阅读说明：本技术 用于制备编码的水凝胶颗粒的方法以及由此制备的编码的水凝胶颗粒 (Methods for making encoded hydrogel particles and encoded hydrogel particles made thereby ) 是由 奉纪完 卢允镐 文炫准 李贤智 金贤雄 文硕骏 于 2018-12-14 设计创作，主要内容包括：本发明涉及一种用于制备用于以高准确度对靶生物分子进行高灵敏度检测的编码的水凝胶颗粒的方法以及由此制备的编码的水凝胶颗粒,且具体来说,涉及一种用于制备编码的水凝胶颗粒的方法以及由此制备的编码的水凝胶颗粒,方法包括合成水凝胶颗粒的步骤以及接着使探针与水凝胶颗粒结合。根据本发明,可以显著提高的高效率装载探针,装载的探针可均匀地分布,且可解决由未反应末端所导致的生物分子检测抑制的潜在问题。此外,本发明可提高检测灵敏度,可通过以高灵敏度对例如核酸和蛋白质的靶生物分子进行多重检测来应用于疾病的诊断或药物的筛选,且可普遍地用于例如分子诊断的医学诊断领域中。(The present invention relates to a method for preparing encoded hydrogel particles for highly sensitive detection of a target biomolecule with high accuracy and the encoded hydrogel particles prepared thereby, and in particular, to a method for preparing encoded hydrogel particles comprising the step of synthesizing hydrogel particles and then binding a probe to the hydrogel particles and the encoded hydrogel particles prepared thereby. According to the present invention, it is possible to load probes with high efficiency, which can be significantly improved, the loaded probes can be uniformly distributed, and the potential problem of biomolecule detection inhibition caused by unreacted ends can be solved. In addition, the present invention can improve detection sensitivity, can be applied to diagnosis of diseases or screening of drugs by multiplex detection of target biomolecules such as nucleic acids and proteins with high sensitivity, and can be generally used in the field of medical diagnosis such as molecular diagnosis.)

1. A method for preparing an encoded hydrogel particle comprising synthesizing a hydrogel particle and conjugating a probe to the hydrogel particle.

2. The method for making an encoded hydrogel particle of claim 1, wherein the probe is conjugated to the synthesized hydrogel particle through an unreacted terminus retained in the hydrogel particle.

3. The method for preparing an encoded hydrogel particle of claim 1, wherein the probe is crosslinked with the synthesized hydrogel particle via radical reaction and spontaneous electron transfer in solution.

4. The method for preparing an encoded hydrogel particle according to claim 3, wherein the radical reaction for crosslinking the probe with the synthesized hydrogel particle is initiated by irradiation with ultraviolet rays in the presence of a photoinitiator or by application of thermal energy in the presence of a thermal initiator.

5. The method for preparing an encoded hydrogel particle according to claim 3, wherein when the probe is crosslinked with the synthesized hydrogel particle via electron transfer in solution, electrons in aqueous solution or polar solvent act as a nucleophile to form a covalent bond.

6. The method for preparing an encoded hydrogel particle of claim 3, wherein the probe is crosslinked at 0 ℃ to 90 ℃.

7. The method for preparing an encoded hydrogel particle of claim 1, wherein the probe is a deoxyribonucleic acid, a ribonucleic acid, or a protein.

8. The method for preparing an encoded hydrogel particle of claim 1, wherein the encoded hydrogel particle is designed such that the code region is integrated with the probe region.

9. The method for preparing an encoded hydrogel particle of claim 8, wherein the code comprises a geometric code.

10. The method for preparing an encoded hydrogel particle of claim 9, wherein the geometric code is comprised of a combination of different types of graphics.

11. The method for making an encoded hydrogel particle of claim 1, wherein the hydrogel particle further comprises a functional nanoparticle.

12. An encoded hydrogel particle prepared by the method for preparing an encoded hydrogel particle of claim 1.

13. An encoded hydrogel particle designed such that a code region is integrated with a probe region.

Technical Field

The present invention relates to a method for preparing encoded hydrogel particles capable of detecting a target biomolecule with high accuracy and high sensitivity, and the encoded hydrogel particles prepared thereby. More particularly, the present invention relates to a method for preparing encoded hydrogel particles comprising synthesizing hydrogel particles and conjugating probes to the hydrogel particles, and encoded hydrogel particles prepared by the method.

