Method for detecting target nucleic acids using plasmonic immunomagnetic nanoparticles

Abstract

To provide a method for detecting target nucleic acids using plasmonic immunomagnetic nanoparticles.SOLUTION: A method for detecting target nucleic acids using plasmonic immunomagnetic nanoparticles according to one aspect is low in costs, allows quick and simple detection of target nucleic acids, and thus can be used in various fields such as molecular biology, medicine, and biological classification.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for detecting a target nucleic acid using plasmonic immunomagnetic nanoparticles. This invention was developed by the Ministry of Science and ICT of Korea. ( Project Number :NRF- 2022R1F1A1070162(1711171631 、1711186785 ) and Project Number: 2023-DD-UP-0007) and the Ministry of Education of the Republic of Korea (Project Number: NRF-2021R1A6A1A03039503(1345362911)). By With support It's finished. [Background technology]

[0002] Immuno-polymerase chain reaction (iPCR) is an immunoassay method for ultrasensitive protein quantification that utilizes the specificity of antigen-antibody interactions and the amplification capabilities of PCR. Generally, iPCR can increase sensitivity by several thousand to several hundred million times compared to enzyme-linked immunosorbent assay (ELISA) due to the exponential amplification capabilities of PCR. However, conventional qPCR thermal cyclers used in iPCR are sophisticated but expensive, time- and energy-consuming Peltier-based thermal block modules, limiting their applicability to point-of-care (POC) diagnostics.

[0003] Recently, photonic PCR, which utilizes the photothermal effect of plasmonic nanomaterials, has attracted attention due to its fast and high photothermal conversion capability. However, photonic PCR still has several problems that limit its widespread application.

[0004] Therefore, to solve the above-mentioned problems, the present inventors developed plasmonic photothermal quantitative iPCR (PPT-qiPCR) using multifunctional plasmonic immunomagnetic nanoparticles (PIMN). Summary of the Invention [Problem to be solved by the invention]

[0005] In one embodiment, the method comprises the steps of: (a) conjugating a capture antibody to plasmonic immunomagnetic nanoparticles (PIMN); (b) binding the detection antibody to the streptavidin-nucleic acid complex; (c) mixing the capture antibody-bound nanoparticles and the detection antibody-bound complex; and (d) separating the mixed nanoparticles and complexes; (e) amplifying the target nucleic acid using the complex, primers, and nucleic acid polymerase; and (f) detecting the target nucleic acid.

[0006] Another aspect is to provide multifunctional plasmonic immunomagnetic nanoparticles comprising iron oxide nanoclusters, gold nanoshells, and antibodies. [Means for solving the problem]

[0007] One embodiment is a method for detecting a capture antibody and a plasmonic immunomagnetic nanoparticle (PIMN). binding the ic immunomagnetic nanoparticles; (b) binding the detection antibody to the streptavidin-nucleic acid complex; (c) mixing the capture antibody-bound nanoparticles and the detection antibody-bound complex; and (d) separating the mixed nanoparticles and complexes; (e) amplifying the target nucleic acid using the complex, primers, and nucleic acid polymerase; and (f) detecting the target nucleic acid.

[0008] The term "streptavidin" refers to a 52 kDa protein purified from Streptomyces avidinii.

[0009] The term "primer" refers to a short gene sequence that serves as the starting point for the creation of another polymer strand complementary to the template during DNA synthesis.

[0010] In one embodiment, the plasmonic immunomagnetic nanoparticles in step (a) may have magnetic and photothermal properties.

[0011] The term "magnetism" means that a magnet has some effect on surrounding objects.

[0012] The term "photothermal property" means the property of being able to specifically stimulate a desired site using light energy.

[0013] The term "Plasmonic immunomagnetic nanoparticles (PIMN)" refers to iron oxide nanoclusters that have magnetic and photothermal properties. The term refers to nanoparticles composed of nanoparticles composed of gold oxide nanoclusters, gold nanoshells, and antibodies.

[0014] In one embodiment, in step (a), the capture antibody may be covalently bound to the surface of the nanoparticle.

[0015] The term "capture antibody" refers to an antibody that can specifically bind to one or more target proteins (antigens).

[0016] The term "covalent bond" refers to the bond formed when atoms share electrons in a chemical bond.

[0017] In one embodiment, the detection antibody in step (b) may be biotinylated.

