KR20090038597A - Signal detecting method for trace of biomolecule using photolytic oligomer and nano particle - Google Patents

Abstract

A signal detecting method for the extremely small amount of bio-molecule using photodegradable oligomer and nano-particles is provided to enable an operator to obtain amplified signals more than those of the actual amount of bio-molecule by labeling the photodegradable oligomer on the nano-particles coupled with bio-molecules. A signal detecting method for the extremely small amount of bio-molecule comprises the following steps of: catching a target bio-molecule in a sample with a probe; coupling the caught target bio-molecule with nano-particles; coupling the nano-particles with a photodegradable oligomer; and decomposing a boding site of the nano-particles and the photodegradable oligomer through light irradiation and detecting signals of target bio-molecule from the oligomer obtained by decomposition.

Description

Signal detection method for trace biomolecules using photodegradable oligomers and nanoparticles {SIGNAL DETECTING METHOD FOR TRACE OF BIOMOLECULE USING PHOTOLYTIC OLIGOMER AND NANO PARTICLE}

The present invention relates to a method for detecting signals for biomolecules present in trace amounts using photodegradable oligomers and nanoparticles. The present invention is derived from research conducted as part of the IT source technology development project of the Ministry of Information and Communication and the Ministry of Information and Communication Research and Development. [Task Management Number: 2006-S-074-02, Title: High-performance biosensor system using nanoparticles ].

Detecting and analyzing traces of biological material is very important for biotechnology and medicine. In particular, in order to save the patient's life from cancer or acute disease and to prevent the spread of the disease, the earlier the time for identifying the cause and possibility of the disease is better. Therefore, techniques have been developed for the detection and analysis of trace biomaterials to determine the extent of disease progression and the likelihood of onset.

Among them, in the case of DNA analysis, polymerase chain reaction (PCR) was devised in the 1980s, so that a part of specific DNA could be easily amplified, so that research on a target gene was easy. However, the detection and analysis of the protein directly causing the disease is completely dependent on the development of equipment and technology, and the detection and analysis of the trace amount of protein is difficult at present. Many studies have been preceded.

Enzyme Linked ImmunoSorbent Assay (ELISA), the most widely used method of analyzing the concentration of free cytokines or kemokines present in normal biological fluids, for example in vivo biological fluids In this case, the antigen and the primary antibody bind in the same molar ratio, and the detection limit is known to be about 6 picomoles. Therefore, it is known that conventional ELISA cannot detect interleukin-8 (IL-8) in normal human plasma. In addition, the ELISA uses a secondary antibody labeled with an enzyme or a luminescent substance such as horseradish peroxidase (HRP), etc., for binding of the antigen and the primary antibody, Acquire and analyze electrical or optical signals. In the case of using the secondary antibody and a separate enzyme as described above, there is a disadvantage in that the time and cost required for the assay are increased, and the results of the assay differ depending on the storage time and method since the materials used are biological materials. In addition, if there is a very small amount of protein (generally less than femto mole / ml), it is impossible to directly analyze, so additional samples are secured and the concentration of the sample is increased by using a chromatography method. There is a disadvantage in that manpower and time are required for this.

In addition, most protein analysis methods including the ELISA may denature the protein to be analyzed during the reaction process, and thus, further analysis of the reacted protein may be almost impossible.

As an example of an attempt to overcome the above detection limit, it has been disclosed to secure an amplified signal using nano-based bio-bar-code DNA (Nam JM et. al . , Science, 2003; 301: 1884). However, since the prior art requires the use of the melting temperature (Tm) for the separation and binding of the bio-barcode DNA, there is a need to raise and lower the temperature of the entire system, thereby disrupting the binding by the antigen-antibody reaction. Can be. In addition, the molecule that binds to the gold magnetic particles is limited to a substance having a sulfhydryl group (-SH), and thus, flexible operation is difficult. In addition, there is a disadvantage that a secondary antibody that recognizes other sites of the target material is necessary for the analysis of the protein.

Photodegradable materials that can be incorporated into DNA are used for linkers of biological materials, and the reason is as follows. When it is necessary to separate the biological material bound to the linker, the general DNA linker requires chemical reaction and heat separation to expose the biomaterial to denaturation, whereas the photodegradable material is irradiated with a range of light. Since it is easily separated, the denaturation of the analyte is minimized. Secondly, when separating the biological material from the beads or substrate, the general DNA connector requires a certain time for hybridization or dehybridization of DNA, whereas the connector including the photodegradable material is the longest moisture. The analyte can be separated within.

