target DNA. Therefore, the present invention is not limited to a specific sequence because the sequence is determined according to the use of the microparticles prepared according to the present invention, that is, according to the sequence of the target DNA to be detected.

Prepared in step (A) The material of the replica mold is a hydrophobic material having a contact angle of 100 to 110 degrees and is preferably a flexible material that can be easily obtained from a silicon wafer. In particular, if the material is flexible, the replication mold can be easily recovered by bending the microparticles from the replication mold and dispersing it in water, but if the material is not flexible, such as sonication or coating of adhesive material, It is more preferable to use a flexible material because additional processing is required. In the exemplary embodiment of the present invention, a replica mold is manufactured using polydimethylsiloxane (PDMS), but synthetic rubber such as polyisobutylene may be used. The size and shape of the micromold in the replication mold may be appropriately selected according to necessity, and for use in DNA hybridization analysis, the length or diameter of the side is preferably about 10 to 200 μm. If the size of the prepared microparticles is too small, there is a problem that it is difficult to identify the fluorescence intensity through a fluorescence microscope, and if the size is too large, the surface area of the microparticles is too small, the usefulness is low.

The composition for the preparation of the microparticles should have a composition such that the probe DNA can form a crosslink with the monomer by photopolymerization. That is, the basic composition is a polyacrylate monomer capable of forming a crosslink by photopolymerization and a probe DNA and a photoinitiator modified at one end thereof to be photopolymerizable with the polyacrylate monomer. In addition, the composition may further contain polyethylene glycol or porogen (porogen) as necessary. PEG or porogen It is well dispersed in polyacrylate monomer and water, and melted when washed with water after the preparation of microparticles to form a porous structure in the corresponding part, so that the target DNA can more effectively penetrate when hybridizing the target DNA. In addition, it is preferable to use a buffer for pH adjustment to stabilize the DNA.

At this time, the probe DNA should be modified at one end such that the end thereof can form a crosslink by copolymerization with a polyacrylate monomer, which is formulated by Acrydite ^™ (Matrix Technologies) by the 5'-end of the prior art. It can be made easily by doing. The formulating process using Acrydite ^™ (Matrix Technologies) is described in detail in a manual provided by Matrix Technologies, and therefore is omitted. However, the modification of the probe DNA is not limited to that by Acrydite ^™ , and if the end is modified to have an acrylate group, it can be said to be included in the scope of the present invention. The concentration of the probe DNA in the composition is preferably 1 ~ 75μM. If the concentration of the probe DNA is too low, the application of the probe DNA in the microparticles is too small, the applicability is lowered, and even if the concentration of the probe DNA is higher than the above range, as can be seen in the examples, the additional advantage due to the high concentration is Since it is economical, it is better to be within the above range.

Examples of polyacrylate monomers include poly (ethylene glycol) diacrylate (PEG-DA), polyethylen glycol dimethacrylate, 4-Hydroxybutyl acrylate (4-HBA), acrylamide or N-isopropylacrylamide, and the like. Can be used. In the examples of the present invention, only the example using only PEG-DA is described, but the main concept of the present invention is to prepare microparticles using a replication mold, and thus DNA-conjugated by the method of the present invention with reference to the following examples. It will be easy to prepare hydrogel microparticles. Particularly, since the monomers are different from each other but the acrylic groups are reacted by the same principle as PEG-DA as a functional group in the photopolymerization reaction, it is natural that the method for producing microparticles of the present invention can be successfully applied. In general, the application of microfluidics is preferable because PEG-DA is preferred because the monomer has high reactivity and the photopolymerization should be completed in a short time. However, in the present invention, photopolymerization is not performed in the state of fluid flow, but photopolymerization is performed in the replication mold. As such, the reactivity of the monomer does not necessarily have to be high. It is preferable that the density | concentration of the polyacrylate monomer in the said composition is 30 to 60% (v / v). If the concentration of the polyacrylate monomer is too low, the formation of microparticles is incomplete, and if the concentration of the polyacrylate monomer is too high, cross-linking by photopolymerization is too dense and the voids of the produced microparticles are too small. have.