Background

There is a need to develop a technology for detecting biomolecules such as nucleic acids and proteins with high accuracy and high sensitivity in the fields of diagnosis, drug screening, and molecular biochemistry.

Encoded hydrogel particles (encoded hydrogel particles) have attracted a great deal of attention because these particles can be used for highly sensitive multiplex detection of biomolecules. The encoded hydrogel particles comprise probes loaded to detect biomolecules and a code to identify the loaded probes. The use of encoded particles enables the simultaneous capture (i.e., multiplexed detection) of multiple biomolecules in a single detection process, as well as the highly sensitive detection of target biomolecules interacting with probes in three-dimensional particle space.

Flow lithography has received much attention as a process for synthesizing encoded hydrogel particles having various functions and shapes. Flow lithography allows for the synthesis of multifunctional asymmetric particles based on patterning microfluidic streams and using UV light through a photomask. According to flow lithography, the fluid in a microchannel flows without mixing with different adjacent flows due to its low Reynolds number (Re), and may be configured as multiple parallel flows. Further, according to the flow lithography, patterning UV is irradiated onto the precursor fluid through the photomask to induce selective polymerization of the precursor fluid, so that particles having the same shape as the photomask can be synthesized. The presence of materials, such as antibodies and nucleic acids, which are capable of binding to biomolecules in the precursor fluid prior to polymerization, enables the synthesis of particles suitable for use in the detection of biomolecules.

Conventional methods for synthesizing encoded hydrogel particles include directly mixing a precursor fluid with probes for biomolecule detection, and crosslinking the probes with the particles during synthesis. However, this method has the following problems. Since the particles are synthesized by flow lithography in a very short time, only about 10% of the monomers in the precursor are polymerized, with the result that only about 10% of the probes are crosslinked with the particles. The low loading yield of the probe results in limited detection performance. Another problem is that most of the probes are not easily dispersed in the precursor. In particular, when a probe (e.g., an antibody) has low compatibility with a precursor, the precursor should be mixed with the probe for a long time, and the probe remaining in the form of an aggregate even after mixing may be crosslinked with the particle. In case the probe is present in the form of (heterogeneous) aggregates, the capture site of the biomolecule is not exposed, resulting in a poor detection capability of the probe. Highly sensitive detection requires uniform loading of the maximum possible number of probes, which inevitably degrades detection performance due to the above-mentioned problems.

According to another conventional method, particles are synthesized by cross-linking of liquid polymers to form a network. As mentioned above, the crosslinking yield of liquid polymers is as low as about 10%, which results in the presence of defects on the network. For example, defects may form when one or more reactive groups of the monomer remain unreacted in the network. The large number of unreacted reactive groups (unreacted ends) remaining in the final particle is a factor that deteriorates the ability to detect biomolecules. The unreacted ends may non-specifically bind to the target biomolecules, resulting in false-positive signals due to their high reactivity. In addition, essential materials (e.g., target biomolecules, fluorescent materials, reporters, and cells) to be used during detection are reacted with the unreacted ends and immobilized on the particles. That is, the presence of unreacted ends may be a factor that deteriorates the ability to detect biomolecules.

Accordingly, the present inventors have earnestly studied to solve the problems of the prior art, and thus found that, when a probe is loaded on an encoded hydrogel particle designed such that a code region is integrated with a probe region, a target biomolecule can be detected with high accuracy and high sensitivity. The present invention has been achieved based on this finding.

Disclosure of Invention

Technical challenge

It is an object of the present invention to provide a method for preparing an encoded hydrogel particle comprising synthesizing a hydrogel particle and conjugating a probe to the hydrogel particle, and an encoded hydrogel particle prepared by the method.

Means for solving the problems

The present invention provides a method for preparing an encoded hydrogel particle comprising synthesizing a hydrogel particle and conjugating a probe to the hydrogel particle, and an encoded hydrogel particle prepared by the method.

The present invention also provides encoded hydrogel particles designed such that the code region is integrated with the probe region.