Therefore, the inventors capture a target biomolecule and bind the nanoparticles, and then, to the nanoparticles, a trace amount of the target biomarker is detected by using a detection method including labeling the photodegradable oligomer incorporated in the oligomer sequence. As a result of analyzing the molecules, the present invention was completed by observing that the conventional detection limit was overcome by signal amplification by the oligomer without concentrating the sample.

Accordingly, an object of the present invention is to capture the biomolecules and bind the nanoparticles, instead of using secondary antibodies, enzymes, and the like to analyze the biomolecules, particularly proteins, present in trace amounts in the sample, and then the nanoparticles. The present invention provides a signal detection method for a trace amount of biomolecules capable of obtaining a signal amplified more than the actual amount of biomolecules by labeling a photodegradable oligomer for signal amplification.

In order to achieve the above object, the present invention

Capturing the target biomolecule in the sample with the probe;

Binding the captured target biomolecule with nanoparticles;

A signal amplifying step of coupling the combined nanoparticles with a photodegradable oligomer for signal amplification;

Signal detection method for a biomolecule comprising a signal detection step of decomposing the bond between the nanoparticles and the photodegradable oligomer for signal amplification by light irradiation, and detecting the signal for the target biomolecule from the oligomer obtained by the decomposition To provide.

Hereinafter, the present invention will be described in detail.

In the detection method according to the present invention, in analyzing a biomolecule present in a trace amount of femto mole / ml level in a sample, after binding the nanoparticles to a target biomolecule, a photodegradable oligomer for signal amplification on the nanoparticles By analyzing the signal amplification oligomers separated by light irradiation, and using conventional DNA analysis, a large amount of sample, denaturation of target biomolecules, high cost, or a long time is required, unlike an analytical method requiring a long time. Accurate analysis of the sample is possible. Therefore, the present invention is a method that can be analyzed easily and cheaply than conventional methods by using a photolysis reaction of a trace amount of biomolecule present in the sample.

First, in order to construct a probe, a connection photodegradable oligomer having magnetic particles coated with a third binding material immobilization means and a third binding material capable of binding to the magnetic particles is prepared.

The magnetic particles are preferably polymers having iron oxide and the like magnetized to an external magnetic field, and more preferably polystyrene.

The binding substance immobilization means according to the present invention is biotin, streptavidin, avidin, aptamer, lectin, DNA, RNA, ligand, coenzyme, inorganic ion, cofactor, sugar It is characterized in that one selected from the group comprising a lipid and a substrate. In one embodiment of the present invention, streptavidin was used as a binder immobilization means, but is not limited thereto.

The linking photodegradable oligomer is an oligomer for connecting the magnetic particles and the trapping agent, and includes a binding material chemically bonded to a binding material immobilization means coated on the magnetic particles at a 3 ′ region, and the photodegradable material in the sequence Characterized in that it comprises a. In one embodiment of the present invention, as the binding material, biotin covalently bonded to the streptavidin as the binding material immobilization means was used, but is not limited thereto, and may be adjusted to a material binding thereto according to the binding material immobilization means. have. The photodegradable material may be a photodegradable material that can be incorporated into DNA, preferably a phosphoramidite compound, more preferably PC-o-nitrophenyl-CE-phosphora having the formula Midite (PC-o-nitrophenyl-CE-phosphoramidite; [o-Nitrophenyl-1-O- (2-cyanoethyl-N, N-diisopropylphosphoramidyl) -3-O- (4,4´-dimethoxytriphenylmethyl) -1,3 -propandiol]).

[Formula]

The sequence of the linking photodegradable oligomer prepared in one embodiment of the present invention may be described as 3′-biotin-AAA-N-AAA-5 ′, wherein A represents adenine and N represents PC -o-nitrophenyl-CE-phosphoramidite. In addition, in one embodiment of the present invention using a light of a wavelength of 200 to 400nm which is a wavelength at which the photo-degradable material PC-o-nitrophenyl-CE-phosphoramidite is decomposed to produce two monophosphate fragments. However, the present invention is not limited thereto, and any light ray having a wavelength sufficient to decompose the photodegradable material used may be used.