The photoinitiator is capable of forming a radical by irradiation of ultraviolet rays, 1-Hydroxy-cyclohexyl-phenyl-ketone, 2-Hydroxy-2-methylpropiophenone, 4- (2-hydroxyethoxy) phenyl- (2-hydroxy-2- propyl) ketone, 2-Methyl-1 [4- (methylthio) phenyl] -2-morpholinopropan-1-one, 1-Hydroxy-cyclohexyl-phenyl-ketone, etc. may be used, but is not limited thereto. The concentration of the photoinitiator is preferably 1 to 2% (v / v). If the concentration of the photoinitiator is too low, the photopolymerization reaction does not proceed efficiently and the microparticles are incompletely formed. If the concentration of the photoinitiator is too high, the photopolymerization reaction proceeds so rapidly that the size of the pores of the microparticles is too small, and its applicability is Limited. The concentration of the photoinitiator may also vary depending on the reactivity of the monomers used. In other words, if the reactivity of the monomer is low, it is natural that a larger amount of photoinitiator than the above range is required.

After filling the composition in a replica mold, the excess composition can simply be recovered by pipette and used for the next reaction. According to the manufacturing method of the present invention, the composition can be recovered in a state in which the composition is not mixed with other solvents or mobile phases and is not contaminated, and thus only the amount necessary for the use of the microparticles is used, thereby eliminating the waste of the sample. In addition, DNA-conjugated is more convenient and economical because there is no need to finely adjust parameters such as the speed of the mobile phase or to use a high-intensity UV light source to complete photopolymerization in a short time in order for the photopolymerization reaction of the composition to proceed. Hydrogel microparticles can be prepared.

When the composition is evaporated during the photopolymerization process of step (C), the resulting particles are not uniformly produced. In particular, since the volume of the micromold formed in the replica mold is very small in nL, evaporation may be affected even during a short time in which photopolymerization occurs. Therefore, the replica mold is preferably formed with a lid so as to minimize evaporation of the composition during photopolymerization. In addition, it is preferable to adjust the relative humidity at the time of photopolymerization to 50 to 90%. As can be seen in the examples below, when the relative humidity is less than 50%, the prepared microparticles were not uniform due to evaporation of the composition.

The DNA-conjugated hydrogel microparticles prepared by the above method can be used in 2D like a conventional DNA chip in a mold after a washing step. Alternatively, the method may further include collecting the photopolymerized microparticles from the replication mold after the step (C), and then collect and wash the prepared microparticles, and use the suspension as a 3D array.

The present invention also relates to a method for analyzing DNA hybridization using DNA-conjugated hydrogel microparticles prepared by the above method. More specifically, the DNA hybridization analysis method of the present invention comprises the steps of: (A) preparing a DNA-conjugated hydrogel microparticle according to claim 1 containing the probe DNA; (B) labeling the target DNA; And (C) hybridizing by mixing the labeled DNA with the DNA-conjugated hydrogel microparticles of (A); (D) washing the hybridized DNA-conjugated hydrogel microparticles; And (E) measuring the intensity of the label of the washed DNA-conjugated hydrogel microparticles; characterized in that it comprises a. According to the present invention, the incubation time required for hybridization can be greatly shortened to 30 minutes or less, and even a small amount of DNA at a concentration of 10 nm can be specifically detected by hybridization analysis.

The label of the target DNA is to be able to detect whether the hybridization, in the embodiment of the present invention was labeled using a fluorescent material, but in addition to labeling using a dye after observing the color or radioactive material labeled to detect radioactivity Those skilled in the art will readily be able to make modifications using existing labeling methods.

In addition, when a label is detected in the step (F), hybridization analysis can be performed quantitatively by measuring the intensity of the label and comparing the label with a standard curve.

As described above, according to the present invention, DNA-conjugated hydrogel microparticles of various shapes can be easily produced in a uniform size and shape using only a minimal sample.