[0018] The term "detection antibody" refers to an antibody that can bind to the target protein (antigen) captured by the capture antibody.

[0019] In one embodiment, the detection antibody is biotinylated to increase detection efficiency. It can be something.

[0020] In one embodiment, in step (c), the nanoparticles and the complex may be mixed in a sandwich structure.

[0021] In one embodiment, the nanoparticles and the complexes in step (d) may be magnetically separated.

[0022] In one embodiment, the polymerase in step (e) may be one or more selected from the group consisting of Taq polymerase, VENT polymerase, DEEPVENT polymerase, PWO polymerase, and Pfu polymerase.

[0023] In one embodiment, the amplification in step (e) may be performed through photothermal cycling amplification.

[0024] In one embodiment, the detection in step (f) may be performed using one or more selected from the group consisting of colorimetric analysis, fluorescent analysis, Raman analysis, and gel electrophoresis.

[0025] The term "colorimetric analysis" refers to testing or quantifying the concentration of a chemical compound or solution by measuring the absorbance of a specific wavelength of light using a color reagent or the like.

[0026] The term "fluorescence analysis" refers to a chemical analysis method that uses the fluorescence of a substance, and refers to converting a non-fluorescent sample into a fluorescent substance through a chemical reaction and then analyzing the fluorescence.

[0027] The term "Raman analysis" refers to the use of light to generate (excite) molecular motion and to analyze such interactions to chemically analyze a sample.

[0028] The term "gel electrophoresis" refers to the separation of DNA, RNA, proteins, etc. by passing an electric current through a gel matrix.

[0029] Another embodiment provides multifunctional plasmonic immunomagnetic nanoparticles comprising iron oxide nanoclusters, gold nanoshells, and antibodies.

[0030] The "iron oxide nanoclusters," "gold nanoshells," "antibodies," "plasmonic immunomagnetic nanoparticles," etc. may be within the scope described above.

[0031] In one embodiment, the iron oxide nanoclusters may be magnetic.

[0032] In one embodiment, the gold nanoshells may have photothermal properties. [Effects of the Invention]

[0033] The method for detecting target nucleic acids using plasmonic immunomagnetic nanoparticles according to one embodiment enables low-cost, rapid and simple detection of target substances, and can be utilized in a variety of fields, including molecular biology, medicine, and biological classification. [Brief explanation of the drawings]