The amount of bonding between the magnetic particles and the photodegradable oligomer for connection is preferably adjusted according to the volume of the magnetic particles.

The oligomers according to the invention are DNA or DNA analogues, said DNA analogues being preferably selected from the group comprising PNA, TNA and GNA.

Next, the phosphate group in the 5 ′ region of the linking photodegradable oligomer is immobilized with a trapping agent that binds to the target biomolecule, thereby completing the probe. In one embodiment of the present invention, the carbodiimide crosslinker EDC (1 -ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride) and imidazole were used to convert the phosphate group at the 5 'site of the linking photodegradable oligomer into phosphoryllimidazolide, followed by anti-DcR3 (NT 1) Immobilized by binding to the amine group of the antibody to complete the probe, but is not limited thereto. In the present invention, the capture agent is a substance for capturing the target biomolecule, and may be any biomolecule that binds to the target biomolecule in an antigen-antibody reaction. In one embodiment of the present invention, anti-DcR3, an antibody of DcR3, was used as a capturer. However, if anti-DcR3 is to be analyzed as a target biomolecule, the antigen DcR3 can be used as a capturer. Thus, the target biomolecule and the trapping agent may be molecules capable of binding to each other by an antigen-antibody reaction, for example, proteins, peptides, haptens and antibodies to the proteins, peptides and haptens that may be immunogens, It is not limited.

Thus, in the present invention, the probe

Photodegradable oligomers comprising a photodegradable substance in a sequence;

Magnetic particles fixed to one end of the photodegradable oligomer; And

It is fixed to the other end of the photodegradable oligomer is characterized in that it comprises a trap to bind to the target biomolecule.

Next, the sample is contacted with the probe under conditions that allow the target biomolecule in the sample to bind with the trap of the probe to generate a probe-target biomolecule complex.

Samples according to the invention are extracts from virus, bacteria, cells or tissues, lysates or purified products, blood, plasma, serum, lymph, bone marrow fluid, saliva, ocular fluid, semen, brain extracts, spinal fluid, joint fluids, thymus Fluid, ascites and amniotic fluid.

Next, in order to bind the nanoparticles of the present invention to the target biomolecules, the target biomolecules bound to the traps are labeled with a first binding agent chemically bonded to the first binding agent immobilization means immobilized on the surface of the nanoparticles. do. The binding substance of the present invention is not limited as long as it is a substance that can be labeled with a functional group on the surface of the target biomolecule, such as a primary amine group, a sulfhydryl group, a carbonyl group, or a carboxyl group. . Some examples of materials capable of labeling a binding material by reacting with the functional group are as follows, but are not limited thereto. Examples of the material that reacts with the primary amine group include an imidoester compound and N-hydroxysuccinimide, and examples of the material that reacts with the sulfhydryl group include maleimide, haloacetyls, Pyridyl disulfide, and the like, and a substance that reacts with a carbonyl group may include carbodiimides, and the like, which may react with carboxyl groups such as primary amines or hydrazides, and the like. Can be mentioned. In one embodiment of the present invention, in adopting biotinylation, biotin, a binding substance, was labeled using N-hydroxysuccinimide reacting with the primary amine group of the target biomolecule. As a result, the present invention improves the signal detection accuracy of the target biomolecule as shown in the embodiment, because the amine group of the capture has already been preempted in the step of binding the capture to the photodegradable oligomer for linkage. This is because biotin can be selectively labeled with the amine group of the target biomolecule. After biotinylation, when washing to remove residual reagents and unnecessary salts, care must be taken to ensure that the binding between the capture and target biomolecules is not separated by pH changes.

Next, the target biomolecule labeled with the first binding agent (for example, biotin) and the first binding agent immobilization means (for example, streptavidin) are chemically bonded to the first binding agent. The nanoparticles are mixed and combined. Thereafter, as an additional step, the photodegradable material in the probe can be decomposed by light irradiation in the above-mentioned range to remove the magnetic particles, thereby producing a captureer-target biomolecule-nanoparticle complex.

As the nanoparticles of the present invention, microspheres including one material selected from the group consisting of gold, silver, silica and polystyrene may be used, and the nanoparticles may be coated with the binder immobilization means of the present invention. It is characterized by. Nanoparticles coated with the binder immobilization means preferably have a diameter of less than 1 μm, but are not particularly limited.