Effects of the invention

The method of the invention ensures loading of probes with significantly improved efficiency and uniform distribution of the loaded probes, while avoiding the potential problem that unreacted ends may inhibit detection of biomolecules. Furthermore, the encoded hydrogel particles of the present invention can be used for highly sensitive multiplex detection of target biomolecules such as nucleic acids and proteins. Thus, the encoded hydrogel particles are applicable in disease diagnosis and drug screening, and thus can be generally used in the field of medical diagnosis such as molecular diagnosis.

In particular, the methods of the invention enable conjugation of at least 8.2 times the number of probes to hydrogel particles as compared to conventional methods. In addition, the encoded hydrogel particle of the present invention prevents non-specific binding (false-positive signal) to a target biomolecule, which may be caused by the presence of unreacted ends, avoids the problem of poor detection performance caused by the immobilization of essential materials to be used for detection, has improved detection sensitivity for the target biomolecule, detects the target biomolecule in a shorter time, has improved specificity for the target biomolecule, and has improved ability to capture the target biomolecule.

Drawings

Fig. 1 shows diagrammatically a process for synthesizing hydrogel particles according to the invention and unreacted ends remaining in the particles after synthesis.

Fig. 2 is a schematic diagram showing a method for synthesizing hydrogel particles by replica molding (replication molding).

Figure 3 schematically shows a process for conjugating probes to unreacted termini remaining after synthesis of hydrogel particles according to the invention.

Fig. 4 is a conceptual diagram illustrating the size of hydrogel particles and code regions according to the present invention.

FIG. 5 shows the time-dependent kinetics (kinetics) a of probe ligation via thiol-ene reaction and compares the fluorescence signals b from conventional and thiol-ene particles.

Figure 6 compares the fluorescence signals a from the conventional particles and thiol-ene particles as a function of salt (NaCl) concentration and shows the specificity test result B.

Figure 7 compares the fluorescence signal a from the conventional and thiol-ene particles as a function of incubation time and shows the detection sensitivity test result b. In b, each of the dashed lines parallel to the X-axis corresponds to 3 times the standard deviation of the signal from the control particle.

Figure 8 compares the extent of antibody polymerization on conventional particles and thiol-ene particles.

Figure 9 compares the fluorescence signal from thiol-ene particles as a function of VEGF concentration with the optical density a of E L ISA and shows the normalized detection signal b from E L ISA and thiol-ene particles as a function of incubation time.

Fig. 10 shows the results of detection sensitivity tests for VEGF, PIGF, and CG beta. The line parallel to the X-axis corresponds to 3 times the standard deviation of the signal from the control particles.

Figure 11 shows the results of a multiplex detection assay for three different proteins and compares the fluorescence signal from thiol-ene particles in a total of 8 cases of proteins.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the terminology used herein is well known and commonly employed in the art.

In one aspect, the invention relates to a method for preparing an encoded hydrogel particle comprising synthesizing a hydrogel particle and conjugating a probe to the hydrogel particle. The hydrogel particles have available pores that are sized to facilitate loading of the probe and to ensure free mass transfer of the target biomolecule.

The probes are conjugated to the synthesized hydrogel particles through unreacted ends remaining in the hydrogel particles. Specifically, the probe is conjugated to the synthesized hydrogel particle through a covalent bond between a carbon-carbon double bond (C ═ C) of an unreacted terminal and a functional group of the probe (fig. 1 to 3).

The probes crosslink with the synthesized hydrogel particles via free radical reactions and spontaneous electron transfer in solution. The radical reaction for crosslinking the probe with the synthesized hydrogel particle is initiated by irradiation with UV in the presence of a photoinitiator or by applying thermal energy in the presence of a thermal initiator. The radical reaction causes the formation of a covalent bond. Alternatively, the probes may be crosslinked to the synthesized hydrogel particles via electron transfer in solution. In this case, the electrons in the aqueous solution or polar solvent act as nucleophiles to form covalent bonds. Preferably, the probe is crosslinked at a temperature of 0 ℃ to 90 ℃, at which temperature the probe remains most stable.