[0034] [Figure 1] FIG. 1 is a schematic diagram of the workflow of plasmonic photothermal quantitative immuno-PCR using plasmonic immunomagnetic nanoparticles for IL-6 detection. [Figure 2a] Figures 2a to 2f show the results of characterizing PIMN. Specifically, Figure 2a shows the results of transmission electron microscopy (TEM), high-angle annular dark-field and elemental mapping images of PMN (Au: red, Fe: green) and magnetic nanocluster cores. [Figure 2b] 2b to 2d show the hydrodynamic diameter distribution (b), zeta potential (c), and UV-Vis spectrum (d) of PMNs at different stages of surface modification. [Figure 2c] Continued from Figure 2b. [Figure 2d] Continued from Figure 2c. [Figure 2e] Figure 2e shows the results of confirming the temperature profile of one photothermal cycling between 72 °C and 95 °C with various optical densities of PIMN. [Figure 2f] Figure 2f shows the results of checking the calculated heating and cooling rates. [Figure 3a]Figures 3a to 3d show images of iron oxide (a), (b) Fe3O4@Au seeds, (c) Fe3O4@Au, and (d) Fe3O4@Au-MPA observed using a scanning electron microscope (scale bar: 500 nm). [Figure 3b] Continued from Figure 3a. [Figure 3c] Continued from Figure 3b. [Figure 3d] Continued from Figure 3c. [Figure 4] Figure 4 shows the EDX pattern of Fe3O4@Au (A) and the XRD patterns of Fe3O4 and Fe3O4@Au (B). [Figure 5] FIG. 5 shows a diagram of a home-build device for PPT-qiPCR. [Figure 6] FIG. 6 shows the results of checking the temperature profile of a solution without PIMM. [Figure 7] FIG. 7 shows the results of checking the thermal stability before and after 35 photothermal cycles. [Figure 8] Figure 8 shows the results of real-time PPT-qiPCR amplification curves using target IL-6 (1,000 pg / ml), different amounts of SG dye (A), 3% agarose gel electrophoresis analysis (B), and fluorescence spectrum (SG7x). [Figure 9] FIG. 9 shows the results of confirming the amplification curves of real-time PPT-qiPCR obtained from IL-6 (1,000 pg / ml) and PIMN at different concentrations. [Figure 10a] Figures 10a to 10g show the results of confirming the characteristics of PPT-qiPCR using PIMN. Specifically, Figure 10a is a schematic diagram of PPT-qiPCR using real-time and endpoint fluorescence dual-readout. [Figure 10b] FIG. 10b shows the results of checking a typical photothermal amplification temperature profile. [Figure 10c]Figure 10c shows the results of PPT-qiPCR real-time amplification curves at different concentrations of IL-6 (1000, 100, 50, 10, 5, and 1 pg mL-1). [Figure 10d] FIG. 10d shows the results of checking the Cq values ​​of the real-time amplification curves. [Figure 10e] Figure 10e shows the results of confirming the endpoint fluorescence curve of PPT-qiPCR for the same sample as in Figure 10c (red and black plots are the endpoint fluorescence signals before and after magnetic separation, respectively). [Figure 10f] Figure 10f shows the fluorescence and gel electrophoresis images of PPT-qiPCR (lanes 1-9: 0, 0.25, 0.05, 0.5, 1, 10, 100, 500, 1000 pg mL-1). [Figure 10g] FIG. 10g shows the results of a selectivity test of PPT-qiPCR [PBS buffer, tumor necrosis factor-α (TNF-α), hemoglobin (Hb)]. [Figure 11] FIG. 11 shows the fluorescence quenching effect of different concentrations of target and fixed concentration of nanoparticles. [Figure 12a] 12a to 12c show the results of colorimetric PPT-qiPCR for IL-6 detection. Specifically, Fig. 12a is a schematic diagram of the workflow of endpoint colorimetric PPT-qiPCR. [Figure 12b] Figure 12b shows an image of the results of end-point colorimetric PPT-qiPCR. [Figure 12c] Figure 12c shows the absorbance plot of the endpoint colorimetric PPT-qiPCR solution at 650 nm. [Figure 13] FIG. 13 shows (A) the change in pH of the iPCR buffer solution due to the addition of MES buffer, and (B) a comparison of the absorbance measured after 35 thermal cycles depending on the TMB concentration. [Figure 14] FIG. 14 is a diagram showing the results of confirming the optimization of the irradiation time. DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention will be described in more detail below with reference to examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples. [Example]

[0036] 1. Experimental materials and methods

[0037] (1) Experimental materials Ferric chloride hexahydrate (FeCl3·6H2O), 3-aminopropyltrimethoxysilane (APTMS), tetrakis(hydroxymethyl)phosphonium chloride (THPC), hydrogen tetrachloroaurate trihydrate (HAuCl4·3H2O), sodium acetate, trisodium citrate dihydrate, potassium carbonate (K2CO3), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich. Formaldehyde (HCHO) was obtained from TCI Company, and 3,3',5,5'-tetramethylbenzidine (TMB) and 3-mercaptopropionic acid (MPA) were purchased from Energy Chemical. Purified anti-human IL-6 antibody (clone MQ2-13A5, rat monoclonal), biotin-anti-human IL-6 antibody (clone MQ2-39C3, rat monoclonal), recombinant human IL-6, and purified streptavidin ( Streptavidin was purchased from BioLegend. The forward primer (5'-CATCGTCTGCCTGTCATGGGCTGTTAAT-3': SEQ ID NO: 1), reverse primer (5'-TCGCCAGCTTCAGTTCTCTGGCATTT-3': SEQ ID NO: 2), and 112-base pair (bp) X-DNA target with a biotin label at the 5' end were purchased from Integrated DNA Technologies (Coralville, IA, USA). Dream Taq DNA polymerase (0.05 U μL) was used. -1 ), reaction buffer (MgCl, 4 mM), dNTP mix (0.4 mM each) and MES buffer (8 μL, BupH TM MES buffered saline packs (28390, 0.1 M MES, 0.9% sodium chloride, pH 4.7) were purchased from Thermo Fisher Scientific, and SYBR® Green I (excitation at 497 nm and emission at 530 nm) was purchased from Molecular Probes, Inc. Quick-Load® purple low molecular weight DNA ladder, Quick-Load® purple 1 kb DNA ladder, and gel loading dye (purple, 6x) were purchased from New England BioLabs, Inc. The morphology and size distribution of PMNs and other intermediates were examined by transmission electron microscopy (TEM, Tecnai 12, Philips) and field emission TEM (FE-TEM, JEM-F200, JEOL). UV-Vis absorption spectra were obtained from Cary. The reaction was monitored using a UV-Vis Spectrophotometer (Agilent Technologies, USA).