Signals are amplified by labeling the capture-target biomolecule-nanoparticle complex with a photodegradable oligomer for signal amplification labeled with a second binding agent chemically bonded to a second binding agent immobilization means coated on the surface of the nanoparticles.

The first binding material immobilization means, the second binding material immobilization means, and the third binding material immobilization means of the present invention may be arbitrarily adjusted to be the same or different. Accordingly, the first binding material, the second binding material, and the third binding material that bind to the binding material fixing means may be arbitrarily adjusted to be the same or different depending on the experiment. In the present invention, preferred first, second and third binder immobilization means are streptavidin and the first, second and third binder are biotin.

The photodegradable oligomer for signal amplification according to an embodiment of the present invention is an oligomer for labeling nanoparticles, and includes a second binding material, preferably biotin, at a 5 ′ region and a photodegradable material in the sequence. It features. The sequence of the signal amplification photodegradable oligomer prepared in one embodiment of the present invention may be described as 5′-biotin-AAA-N-GGGGGGGGGGGGG-3 ′, wherein G represents guanine, and A is Adenine, N represents a photodegradable substance, ie PC-o-nitrophenyl-CE-phosphoramide.

In the present invention, the term "signal for biomolecule" refers to the conversion of quantitative values of a plurality of oligomers labeled on nanoparticles bound to a target biomolecule into optical, electrochemical, electronic signals, and the like. The qualitative and / or quantitative detection of target biomolecules is possible by analyzing the degree of detection.

In one embodiment of the present invention to remove the nanoparticles to separate only the signal amplification oligomer, the photo-degradable material PC-o-nitrophenyl-CE-phosphoramidite is decomposed two monophosphate fragments Light having a wavelength of 200 to 400 nm, which is a generated wavelength, is used, but is not limited thereto. Any light having a wavelength sufficient to decompose the photodegradable material used may be used.

The collected signal amplification oligomers can be detected and analyzed using a detector such as a UV-VIS spectrophotometer for conventional DNA quantification, or using LC-MS or MALDI-TOF. Since more DNA can be obtained than the number of moles of the target biomolecule, there is an advantage of detecting the target biomolecule as an amplified signal.

Through the detection method of the present invention, when the target biomolecule to be analyzed is present in a very small amount of femto mole / ml level, the degeneration of the target biomolecule is almost induced without additionally increasing the concentration or increasing the concentration of the sample. Without this, there is an effect that can detect the signal to the target biomolecule. Further, even after the above detection is completed, the above detection result can be stored as an oligomer for signal amplification, so that a reliable analysis result can be secured for a long time.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are provided to illustrate the present invention, and the present invention is not limited to the following examples and may be variously modified and changed.

Reference Example . Oligomer  synthesis

1-1. PC -o- nitrophenyl - CE - phosphoramidite For connection with Oligomer  Synthesizing

Synthesis was requested to Bioneer (Daejeon, Korea) to prepare a photodegradable oligomer having a sequence of 3′-biotin-AAA-N-AAA-5 ′. In the sequence, A means adenine and N means photo-degradable PC-o-nitrophenyl-CE-phosphoramidite (P / N 212393, Bruker daltonics).

1-2. PC -o- nitrophenyl - CE - phosphoramidite For signal amplification Oligomer  Synthesizing

By requesting synthesis to Bioneer (Daejeon, Korea), a photo-degradable oligomer for signal amplification having a sequence of 5′-Biotin-AAA-N-GGGGGGGGGGGGG-3 ′ was prepared. In the sequence, A means adenine, G means guanine, and N means photo-degradable PC-o-nitrophenyl-CE-phosphoramidite (P / N 212393, Bruker daltonics).

Example .

1-1. On target biomolecules  About Probe ( probe ) Produce

A. For connection Oligomer Magnetic particle complex

100 μl of a suspension of Dynabeads ^® M-280 (Invitrogen Dynal AS, Oslo, Norway), a magnetic particle coated with streptavidin on the surface, was added to 2X B & W buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2.0 M NaCl) 100 Wash three times with μl. In this case, the Dynabeads ^® M-280 100㎕ is capable of binding both with biotinylated oligomers of 200 picomolar (picomoles). 100 µl of the magnetic particles after the washing was suspended again with 100 µl of 2X B & W buffer to prepare a magnetic particle solution.