The term "probe" as used herein refers to a reagent capable of specifically binding to target material in a sample to specifically identify the presence of target material in the sample.

The probe may comprise a chemically reactive group that binds to DNA, RNA, protein or later probes.

Hydrogel particles can be synthesized by various processes such as stop flow lithography (SF L) (FIG. 1) and replica molding (FIG. 2.) according to one embodiment of the present invention, hydrogel particles are synthesized by stop flow lithography (SF L). in this embodiment, a precursor fluid is allowed to flow into the channel or irradiated with UV in the channel.

The precursor fluid may comprise a photocurable monomer containing a methacrylate or acrylate group and a photoinitiator. The photocurable monomer may be, for example, a polyethylene glycol monomer, a 2-Methacryloyloxyethyl Phosphorylcholine (MPC) monomer, or a mixture thereof. The precursor fluid contains a porogen, such as polyethylene glycol and/or Deionized (DI) water. Alternatively, the precursor fluid may comprise a plurality of different photocurable monomers.

The encoded hydrogel particles are designed such that the code regions are integrated with the probe regions. The code comprises a geometric code. The geometry code may be composed of a combination of different types of graphics. In a conventionally encoded hydrogel particle designed such that the code region is separated from the probe region, the signal from the code region may overlap with the detection signal due to the distribution of unreacted ends over the hydrogel particle. This problem can be avoided by the design of the encoded hydrogel particles according to the invention, wherein the code region is integrated with the probe region.

The hydrogel particles may further comprise functional nanoparticles.

In another aspect, the invention relates to an encoded hydrogel particle prepared by the method.

In yet another aspect, the present invention relates to an encoded hydrogel particle designed such that the code region is integrated with the probe region.

Modes for carrying out the invention

[ examples ]

The present invention will be explained in more detail with reference to the following examples. It will be appreciated by those skilled in the art that these examples are illustrative only and that the scope of the invention is not to be construed as being limited thereto. The substantial scope of the present invention should, therefore, be defined by the appended claims and equivalents thereof.

Example 1: synthesis of encoded hydrogel particles, DNA-loaded probes, and detection of target microRNAs

1-1: fabrication of microfluidic devices

The microfluidic device was designed using AutoCAD (Autodesk, usa) and printed on a photomask film (Han & All Technology, korea) particles were designed to have a size of 50 micrometers (width) × 50 micrometers (length) × 20 micrometers (height) which can be divided into 9 zones to generate a bright detection signal for encoding (fig. 4) using a silicon wafer coated with SU-825 (Microchem), usa) to create a SU-8 master by photolithography which acts as a negative photoresist, the thickness of the SU-8 master containing the pattern of the microfluidic device was adjusted to 24 micrometers, Polydimethylsiloxane (polydimethysiloxane; PDMS) mixed with a curing agent was poured onto the SU-8 and cured at 70 ℃ for 8 hours, then the master was punched with a punch (1 mm) to create a PDMS slab, PDMS slab was peeled off the master and the PDMS slab was then baked to create a slab with a final slide of PDMS, and the PDMS was attached to a glass slide and the PDMS slab was cured at 70 ℃ for 8 minutes.

1-2: synthetic hydrogel particles

Synthesis of hydrogel particles via stop-flow lithography (SF L) (k.w. bang et al, lab-on-a-chip (L abcip), 2011, vol 11, p 743 to p 747.) briefly, to perform SF L, a UV light-emitting diode (sorlebs, uk) was used as the light source and a pressure regulator (ITV0031-3B L pneumatic (SMCpneumatics), japan) was used to control the flow of fluid in a microfluidic device, UV L ED and pressure regulator were controlled in a synchronized manner by custom circuit boards and L abView (National instruments, usa) codes, a hydrogel particle was synthesized by UV L ED through a film photomask (Hanall technology, korea) mounted on an inverted microscope (Axiovert 200, Zeiss, germany), a) a periodic 50 ms of UV particles were synthesized by UV L ED while the precursor solution was stopped in the channel during SF L.