The DNA-conjugated hydrogel microparticles prepared by the present invention hybridize with a low concentration of target DNA because a porous network of probe DNA is formed on the surface unlike microparticles prepared by applying microfluidics. Analysis is possible. Therefore, since a small amount of the detection of a genetically modified substance or a labeled gene such as cancer can be detected, it can be usefully used for diagnosing a disease or detecting a biohazardous substance.

In addition, since the DNA-conjugated hydrogel microparticles of the present invention can be manufactured in various shapes, hybridization of target DNA to various probe DNAs can be analyzed at a time.

1 is a conceptual diagram showing the manufacturing process and application of the DNA-conjugated hydrogel microparticles of the present invention.
2 and 3 are brightfield and fluorescence microscopy images showing the effect of relative humidity in the preparation of DNA-conjugated hydrogel microparticles.
FIG. 4 is a graph showing fluorescence microscopy, confocal images and fluorescence intensity measured from them and penetration depth of target DNA showing the effect of PEG-DA concentrations in the preparation of DNA-conjugated hydrogel microparticles.
5 is a fluorescence micrograph showing the effect of probe DNA concentration on the preparation of DNA-conjugated hydrogel microparticles and a graph showing the fluorescence intensity measured from them.
FIG. 6 is a graph showing brightfield and fluorescence microscopy images showing the effect of photoinitiator concentration in the preparation of DNA-conjugated hydrogel microparticles and the fluorescence intensity measured therefrom.
7 is a graph showing brightfield and fluorescence microscopy images showing the sequence specificity upon hybridization with target DNA using DNA-conjugated hydrogel microparticles according to an embodiment of the present invention and fluorescence intensities measured therefrom.
8 is a fluorescence microscopy image showing the difference in fluorescence intensity according to the concentration of the target DNA when hybridizing with the target DNA using the DNA-conjugated hydrogel microparticles according to an embodiment of the present invention and the fluorescence intensity measured from them Showing graph.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings and examples. However, the drawings and the embodiments are only illustrative of the contents and scope of the technical idea of the present invention, and 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 Fabrication of Duplicate Molds

The replica mold was prepared to have 1600 micro molds with a diameter of 50-100 μm per 1 cm × 1 cm. A negative photoresist (SU-8, Microchem Co., USA) was evenly applied on the silicon wafer, followed by spin coating at 2,000 rpm to coat the photoresist at a height of 50 μm. Masks were prepared using an AutoCAD program to place a circle, triangle, or square pattern of 1600 to 50-100 μm in diameter per 1 cm × 1 cm. UV was irradiated to the photosensitive coating layer coated on the silicon wafer through the mask to produce a master mold having the pattern embossed. Thereafter, PDMS (Polydimethylsiloxane) (Sylgard 184; Dow Corning, Midland, MI) was poured into the prepared master mold, and cured at 65 ° C. for 48 hours to produce a replica mold.

Meanwhile, PDMS was spin-coated on slide glass at 2000 rpm for 30 seconds. Then, the cover of the replica mold is removed by removing PDMS in the rectangular region corresponding to the region where the micro mold of the replica mold is generated in the spin-coated slide glass to form a predetermined gap with the top surface of the replica mold and the slide glass cover. It was.

Example 2 Preparation of DNA-Conjugated Hydrogel Microparticles

1) Preparation and Hybridization of DNA-Conjugated Hydrogel Microparticles

Methods for preparing and hybridizing the standardized DNA-conjugated hydrogel microparticles used in the Examples below are as follows.

30% (v / v) Poly (ethylene glycol) diacrylate (PEG-DA, Mn = 700, Sigma-Aldrich), 10% (v / v) Poly (ethylene) to prepare DNA-conjugated hydrogel microparticles glycol) (PEG, Mw = 200, Sigma-Aldrich), 2% (v / v) 2-hydroxy-2-methylpropiophenone (Darocur 1173, photoinitiator (PI), Sigma-Aldrich), 50 μM 5'-terminus Acrydite ^TM Probe DNA (Integrated DNA Technologies), modified with 58% (v / v) TE Buffer (10 mM Tris, pH 8.0, Sigma-Aldrich and 1 mM EDTA, Sigma-Aldrich) containing 0.02% (v / v) of 10% (w / w) precursor composition consisting of sodium dodecyl sulfate (SDS, Sigma-Aldrich) was prepared. Table 1 shows the cross-sectional shape of the replication mold and the sequence of the probe DNA used in the following examples.