The oligomer solution was prepared by diluting 10 μl (1,000 picomolar) of the biotinylated linkage oligomer prepared in Reference Example 1-1 in 190 μl of ultrapure water.

100 μl of the magnetic particle solution and 100 μl of the oligomer solution were mixed and reacted with slow stirring at room temperature for 10-20 minutes. Mix gently, occasionally during the reaction. In the mixed solution, the oligomer is present in 500 picomol / 200 μl (ie, 2.5 picomol / μl). After completion of the reaction, 1.5 picomolar / μl of the linking oligomer is present in the supernatant. To determine the progress of the reaction, it was analyzed by NanoDrop (ND-1000 spectrophotometer, NanoDrop, USA).

After confirming that the reaction is completed, the magnetic particles bound to the oligomer for coupling using a magnet, separated, washed three times with 100 μl of 1 × B & W buffer, and suspended again with 75 μl of B & W buffer.

B. Probe (For connection Oligomer Magnetic Particles Catcher  Complex)

Anti-DcR3 (NT) (DcR3 (3H5 / DcR3), Cat No. 2, which is a capturer (antibody) to the DcR3 (NT) peptide (905-219, Assay Design, Inc., USA) antigen, which is the target analyte of this example. sc-53685, Santa Cruz, USA) 10 μl (100 μg IgG / 1.0 mL PBS) was dissolved in 90 μl of 0.5 mM imidazole solution (pH 6.0).

Using the following method, anti-DcR3 (NT) was bound to the 5′-phosphate group of the linking oligomer in the complex prepared in B.

1.25 mg of EDC (1-ethyl-3 [3-dimethylaminopropyl] carbodiimide hydrochloride, # 22980, PIERCE, USA) was added to a microcentrifuge tube containing 75 μl of the oligomeric-magnetic particle solution prepared in A. Inject and vortex to completely dissolve the EDC. Immediately thereafter, 50 µl of the anti-DcR3 (NT) / imidazole solution prepared above was added thereto. The contents were vortexed until the contents were completely dissolved and then centrifuged to collect the contents. 200 µl of 0.5 M imidazole solution (pH 6.0) was added thereto and allowed to react at room temperature for 24 hours. The unreacted EDC was removed, and the antibody-linked oligomer-magnetic particles were separated using a magnet, washed three times with PBS, and then dissolved in 100 μl of PBS buffer. In this way, a probe having a structure as shown in FIG. 3 was provided.

To check if the antibody is bound to the probe, a standard curve is prepared using Biorad protein assay standard II (CAT No: 500-0007, USA) and the micro-BCA method and accordingly Quantification was carried out and the results are shown in FIG. 7B.

1-2. Probe - Target biomolecule  Composite manufacturing

The target biomolecule DcR3 (NT) antigen of this Example was cited by ELISA technique (Santa-Cruz, Inc., USA), which is known to those skilled in the art as an antibody supplier. It was bound to the antibody of the probe prepared in.

First, the antigen DcR3 (NT) peptide (2 mg / ml) was diluted with PBS to 10 ng / ml, 100 pg / ml, 1.0 pg / ml. Each 25 μl of the diluted solution and 25 μl of the probe prepared in B of 1-1 were mixed, and then reacted at room temperature for 1 hour. Thereafter, in order to remove other unreacted substances and residues, they were separated by using a magnet and dissolved in each 50 µl of PBS.

To check the binding of the probe to the target biomolecules, a standard curve was created using Biorad's Protein-Analysis Standard Solution (Bio-rad protein assay standard II, CAT No: 500-0007, USA) and the micro-BCA method. It was quantified according to the results, it is shown in Figure 7c.

1-3. Target biomolecule  Selective labeling

Biotinylation kit for labeling the binding material for binding the nanoparticles (silica microspheres) coated with the binding material immobilization means (streptavidin) to the probe-target biomolecule complex of Example 1-2, Using EZ-Link sulfo NHS biotinylation kit, P / N 21217, PIERCE, USA), biotinylation was performed as described below according to the manufacturer's instructions.