The precursor solution consisted of 20% (v/v) polyethylene glycol diacrylate 700(PEG700DA, Sigma Aldrich, usa), 40% (v/v) polyethylene glycol 600 as a porogen (PEG200, Sigma Aldrich), 5% delta-firm (Darocur)1173 as a photoinitiator (Sigma Aldrich), and 35% Deionized (DI) water after synthesis, hydrogel particles were collected in microtubes pre-filled with 200 microliters of a mixture (5 × PBST buffer) consisting of 5 o × PBS buffer (Sigma Aldrich) and 0.05% (v/v) Tween (Tween) -20 (Sigma Aldrich) — this collected particles were vortexed and centrifuged for about 1 minute.

Hydrogel particles for miRNA detection were synthesized by conventional methods (y.h. lo (Roh) et al, Analyst (Analyst), 2016, vol 141, p 4578 to p 4586.) a precursor solution consisting of 20% (v/v) PEG700DA, 40% (v/v) PEG200, 5% (v/v) photoinitiator and 35% (v/v)3 × Tris aminomethane EDTA (Tris EDTA; TE) buffer (sigma aldrich) was mixed with acrylamide (acrydite) modified ssDNA oligomers (Integrated DNA technologies) in a volume ratio of 9: 1.

1-3: DNA Probe Loading

Thiol-modified ssDNA oligomers (integrated DNA technology, USA) are reduced in 0.5 moles per liter of tris [ 2-carboxyethyl ] phosphine (Thermo Fisher Scientific, USA) to form active thiol groups after which the reduced oligomers are mixed at a concentration of about 7 particles/microliter with hydrogel particles suspended in 140 microliters of buffer, China the final mixed solution is incubated in a thermal shaker (constant temperature shaker, Hangzhou Instruments Co., L td), China) at a constant temperature of 37 ℃ while stirring at 1,500 revolutions per minute.

1-4: MiRNA detection assay

First, 40 microliters of 1 × TET buffer consisting of 1 × TE buffer (sigma aldrich) and 0.05% (v/v) tween-20 was applied to each microtube, followed by the addition of 5 microliters of target miRNA.

To observe the effect of NaCl concentration, the final concentration of NaCl in the hybridization buffer was adjusted to 50 to 350 mmol per liter, the assay was performed at 55 ℃ for 90 minutes, after hybridization, three times with 350 microliters of 1 × TET hydrogel particles containing 50 mmol per liter of NaCl, and then 245 microliters of ligation reaction mixture was added, this ligation reaction mixture consisted of 1350 microliters of 1 × TET, 150 microliters of 10 × NE buffer 2 (new england Biolabs), usa), 250 nmoles per liter ATP (new england biologies), 40 nmoles per liter of universal linker (integrated DNA technology), and 800 units/ml of T4 DNA ligase (seemer femtology technology), then the mixture was hybridized in a thermal shaker for 45 minutes at 21.5 ℃, after another washing step, avidin-phycoerythrin 1 × TET diluted 10 times (SA-PE; streptavidine technology (streptazif technology) was added for final analysis at 50. 5 ℃ after incubation of the hydrogel particles at 50. 595 minutes, the technology step was performed for 50. micro-gel analysis at 50 minutes.

1-5: image analysis

RGB fluorescence images of hydrogel particles were acquired using a digital single-lens reflex (DS L R) camera (EOS 6D, Canon, japan.) for monochromatic fluorescence images used to analyze the intensity of target signals from the probe region, a scientific complementary metal-oxide-semiconductor (sCMOS) camera (Prime, optical properties (Photometrics), usa) was used, both cameras were connected to an inverted microscope (Axiovert 200, zeiss) and a L ED lamp (HXP 120V, zeiss) was filtered through a cube set to image the particles.

1-6: measurement results

At least 8.2 times greater number of probes (single-stranded DNA) were conjugated in the encoded hydrogel particles (hereinafter referred to as "thiol-ene particles") prepared by synthesizing the hydrogel particles and loading the probes on the hydrogel particles, compared to in the encoded hydrogel particles prepared from a precursor containing the probes according to the conventional method (hereinafter referred to as "conventional particles") (fig. 5).

Nucleic acids were detected by varying the concentration of NaCl from 50 to 350 millimoles per liter. Analysis showed that detection sensitivity was improved, but detection specificity decreased with increasing NaCl concentration. Unlike conventional particles, thiol-ene particles produced the best signal at low NaCl concentrations (200 mmol per liter) (a of fig. 6).