[0038] (2) Synthesis of magnetic nanoparticles and plasmonic magnetic nanoparticles (PMN)

[0039] First, 0.34 g of FeCl3·6H2O, 0.6 g of sodium acetate, and 0.11 g of trisodium citrate dihydrate were dissolved in ethylene glycol (10 mL). The mixture was stirred and sonicated for 2 hours, then sealed in a Teflon-lined stainless-steel autoclave. The autoclave was heated to 200 °C for 12 hours. The magnetic product was collected by magnetic separation, washed three times with ethanol / deionized water (DIW), and dried at 60 °C before use. For PMNs, we used the same synthesis method as previously described [Fast and sensitive immuno-PCR assisted by plasmonic magnetic nanoparticles, Appl Mater Today, 23 (2021)].

[0040] (3)PPT-qiPCR optical device

[0041] Infrared LED (850nm peak wavelength, mounted on a metal-core PCB, 8.5W rated power, 3.8W radiant flux at 700mA forward current, 12.4V forward voltage, LZ4-40R608, LED Engine, CA, USA), blue LED (460nm peak wavelength, mounted on a metal-core PCB, 10W power rating, 3.9W radiant flux at 700mA forward current and 14V forward voltage, LZ4-40B208, LED Engine, CA, USA), IR thermometer (OPTCSTCLT15), blue LED (5 mm, 480 nm peak wavelength, 3.2 V forward voltage, 20 mA forward current, 4.1 cd, C503B-BCN-V0Z0461, CREE, Inc., NC, USA), FITC emission filter (center wavelength = 475 nm, bandwidth = 35 nm, MF475-35, Thorlabs, Inc., NJ, USA), spectrophotometer (CCS200, Thorlabs, Inc., NJ, USA), and optical fiber (M0065-51-0034, Thorlabs, Inc., NJ, USA). The PPT-qiPCR optical system was constructed using a NIRS (NIRS Corporation, USA). A LabVIEW program controlled the power supply, LED-based thermal cycling, cooling fan, fluorescence measurement, and temperature measurement.

[0042] (4) Surface carboxylation of PMN (PMN-MPA)

[0043] The synthesis of magnetic nanoparticles and PMNs was carried out as in a previous study (Fast and sensitive immuno-PCR assisted by plasmonic magnetic nanoparticles, Appl Mater Today, 23 (2021)). PMNs (0.25 mL, 1 mg mL) were used. -1 in DIW) and 3-mercaptopropionic acid (0.5 μL, MPA) were mixed, and then the mixture was sonicated for 30 min and centrifuged at 1,500 g for 8 min, washed with DIW, and finally washed with DIW (1 mg mL -1 ) and redispersed.

[0044] (5) Production of Plasmonic Immunomagnetic Nanoparticles (PIMN)

[0045] The capture antibody was conjugated to PMNs via carbodiimide coupling. Specifically, PMN-MPA (20 μL, 1 mg mL) -1 in DIW), N-(3-Dimethylaminopropyl)-N'-ethylcarbodimide hydrochloride (25 μL, 10 mg mL -1 in pH 5.5 MES buffer) and anti-human IL-6 Ab (10 μL, 0.5 mg mL -1 The nanoparticles were mixed in a pH 5.5 MES buffer for 1 hour. The remaining active sites on the nanoparticles were blocked with bovine serum albumin (BSA, 2.0 wt%), and the product was washed with PBS (0.05 wt% Tween-20) to remove excess capture antibody and BSA. The product was then resuspended in PBS and stored at 4°C.