First, 2.2 mg of Sulfo-NHS-biotin was dissolved in 500 µl of ultrapure water, and 10 µl of the solution was dispensed into each of the solutions prepared in 1-2 above, and reacted at room temperature for about 1 hour. A roller mixer was used to achieve gentle stirring during the reaction. When the reaction was completed, separated by a magnet and washed three times with 100 μl PBS solution, and dissolved in each 50 μl PBS solution. At this time, the antigen-selective biotinylation is performed in the present invention because the amine group of the antibody has preempted when the antibody binds to the linking oligomer. After biotinylation was performed, the probe-target biomolecule complex was separated using a magnet to remove remaining reagents and unnecessary salts, and then washed three times using 2M NaCl TE buffer. At this time, to prevent separation of the antigen and antibody, care should be taken not to change the pH. After washing it was dissolved in 2M NaCl TE buffer (100 μl).

1-4. Probe - Target biomolecule -Nanoparticle Complex

Streptavidin-coated silica microspheres (# CS01N, Bang's Laboratories, Inc., USA), the nanoparticles of the present invention, were appropriately washed in a suitable manner in advance and then washed in ultrapure water (100 μl). Melted. This was mixed in the same amount with the target biomolecule-probe complex solution dissolved in the 2M NaCl TE buffer. After mixing for 20 minutes at room temperature in an up-down manner, the probe-target biomolecule-nanoparticle complex was separated using a magnet. The separated complex was washed three times with 2M NaCl TE buffer and dissolved in 2M NaCl TE buffer (100 µl).

1-5. Lightly Probe  Remove Magnetic Particles

The probe-target biomolecule-nanoparticle complex solution (50 μl for each concentration) prepared in Example 1-4 was placed in a quartz reaction vessel and 365 nm and 254 in an ultraviolet illuminator (UV illuminator, Longlife filter, Spectroline). Light was irradiated for 15 minutes using the wavelength of nm simultaneously. During the irradiation, the particles were occasionally shaken to prevent the particles from settling. Then, after separating the magnetic particles M-280 using a magnet, the supernatant was taken. The separated supernatant was washed three times with 2M NaCl TE buffer, and then dissolved in 10 μl of 2M NaCl TE buffer.

1-6. Signal amplification Photodegradable With oligomer  Nanoparticle Label

To 10 µl of the antibody-antigen-nanoparticle complex solution 1-5 above, a photodegradable oligomer (100 µl) synthesized in Reference Example 2 was added and allowed to react for 20 minutes. After labeling using the streptavidin and biotin binding properties, centrifugation was performed to remove unlabeled oligomers and finally dissolved in 10 μl of ultrapure water.

1-7. Lightly  Antibody-antigen-nanoparticle complex removal

10 μl of the antibody-antigen-silica microsphere complex solution prepared in Example 1-6 was placed in a quartz reaction vessel and irradiated with light for 5 minutes using a wavelength of 365 nm and 254 nm in an ultraviolet irradiator. Subsequently, the antigen-antibody-nanoparticle complex was removed by centrifugation at 14,000 rpm, and the supernatant was collected to collect oligomers for signal amplification.

Test Example . On oligomers  by Signal amplification  Confirm

The supernatant separated in Example 1-7 was supplied using the UV-VIS spectrophotometer (UV-VIS spectrophotometer, model number, manufacturer, country), the amount supplied from Bionia (Daejeon, Korea), a synthesis company of oligonucleotides As a standard, a standard curve was prepared at 260/280 nm, and quantification was performed accordingly, and the results are shown in FIG. 6.

As shown in Figure 6, even when treated with 1 picogram / ml of DcR3 when using the analysis method of the present invention, it can be seen that the oligomer value is clearly displayed compared to the control, antigen 100 picogram / ml Even when treated with, the linearity (linearity) is good, it can be seen that the analysis method of the present invention is effective in amplifying the signal for a trace amount of biomolecules, especially proteins.

1 is a schematic diagram showing an embodiment of a detection method of the present invention.

Figure 2 is a schematic diagram of the oligomer having a photodegradable material of the present invention, the photodegradable material is PC-o-nitrophenyl-CE-phosphoramidite (PC-o-nitrophenyl-CE-phosphoramidite).

3 is a schematic diagram in which an oligomer having a photodegradable substance and an associated antibody are bound to magnetic particles.

4 is a schematic diagram showing the binding of antigen DcR3, a target biomolecule to be measured, to a probe used in the present invention.

5 is a schematic view of the signal immediately before the photolytic oligomer for separation by light irradiation.

6 is a graph showing the results of detecting and analyzing a signal for antigen DcR3 while varying the amount of antigen DcR3. Compared to the control PBS buffer, it shows very high reactivity in the presence of 1.0 pg / ml of antigen and linearity up to about 100 ng / ml.

