DETECTION METHOD OF TARGET ANALYTE USING GOLD NANOPROBE THROUGH OVERGROWTH OF COPPER CRYSTAL

Abstract

The present invention relates to a method for detecting a target analyte using a gold nanoparticle, comprising growing a copper crystal specifically on a gold nanoparticle by treating the gold nanoparticle with a solution comprising a copper ion, a polymer having a primary or secondary amine group, and a reducing agent, a composition for amplifying a signal used in the detection method above, and a kit for detecting a target analyte comprising the composition for amplifying a signal above.

Claims

1. A method for detecting a target analyte using a gold nanoparticle, comprising: Step 1 of preparing a substrate on which a target analyte is immobilized directly or through a material capable of capturing the target analyte; Step 2 of forming a complex with the target analyte by contacting a gold nanoparticle-labelled material that specifically binds to the target analyte with the substrate of Step 1; and Step 3 of growing a copper crystal specifically on the gold nanoparticle by treating the substrate, on which the complex of Step 2 is formed, with a solution comprising a copper ion, a polymer having a primary or secondary amine group, and a reducing agent.

2. The method of claim 1, wherein the polymer having a primary or secondary amine group is a polymer that can form a ligand complex with a copper ion.

3. The method of claim 2, wherein the polymer having a primary or secondary amine group is polyethyleneimine.

4. The method of claim 1, wherein the reducing agent is ascorbic acid, hydroxylamine or hydroquinone.

5. The method of claim 1, wherein Step 3 is performed at 10 C. to 35 C. for 3 to 20 minutes.

6. The method of claim 1, wherein a particle with grown copper crystal has a diameter of 100 to 1000 nm.

7. The method of claim 1, wherein the target analyte can be detected by the naked eye or using photos.

8. The method of claim 1, wherein the target analyte can be detected by a signal-to-noise ratio of 20 to 100.

9. A composition for amplifying a signal used in the method for detecting a target analyte using a gold nanoparticle of claim 1, comprising a compound capable of providing a copper ion in solution, a polymer having a primary or secondary amine group, and a reducing agent.

10. The composition of claim 9, wherein the compound capable of providing a copper ion in solution is copper chloride.

11. The composition of claim 9, wherein the polymer having a primary or secondary amine group is a polymer that can form a ligand complex with a copper ion.

12. The composition of claim 9, wherein the polymer having a primary or secondary amine group is polyethyleneimine.

13. The composition of claim 9, wherein the reducing agent is ascorbic acid, hydroxylamine or hydroquinone.

14. A kit for detecting a target analyte using a gold nanoparticle, comprising a compound capable of providing a copper ion in solution, a polymer having a primary or secondary amine group, and a reducing agent.

15. The kit of claim 14, further comprising a solution containing water as a solvent.

16. A method for preparing a copper crystal, comprising growing a copper crystal on a gold nanoparticle by treating the gold nanoparticle with a solution comprising a copper ion, a polymer having a primary or secondary amine group, and a reducing agent.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0038] FIG. 1 is a schematic representation of specific copper crystal overgrowth on gold nanoprobes according to the present invention.

[0039] FIG. 2 is a schematic representation of a method for detecting an analyte labeled with gold nanoprobes using copper crystal overgrowth.

[0040] FIG. 3 is a diagram showing DNA detection results using the number of gold nanoprobes and specific copper crystal overgrowth on the gold nanoprobes according to the present invention. As the control, the analysis result using silver instead of copper was used.

[0041] FIG. 4 is a diagram showing the results of norovirus detection using specific copper crystal overgrowth on the gold nanoprobes according to the present invention.

[0042] FIG. 5 is a diagram showing specific overgrowth of a copper crystal by a polymer having primary or secondary amine groups on a gold nanoparticle according to the present invention.

[0043] FIG. 6 is a diagram illustrating a DNA detection method using copper crystal overgrowth according to the present invention.

[0044] FIG. 7 is a diagram showing a norovirus detection method using copper crystal overgrowth according to the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS

[0045] The present invention will be described in more detail through exemplary embodiments. However, these exemplary embodiments are provided for illustrative purposes only, and are not intended to limit the scope of the invention.