About 75 μl of the precursor composition was dropped onto the surface of the PDMS replication mold prepared in Example 1 and lightly tapped to remove air bubbles in the micro mold using the tip of a disposable plastic pipette. The mold was filled and the excess precursor solution remaining was recovered again using a pipette. The replica mold filled with the precursor solution was also covered with a slide glass cover coated with PDMS prepared in Example 1.

Covered replication molds were placed on aluminum mirrors (Thorlabs, Newton, NJ) and irradiated with 365 nm ultraviolet light for 15 minutes using an 8 W small UV lamp (Spectronics Corp., Westbury, NY). Thereafter, the mold was bent to separate the photopolymerized microparticles from the replica mold, and a 0.5% (v / v) Tween 20 aqueous solution was dropped and collected by a pipette into a vial. The microparticles were collected repeatedly using aqueous Tween 20 solution until all of the microparticles were recovered.

In order to confirm the uniformity of the prepared DNA-conjugated hydrogel microparticles by measuring fluorescence images of the microparticles, the target DNAs were labeled using fluorescently labeled target DNA. For hybridization of the target DNA, 5 × SSC buffer (75 mM sodium citrate, 750 mM sodium chloride, pH 7.0) containing 0.05% (v / v) Tween 20 was added to about 1000 microparticles recovered by the method described above. After stirring, the mixture was washed four times in a manner of removing the supernatant. 100 μl of 200 nM target DNA of the sequence shown in Table 2 was added to the residue from which the supernatant was removed, and hybridized at room temperature for 30 minutes. Target DNA was 5'-terminally modified with fluorescein isothiocyanate (FTTC) and purchased from Gene Probe Technologies. The unhybridized target DNA was removed by adding 5 × SSC buffer (75mM sodium citrate, 750 mM sodium chloride, pH 7.0) containing Tween 20, stirring, and removing the supernatant.

2) Preparation of DNA-Conjugated Hydrogel Microparticles According to Humidity

In order to minimize the effects of evaporation of the precursor composition during the preparation of the microparticles 15%, DNA-conjugated hydrogel microparticles were prepared according to the above method at various relative humidity conditions of 30%, 50%, 70% or 90%. More specifically, after the lid closed in the replicated mold precursor composition is filled in accordance with the method described in 1) put in a plastic glove box (Thermo Fisher Scientific), determine the humidity in standard Traceable ^TM humidity / temperature (VWR), and 0.5 gallons Ultrasonic humidifier was used to manually control and maintain the humidity and perform a photopolymerization reaction. Humidity without humidity control was 15%, and the temperature was 21 ° C.

Brightfield microscopic images of the DNA-conjugated hydrogel microparticles hybridized by the method of 1) were measured using an Olympus BX51 microscope equipped with the U-N31001 standard green filter. Captured using. In addition, the microparticles were hybridized to the target DNA by the same method as described above in the state of being in the mold without recovering from the replication mold, thereby obtaining a fluorescence microscopic image.

FIG. 2 shows brightfiled microscopic images (a, d) and microscopic images (b, c, e, f) measured at conditions of 15% and 90% relative humidity, respectively. (A) and (d) of FIG. 2 are brightfield microscopic images of microparticles prepared at 15% and 90% relative humidity, respectively, and it can be seen that similarly uniform particles are prepared under both 15% and 90% relative humidity conditions. . However, comparing (b) and (e) showing fluorescence images of microparticles prepared at 15% and 90%, respectively, compared to 15% relative, whereas very uniform microparticles were produced at 90% relative humidity. In the humidity conditions, the size and shape of the microparticles are uniform, but the fluorescence intensity is not uniform.