The same procedure for miRNA detection was used for specificity testing except that the initial amount of 1 × TET was 25. mu.l, four microRNAs (let-7a, let-7B, let-7c and let-7d) were added in an amount of 1000amol, and the NaCl concentration was 200 mmoles per liter, therefore, the maximum cross-reactivity decreased from 25% to 15% (B of FIG. 6).

The measurement is performed by changing the measurement time for nucleic acid detection. Analysis showed that thiol-ene particles produced the same fluorescence signal at a rate at least 3 times higher than the conventional particles (a of fig. 7).

The initial amount of 1 × TET was adjusted to 40. mu.l, signals were detected by adding 50, 100, 500, 1000 and 5000amol of conventional particles and thiol-ene particles having NaCl concentrations of 350 and 200 mmole per liter, respectively, and thereafter, the detection limit was obtained by extrapolating the curve of the fluorescence signal against the target concentration, and therefore, the detection limit of thiol-ene particles (3.4amol) was lower than that of conventional particles (4.9amol) (FIG. 7 b).

For protein detection, the antibody was easily aggregated in the conventional particles due to poor compatibility with the precursor, and no antibody polymerization was observed in thiol-ene particles, so that the detection sensitivity was improved (fig. 8).

Example 2: synthesis of encoded hydrogel particles, antibody-loaded probes, and detection of target proteins

2-1: fabrication of microfluidic devices

A microfluidic device for synthesizing hydrogel particles was manufactured by the same method described in example 1.

2-2: synthetic hydrogel particles and antibody-loaded probes

Particle synthesis and probe loading were carried out in two unconnected (discrete) steps precursor solutions consisting of 20% (v/v) polyethylene glycol diacrylate 700(PEG700DA, sigma aldrich, usa), 40% (v/v) polyethylene glycol 600(PEG200, sigma aldrich) as a porogen, 5% delta-solid 1173 (sigma aldrich) as a photoinitiator, and 35% Deionized (DI) water one cycle of particle synthesis via stop flow lithography consists of 400 milliseconds of flow, 200 milliseconds of stop, 75 milliseconds of UV exposure, and 200 milliseconds of hold time for each microliter of protein assay, 1 hour of synthesis using a 1-D photomask to produce approximately 4000 particles using 1-D photomasks.

2-3: protein detection

For protein detection, 50 particles in 25 microliters of 1 × PBST and 25 microliters of 2 × target protein in 2% BSA in 1 × PBS are placed in a microtube. after incubating the particles for 2 hours at 1500 rpm at 25 ℃, three washes in 1 × PBST, 10 microliters of reconstituted secondary antibody (12.5 micrograms/microliter for I L-6, 25 micrograms/microliter for VEGF, 15 micrograms/microliter for PIGF, and 125 micrograms/microliter for CG beta) is added to the protein-binding particles in 40 microliters 1 × PBST. after incubating the mixture for 1 hour at 1500 rpm at 25 ℃, three washes in 1 × PBST, 10 microliters of streptavidin-phycoerythrin (SA-PE, life technology) diluted 50-fold in 5% BSA in 1 × PBST is added to the particles in 40 microliters 1 × PBST.

2-4: image analysis

The same system as described in example 1 was used for image analysis. Images were taken with an exposure time of 50 milliseconds. The intensity of the light source was fixed at 1100 milliwatts per square centimeter. The fluorescence intensity of the images retrieved in TIFF format was measured using the image J program.