Figure 7a is a graph showing the measurement of the amount of the photodegradable oligomer for the remaining in the reaction vessel before and after the reaction to determine whether the linking photodegradable oligomer to the magnetic particles.

Figure 7b is a graph showing the measurement of the amount of antibody remaining in the reaction vessel before and after the reaction to determine whether the antibody bound to the probe.

Figure 7c is a graph showing the measurement of the amount of antigen remaining in the reaction vessel before and after the reaction, in order to determine whether the antibody and the antigen binding.

Claims (17)

Capturing the target biomolecule in the sample with the probe; Binding the captured target biomolecule with nanoparticles; A signal amplifying step of coupling the combined nanoparticles with a photodegradable oligomer for signal amplification; A signal detection step of decomposing a binding site of the photodegradable oligomer for signal amplification combined with the nanoparticles by light irradiation and detecting a signal for the target biomolecule from the oligomer obtained by the decomposition; Signal detection method for a biomolecule comprising a. The method according to claim 1, The binding between the target biomolecule and the nanoparticles and the binding of the nanoparticle and the photodegradable oligomer for signal amplification are achieved by chemical bonding between a binding material immobilization means and a binding material bound thereto. Signal detection method. The method according to claim 2, The binder immobilization means, biotin, streptavidin, avidin, aptamer, lectin, DNA, RNA, ligand, coenzyme, inorganic ion, enzyme cofactor, sugar, lipid And a substrate comprising a substrate, The binding material is a signal detection method for a biomolecule, characterized in that selected from the substance chemically bonded to the binding material immobilization means. The method according to claim 2, The binding between the target biomolecule and the nanoparticles is made by chemical bonding between the first binding material immobilized on the captured target biomolecule and the first binding material immobilization means immobilized on the nanoparticle, Bonding between the nanoparticles and the signal-amplifying photodegradable oligomer is made by a chemical bond between a second binding material immobilized on the nanoparticles and a second binding material immobilization means immobilized on the signal-amplifying photodegradable oligomer A signal detection method for a biomolecule. The method according to claim 4, wherein the first and second binder immobilization means is streptavidin, and the first and second binder are biotin. The method of claim 1, wherein the nanoparticles are coated with a binder immobilization means. The method according to any one of claims 1 to 6, wherein the nanoparticles comprise one material selected from the group consisting of gold, silver, silica and polystyrene. The method according to claim 1, wherein the probe Photodegradable oligomers comprising a photodegradable substance in the sequence; Magnetic particles fixed to one end of the photodegradable oligomer; And A capturer fixed to the other end of the photodegradable oligomer to bind to the target biomolecule; Signal detection method for a biomolecule, characterized in that it comprises a. The method according to claim 8, The magnetic particles and the photodegradable oligomer are fixed to each other by a third binder and a third binder immobilization means, And said third binding agent immobilizing means is streptavidin and said third binding agent is biotin. The method according to claim 8, Before the signal amplification step, further comprising the step of decomposing the photodegradable material in the oligomer in the probe by light irradiation to remove the magnetic particles, signal detection method for a biomolecule. The method of claim 1 or 8, wherein the oligomer is a DNA or a DNA analogue, signal detection method for a biomolecule. The method of claim 8, wherein the photodegradable material is a photodegradable material that can be incorporated into DNA. The method of claim 12, wherein the photodegradable material comprises a phosphoramidite compound. The method according to claim 13, wherein the phosphoramidite compound is PC-o-nitrophenyl-CE-phosphoramidite, characterized in that the signal detection method for a biomolecule. The method of claim 14, wherein the light irradiation is performed using light having a wavelength of 200 to 400 nm, which is a wavelength at which the phosphoramidite compound is decomposed. The signal detection method of claim 1 or 8, wherein the target biomolecule or the capture agent is selected from the group comprising a protein, a peptide, a hapten and an antibody against the protein, the peptide and the hapten, respectively. Way. The method of claim 1, wherein the sample is a virus, bacteria, cell or tissue-derived extract, lysate or purified product, blood, plasma, serum, lymph, bone marrow fluid, saliva, ocular fluid, semen, brain extract, spinal fluid, joint fluid , Thymic fluid, ascites and amniotic fluid, characterized in that the signal detection method for biomolecules.

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