(C) and (f) of FIG. 2 are hybridized in the replication mold without collecting the prepared microparticles to confirm whether the uniformity of the prepared microparticles differs depending on the position in the replication mold. ) Is 15%, (f) is a fluorescence microscopic image of the distal end of the replica mold after hybridization of microparticles prepared at 90% relative humidity. While the fluorescent image of FIG. 2 (f) shows uniform fluorescence intensity, in the case of (c), the intensity of fluorescence is greatly reduced at the outer portion of the mold. This is because the volume of the micro-mould in the replica mold is very small, even when the humidity is low, even if the precursor composition evaporates during the photopolymerization, it is determined that the formation of the hydrogel is not effective because of its influence.

Figure 3 shows the results of measuring the fluorescence microscopy image after hybridizing the microparticles prepared in each humidity condition with the target DNA in the mold. When the relative humidity is 50% or more from Figure 3 it can be seen that the microparticles are uniformly produced.

3) Preparation of DNA-Conjugated Hydrogel Microparticles According to PEG-DA Concentration

When hybridizing to the target DNA using microparticles prepared according to the PEG-DA concentration, the concentration of PEG-DA was changed to 20%, 30%, 40% to determine whether there was a difference in the intensity of fluorescence and the penetration depth of the target DNA. DNA-conjugated hydrogel microparticles were prepared and hybridized with the target DNA by the same method as 1) except using 50% or 60%.

Confocal images were collected using a Leica DMIRE2 microscope (Wetzlar, Germany) equipped with a TCS SP2 scanner after 63x (NA 1.2) water immersion of hybridized microparticles. At this time, the excitation wavelength was 488 nm, and the emission wavelength was 500 to 530 nm. The depth scan increment for the z-scan image was 3 μm. Confocal images (e-h) measured according to PEG-DA concentrations are shown in FIG. 4 along with fluorescence microscopy images (a-d). In addition, the fluorescence intensity was measured from the measured fluorescence image using ImageJ software, and the results are shown together in (i) of FIG. 4.

As can be seen from Figure 4, the lower the concentration of PEG-DA, the higher the fluorescence intensity, the microparticles prepared from 30% PEG-DA showed the brightest fluorescence, but the fluorescence of the microparticles prepared from 60% PEG-DA was Appeared very weak. Irrespective of the intensity of fluorescence, the shape, size and fluorescence of the microparticles prepared in the range of 30-60% PEG-DA were uniform. Yellow bars in FIGS. 4A to 4D represent 100 µm and red circles represent areas used for fluorescence intensity measurement of (i). When 20% PEG-DA was used, the formation of microparticles was incomplete and no fluorescence microscopy image was shown.

As can be seen from the confocal images of (e) to (h) of FIG. 4, the penetration depth of the target DNA upon hybridization of the target DNA largely depends on the concentration of PEG-DA when preparing the microparticles. In FIG. 4 (e)-(h), a yellow bar represents 100 micrometers. When 30% PEG-DA was used, the target DNA penetrated about 6 μm, but when 60% PEG-DA was used, hybridization occurred only on the surface of the microparticles. This is considered to be difficult to penetrate the target DNA when the PEG-DA concentration is high because the density of crosslinking by photopolymerization is high and the pore size is reduced.

It can be seen from FIG. 4 that hybridization of the target DNA and the microparticles occurs mostly on the surface. In the case of preparing microparticles using the microfluidics according to the prior art, since the continuous process, the amount of ultraviolet irradiation is limited, and photopolymerization at the particle surface is inhibited by the transmission of oxygen through the porous PDMS layer. Probe DNA density is inevitably limited. However, according to the replication molding of the present invention, since the amount of ultraviolet irradiation is freely controlled, photopolymerization at the surface can be sufficiently progressed, so that high density of probe DNA can be conjugated to the surface.