Preparation Example 1: Synthesis of Gold Nanoprobe Labeled with Gold Nanoparticles on Antibodies for Norovirus Detection

[0046] Gold nanoparticles (1 mL) having a diameter of 50 nm dispersed in distilled water were reacted with polyethylene glycol (PEG) polymers (MW 5K, 200 M, 0.1 mL) having each of a thiol group (SH) and a carboxyl group (COOH) at both ends for 3 hours at room temperature to allow PEG polymers to bind to the surface of gold nanoparticles through a thiol group. The supernatant was removed by centrifugation at 6000 rpm for 10 minutes, then suspended in 0.25 mL of 50 mM MES buffer (2-(N-orpholino) ethanesulfonic acid, pH 4.8) and allowed to react with 0.1 mL of 1-Ethyl-3-(3-dimethylaminopropyecarbodiimide (EDC) at a concentration of 1 mg/mL and 0.1 mL of N-hydroxysulfosuccinimide (sulfo-NHS) at a concentration of 0.1 mg/mL at room temperature for 5 minutes to activate the carboxyl group. Again, the supernatant was removed by centrifugation at 6500 rpm for 15 minutes, suspended in 0.25 mL of 10 mM PBS (phosphate buffered saline, pH 7.2) and reacted with an anti-norovirus capsid protein VP1 antibody (Abeam, UK, # ab92976) at pH 7.2 for 3 hours at room temperature to allow the PEG-modified gold nanoparticles to bind through the amine group of the antibody using the activated carboxyl group. After repeating centrifugation twice at 6500 rpm at 4 C. for 10 minutes, the supernatant was removed and suspended in 0.05% PBST (phosphate buffered saline with Tween-20) to obtain a gold nanoprobe solution having a concentration of 200 pM.

Example 1: Norovirus Detection Method Using Gold Nanoparticle-Specific Copper Crystal Overgrowth

[0047] 1 L of a solution prepared by mixing a virus sample and 10 mM Tris buffer in a 1:1 volume ratio was loaded onto an aldehyde-modified glass substrate (Aldehyde glass, Luminano) and reacted at room temperature for 2 hours to allow the virus sample to bind to the glass substrate. After washing the glass substrate three times with 2 mL of 0.05% PBST, the exposed surface was blocked by treatment with 1% BSA solution for 30 minutes at room temperature. After washing the glass substrate three times with 2 mL of 0.05% PBST, 8 L of the gold nanoprobe solution prepared according to Preparation Example 1 was added thereto, and allowed to react at room temperature for 1 hour and washed three times with 2 mL of 0.05% PBST. Then, the glass substrate in which the reaction was completed was soaked into 35 mL of a copper enhancer solution prepared by mixing 5 mL of 0.1 M copper chloride (CuCl2), 5 mL of 1% polyethyleneimine (PEI), and 25 mL of 0.5 M ascorbic acid and allowed to react at room temperature for 10 minutes to overgrow the copper crystals on the gold nanoprobe. After the reaction was completed, the glass substrate was washed with water and dried. The substrate, on which the copper crystals were overgrown, was analyzed by ImageJ software to quantify the signal values, and the results are shown in FIG. 4.

Preparation Example 2: Synthesis of Gold Nanoprobe Labeled with Gold Nanoparticles on DNA for Detection of Bacillus anthracis Gene

[0048] 5-end thiolated Bacillus anthracis probe oligonucleotides (SEQ ID NO: 1: 5-SH-A.sub.10-PEG.sub.18-AAT GCT TTA TTC CAT TCC TGA TTT ATA TTT AAC TGT GCT T-3) was added in excess into a 10 nm diameter-gold nanoparticle solution at a concentration of 10 nM. The salt concentration was gradually increased to reach a final concentration of 0.15 M by adding 2.0 M NaCl solution using a double boiler containing hot water (95 C.). After centrifugation at 17000 rpm for 40 minutes, the supernatant was removed and suspended in 0.5 mL of 0.1% SDS, 1PBS to prepare a DNA-modified gold nanoparticle solution at a concentration of 500 pM, which was then used as a gold nanoprobe.

Example 2: Detection of Bacillus anthracis Gene Using Gold Nanoparticle-Specific Copper Crystal Overgrowth