2-5：ELISA

The process followed the general E L ISA protocol provided by the manufacturer 96-well microplates (addy & D Systems)) were coated with 100 microliters per well of diluted VEGF capture antibody (1 microgram/microliter) at 25 ℃ and incubated overnight.the wells were rinsed three times with 400 microliters of 1 × PBST to block free sites, the plates were incubated with 300 microliters of 1% BSA in 1 × PBS for 1 hour after two washes, the plates were incubated with 100 microliters of target protein diluted in 1% BSA in 1 × PBS for 2 hours after two washes, the plates were washed three times before adding 100 microliters of diluted detection antibody (100 ng/ml), the plates were incubated for 2 hours at 25 ℃, after three washes, 100 microliters of 40-fold diluted streptavidin-HRP (addy bioanals) were added to each well and 20 minutes, the plates were washed three times, and 100 microliters of substrate solution (addy andy catalog, 99570 min and 25 ℃ after adding 20 minutes of diluted streptavidin-HRP (addy) to each well and reading in a 450 μm optical catalog (addy) to correct for defects in the plate using a micrometer densitometer, a densitometer reading from a micrometer densitometer, a densitometer, read from a densitometer, a densifier, a dyno. was added at 25 ℃ after adding 99570, a.

2-6: multiplex assays

In each case, 150 particles (50 per protein) in 100 microliters of 1 × PBST were mixed with 100 microliters of 2 × target protein in 2% BSA in 1 × PBS premixture was prepared by mixing 25 microliters of 2% BSA in 1 × PBS with 25 microliters of 8 × target protein (in the presence of the target) in 2% BSA in 1 × 2PBS and 25 microliters of 2% BSA in 1 × PBS (in the absence of the target). particles in 8 different microliter target combinations were incubated at 25 ℃ for 2 hours 8658 microliters in 1 × PBST, and 10 microliters of three secondary antibodies (25 micrograms/microliter for VEGF, 15 micrograms/microliter for PIGF, and 125 micrograms/CG for beta) were each added to 20 microliters 1 × PBST and the particles were washed in 1500 microliters/1 PBST for three times 6330 minutes at 25 ℃ and diluted with streptavidin labeled 50 times the fluorescent dye in streptavidin 1 PBST 364 after washing the three times of PBST 3-streptavidin × PBST.

2-7: measurement results

According to conventional methods for preparing encoded hydrogel particles from probe-containing precursors, the presence of a hydrophobic initiator causes the antibody to aggregate. In contrast, the method of the present invention for preparing encoded hydrogel particles by synthesizing hydrogel particles and loading probes on hydrogel particles prevents antibody aggregation and ensures uniform conjugation of probes to the particles (fig. 8).

In a comparative experiment using E L ISA, which is a procedure commonly used for protein detection, the thiol-ene particle assay has a detection range of 17.7 to 60000 pg/ml, yielding a 3.5 recorded range, compared to E L ISA yielding a 1.8 recorded (log) range with a detection range of 31.2 to 2000 pg/ml, the lower detection limit (17.7 pg/ml) exhibits greatly improved performance of the thiol-ene particle assay compared to E L ISA (fig. 9 a).

Normalized fluorescence signals of the E L ISA and thiol-ene particle assays were observed as a function of incubation time therefore, slightly less than 1 hour of incubation of the thiol-ene particle assay yielded signals comparable to 2 hours of incubation in the E L ISA (fig. 9 b).

To obtain calibration curves for the three proteins (VEGF, PIGF, and CG beta), the proteins were added at different concentrations to detect the signal, and then the curves of fluorescence signal versus protein concentration were extrapolated to determine the limit of detection, therefore, the limits of detection (limits of detection; L OD) were 17.7, 17.5, and 4.2 pg/ml for VEGF, PIGF, and CG beta, respectively (FIG. 10).

To confirm whether multiplex detection of three proteins is possible, a total of eight cases depending on the presence or absence of proteins were used. In all cases the multiplex assay was performed successfully (FIG. 11).

Although the details of the present disclosure have been described in detail, it will be apparent to those skilled in the art that such details are merely preferred embodiments and are not intended to limit the scope of the invention. Therefore, the true scope of the invention is defined by the following claims and their equivalents.

Industrial applicability

The method of the invention ensures loading of probes with significantly improved efficiency and uniform distribution of the loaded probes, while avoiding the potential problem that unreacted ends may inhibit detection of biomolecules. Furthermore, the encoded hydrogel particles of the present invention can be used for highly sensitive multiplex detection of target biomolecules such as nucleic acids and proteins. Thus, the encoded hydrogel particles are applicable in disease diagnosis and drug screening, and thus can be generally used in the field of medical diagnosis such as molecular diagnosis.