4) Preparation of DNA-Conjugated Hydrogel Microparticles According to Probe DNA Concentration

Depending on the probe DNA concentration used in the preparation of the microparticles, the concentration of the probe DNA is 1 to 1 as shown in FIG. 4 to confirm whether there is a difference in fluorescence intensity when hybridizing to the target DNA using the prepared microparticles. DNA-conjugated hydrogel microparticles were prepared and hybridized with the target DNA by the same method as 1) except the change to 100 μM. Fluorescence microscopy images of the microparticles hybridized with the target DNA were measured and the results are shown in FIG. 5.

5 (a) to 5 (f) are fluorescence microscopy images obtained after hybridizing the microparticles prepared for each probe DNA concentration with the target DNA, and (g) shows the graphs by measuring the fluorescence intensity from the image. In FIG. 5 (a)-(f), a yellow bar represents 200 micrometers.

As shown in FIG. 5, the fluorescence intensity of the microparticles hybridized with the target DNA increased in proportion to the concentration of the probe DNA used in the preparation of the microparticles, and then saturated when the concentration of the probe DNA was 50 μM or more.

5) Preparation of DNA-Conjugated Hydrogel Microparticles According to Photoinitiator (PI) Concentration

Depending on the PI concentration used in the preparation of the microparticles, the concentration of the PI is 0.5 to 3% as shown in FIG. 5 to confirm whether there is a difference in fluorescence intensity when hybridizing to the target DNA using the prepared microparticles. DNA-conjugated hydrogel microparticles were prepared and hybridized with the target DNA by the same method as 1) except for changing to (v / v). In FIG. 6, brightfield microscopy images of microparticles hybridized with target DNA (FIG. 6 (a) and (b)) and fluorescence microscopy images (C) to (f) of FIG. The results are shown in conjunction with (g) of FIG. 6.

As shown in (a) and (d) of FIG. 6, when the concentration of PI was 0.5%, the formation of microparticles was incomplete and the fluorescence was also weak, indicating that the concentration of the photoinitiator was not sufficiently included in the photopolymerization. . When the concentration of PI was 1%, the formation of microparticles showed a perfect form, the intensity of fluorescence was also strong, and the intensity of fluorescence increased significantly as the concentration of PI increased to 2%. On the other hand, when the concentration of PI was increased to 3%, the fluorescence decreased significantly. This is because the size of the pores formed in the microparticles is greatly reduced as the photopolymerization progresses rapidly due to the increase of the concentration of the photoinitiator, making it difficult for the target DNA to penetrate into the pores.

Example 3: Hybridization Analysis of Target DNA

1) Target sequence specificity confirmation

The specificity for the target sequence was confirmed in order to apply the DNA-conjugated hydrogel microparticles prepared by the method of Example 2 to the hybridization analysis of the target DNA.

By the method of 1) of Example 2, hydrogel microparticles of circular, triangular and rectangular hydrogels were prepared in which the three probe DNAs of Table 1 were conjugated, respectively. The prepared microparticles were recovered and washed by the method also described in 1).

After mixing the three microparticles, divided into three groups to contain about 1000 mixed microparticles, and to each group, 100 μl of 200 nM target DNA modified with 5'-terminal FTTC as shown in Table 2 was added to each group, and at room temperature. Hybridization was done for 30 minutes. Then, the hybridized target DNA was added to 5 × SSC buffer (75mM sodium citrate, 750 mM sodium chloride, pH 7.0) containing Tween 20, stirred, and washed by removing the supernatant.

Brightfiled microscopic images and fluorescence microscopic images of the hybridized microparticles of each group were measured, and the results are shown in FIGS. 7A to 7C and FIGS. In addition, the fluorescence intensity was measured from the fluorescence microscopy image and is shown in FIG. Yellow bars in FIG. 7 represent 200 μm.

As can be seen in Figure 7, each target DNA hybridized only with microparticles containing the probe DNA of the complementary sequence, so that only microparticles having a circular, square or triangular shape in each group showed fluorescence. In addition, regardless of the sequence of the target DNA used, the detection sensitivity for the same concentration of the target DNA sequence was shown to be the same within the margin of error. From these results, it was confirmed that the DNA-conjugated hydrogel microparticles according to the present invention can be applied to the hybridization analysis of nucleic acids.