[0049] Capturing oligonucleotides (SEQ ID NO: 2: 5-CTT GAA TTT TTG TAT CTA TTT TAC TCT TTG GCA CTA CTT T-PEG.sub.18-C.sub.6 Amine-3) were diluted to 5 M with carbonate buffer at pH 10 containing 0.15 M NaCl, 0.01% SDS and 5% glycerol. Using a microarray, the capturing oligonucleotide solution was directly spotted with a diameter of about 500 m on an aldehyde-modified glass substrate and allowed to stay overnight. The substrate spotted with the capturing oligonucleotide solution was washed with 0.1% SDS, 1PBS. A silicon chamber for hybridization was attached to the thus-washed substrate. 20 L of a target oligonucleotide solution (SEQ ID NO: 3: 5-AAA GTA GTG CCA AAG AGT AAA ATA GAT ACA AAA ATT CAA GAA GCA CAG TTA AAT ATA AAT CAG GAA TGG AAT AAA GCA TT-3) dissolved in 0.1% SDS, 1PBS was added thereto at concentrations of 800 fM, 80 fM, and 8 fM. Then, the substrate was hybridized by incubating for 2 hours in a wet environment of 30 C. When the reaction was completed, the substrate was washed with 0.1% SDS, 1PBS and added with 18 L of a gold nanoprobe solution containing 500 pM probe oligonucleotides prepared according to Preparation Example 2. Then, the substrate was hybridized by incubating for 1 hour 30 minutes in a wet environment of 30 C. and washed three times with 0.1% SDS, 1PBS. After further washing with 1PBS, the copper enhancer solution prepared in Example 1 was added thereto and allowed to react for 5 minutes at room temperature to grow copper crystals. After the reaction was completed, the substrate was washed with water and dried.

Comparative Example 1: Nonspecific Growth of Copper Crystals on Substrate Not Containing Gold Nanoprobe

[0050] A copper enhancer solution was added to the substrate treated in the same manner as in Example 1 or 2 and allowed to react for 30 minutes to confirm the formation of copper crystals, except that the gold nanoprobe solution was not added (see right side of FIG. 3).

Comparative Example 2: DNA Detection Using Silver Crystal Growth

[0051] The substrates with or without gold nano probe according to Examples 1 and 2, and Comparative Example 1 were incubated with a silver enhancer solution (SE100Silver Enhancer Kit) manufactured by Sigma-Aldrich for 15 minutes (Examples 1 and 2) or 30 minutes (Comparative Example 1), and washed to observe formed silver crystals. As a result, as shown in FIG. 3, when the copper crystals were grown by treating the gold nanoprobes introduced on the substrate with a copper enhancer solution, a significant signal enhancement effect was observed compared to the case where the silver crystals were formed, thereby confirming that the target analytes could be detected at lower concentrations (left side of FIG. 3). Further, when no gold nanoprobe was included, no signal was detected in the substrate treated with the copper enhancer solution. In other words, it was confirmed that no copper crystals were formed when no gold nanoprobe was present, however, a signal was detected in the substrate treated with the silver enhancer solution (right side of FIG. 3). These results indicate that silver nanoparticles are formed nonspecifically even in the absence of gold nanoprobe serving as a seed when treated with the silver enhancer solution, and also that specific crystal growth is only possible in the presence of gold nanoprobe when treated with the copper enhancer solution, resulting in higher selectivity for analysis (center of FIG. 3).

Experimental Example 1: Reproducibility for Repeated Experiments

[0052] Substrates reacted with samples each containing 5, 10, and 20 10.sup.4 noroviruses according to Example 1 were treated with gold nanoprobes labeled with an anti-norovirus capsid protein VP1 antibody. A copper enhancer solution was added to the substrates, on which the gold nanoprobes were immobilized, and incubated for 10 minutes to grow copper crystals. Subsequently, the substrates were washed, and the thus-formed copper crystals were visually observed and photographed for analysis. The experiment was repeated three times each and the measured signals were digitized to derive the means and standard deviations. As a result, as shown in FIG. 4, the detection method using the gold nanoprobes through the copper enhancement according to the present invention showed excellent reproducibility in the repeated experiments, and further, it was confirmed that the measured signals were proportionally increased according to the concentration of the gold nanoprobes bonded to the analytes.

SEQUENCE LISTING

[0053] 1; DNA; thiolated-probe oligonucleotide; 5-SH-A.sub.10-PEG.sub.18-AAT GCT TTA TTC CAT TCC TGA TTT ATA TTT AAC TGT GCT T-3; 50 nucleotides [0054] 2; DNA; capturing oligonucleotide; 5-CTT GAA TTT TTG TAT CTA TTT TAC TCT TTG GCA CTA CTT T-PEG.sub.18-C.sub.6 Amine-3; 40 nucleotides [0055] 3; DNA; target oligonucleotide; 5-AAA GTA GTG CCA AAG AGT AAA ATA GAT ACA AAA ATT CAA GAA GCA CAG TTA AAT ATA AAT CAG GAA TGG AAT AAA GCA TT-3; 80 nucleotides