2) Hybridization Analysis According to Target DNA Concentration

In order to confirm whether quantitative hybridization analysis according to the target DNA concentration is possible, using the microparticles prepared using the probe DNA of SEQ ID NO: 1 of Table 1 according to the method of Example 2, SEQ ID NO of Table 2 Hybridization was performed while changing the concentration of the target DNA of 4 to 0˜2 μM, and the fluorescence intensity was measured from the fluorescence microscopy image. 8 shows a graph of fluorescence microscopy images and fluorescence intensities. It can be seen that the fluorescence microscopy image of the microparticles appeared uniformly in the concentration range of all target DNA.

8 shows that in the range where the concentration of the target DNA is low, the fluorescence intensity is linearly proportional to the concentration of the target DNA, but when 1 μM or more, the fluorescence intensity shows a saturation curve. In FIG. 8, saturation of fluorescence intensity at high target DNA concentrations indicates that hybridization has reached saturation, and the sensitivity of the device is not saturated.

From the results of FIG. 8, it was confirmed that the DNA-conjugated hydrogel microparticles of the present invention were capable of quantitative analysis as well as hybridization analysis of qualitative target DNA even when a small amount of target DNA was used.

Attach an electronic file to a sequence list

Claims (12)

(A) manufacturing a replica mold in which a micro mold having a predetermined shape and size is formed in a predetermined pattern;
(B) filling the replication mold with a composition comprising a polyacrylate monomer, a probe DNA modified at one end with an acrylate group to be copolymerizable with the monomer, and a photoinitiator; And
(C) photopolymerizing the composition by irradiating the replica mold with ultraviolet light;
Method for producing a DNA-conjugated hydrogel microparticles comprising a.
The method of claim 1,
The replication mold is a method of producing a DNA-conjugated hydrogel microparticles, characterized in that the cover is formed.
The method of claim 1,
After the step (C),
(D) collecting the photopolymerized microparticles from the replication mold, the method of producing a DNA-conjugated hydrogel microparticles.
The method of claim 1,
The replication mold is a method of producing a DNA-conjugated hydrogel microparticles, characterized in that consisting of polydimethylsiloxane (PDMS) or polyisobutylene.
The method according to any one of claims 1 to 3,
The relative humidity at the time of photopolymerization is a method for producing a DNA-conjugated hydrogel microparticles, characterized in that 50 ~ 90%.
The method according to any one of claims 1 to 3,
The composition is a method for producing a DNA-conjugated hydrogel microparticles, characterized in that it further comprises polyethylene glycol or porogen (porogen).
The method according to any one of claims 1 to 3,
The concentration of the polyacrylate monomer in the composition is a method of producing a DNA-conjugated hydrogel microparticles, characterized in that 30 to 60% (v / v).
The method according to any one of claims 1 to 3,
The method of producing a DNA-conjugated hydrogel microparticles, characterized in that the concentration of the probe DNA in the composition is 1 ~ 75μM.
The method according to any one of claims 1 to 3,
The concentration of the photoinitiator is a method for producing a DNA-conjugated hydrogel microparticles, characterized in that 1 ~ 2% (v / v).
(A) preparing a DNA-conjugated hydrogel microparticle according to claim 1 containing probe DNA;
(B) labeling the target DNA; And
(C) mixing and hybridizing the labeled DNA with the DNA-conjugated hydrogel microparticles of (A);
(D) washing the hybridized DNA-conjugated hydrogel microparticles; And
(E) measuring the intensity of the label of the washed DNA-conjugated hydrogel microparticles;
Hybridization analysis method of DNA, characterized in that comprises a.
11. The method of claim 10,
Said DNA-conjugated hydrogel microparticles are in a 3D array state suspended in a 2D array or suspension arranged in a replication mold.
The method of claim 10 or 11,
The hybridization analysis method of DNA, characterized in that for quantitative analysis by measuring the intensity of the label and comparing with a standard curve.

Images