[0046] (6) PCR tube immunoassay

[0047] The functionalized nanoparticle-based immunoassay was performed as follows: 25 μL IL-6 sample was added at various concentrations (1000, 500, 100, 10, 1, 0.1, 0.05, 0.025, 0 pg mL -1 ) in PIMN (12.5 μL, 0.2 mg mL -1 , 6OD) and biotin anti-human IL-6 detection antibody (12.5 μL, 0.005 mg mL -1 After incubation on a shaker for 1 hour, the samples were washed three times with PBS (0.05 wt% Tween-20) using magnetic separation. The pre-conjugated streptavidin-dsDNA complexes were prepared by mixing streptavidin (2.5 μL, 10 μg mL -1 The dsDNA template was mixed with biotin-dsDNA (9.4 μL, 0.1 μM) at a 1:2 ratio for 30 minutes at room temperature. The pre-conjugated streptavidin-dsDNA complex was then mixed for another 30 minutes. The sandwich immunocomplex formed with the dsDNA template was washed several times with PBS (0.05 wt% Tween-20) to reduce background noise and prevent nonspecific interactions. The complex solution was then resuspended in 5 μL of PBS for further PCR reactions.

[0048] (7) Preparation of PCR mixture for PPT-qiPCR

[0049] The 10 μL reaction mixture for PCR amplification is as follows: PCR buffer (1 μL, 10×), forward primer (0.5 μL, 10 μM), reverse primer (0.5 μL, 10 μM), dNTPs (0.2 μL, 10 μM), DNA polymerase (0.3 μL, 5 U μL), and 5 μL of dNTPs (0.3 μL, 5 U μL). -1 ), SYBR Green I (0.5 μL, 140x in DMSO), PIMN-sandwich (5 μL, 15 OD), nuclease-free water (2 μL ) and mineral oil (10 μL, mixture). PPT-qiPCR was performed in two steps: initial denaturation at 95°C (5 s), followed by 35 photothermal cycles between 95°C (1 s) and 72°C (8 s). The reaction was monitored by measuring the fluorescence emission signal at 530 nm in real time during annealing / extension at 72°C for 1 s. After amplification, the product solution was mixed with TBE buffer (5 μL, 0.5x) and loading buffer (3 μL, 6x) and subjected to agarose gel (3%) electrophoresis.

[0050] (8)Colorimetric assay

[0051] After PPT-qiPCR, MES buffer (9 µL, 0.1 M, pH 4.7) and TMB (1 µL, final concentration 3 mM) were added to 10 µL of the amplified PCR reaction mixture. The resulting mixture was illuminated for 10 min using Blue-Led.

[0052] 2. Experimental Results

[0053] (1) Multifunctional plasmonic immunomagnetic nanoparticles and plasmonic photothermocycle

[0054] 1) Multifunctional plasmonic immunomagnetic nanoparticles

[0055] Multifunctional plasmonic immunomagnetic nanoparticles (PIMNs) consist of Fe3O4 nanoclusters for magnetic properties, gold nanoshells for plasmonic photothermal conversion, and antibodies for immunoreaction (Figure 1). Magnetite Fe3O4 nanoclusters were prepared via a solvothermal reaction (Figures 2a and 3a). Colloidal gold seeds were then attached to the Fe3O4 nanoclusters for the growth of the gold shell layer (Figure 3b). The attached gold seeds (Fe3O4@Au seeds) served as nucleation sites for the formation of a continuous gold shell. The resulting plasmonic magnetic Fe3O4@Au nanoparticles were labeled PMNs (Figures 2a and 3c). Electron microscopy images confirmed that the magnetic core was completely covered by the gold shell (Figure 2a). X-ray powder diffraction (XRD) patterns confirmed the crystallinity of the magnetic core and gold shell, and other patterns between the magnetic core and PMNs further supported the core-shell structure of PMNs (Figure 4). Furthermore, particle analysis using electron microscopy and dynamic light scattering confirmed that PMNs were monodisperse, with a uniform size of 108.7 ± 10.4 nm (Figures 2b and 3c). To immobilize the capture antibody on the nanoparticle surface, bifunctional 3-mercaptopropionic acid (MPA), which contains sulfhydryl and carboxyl groups, was introduced as a linker between PMNs and antibodies. PMNs were first modified with MPA through a semicovalent gold-sulfur (Au-S) bond, and then the antibody was covalently bound to the nanoparticle surface via carbodiimide coupling. The modification process was monitored through UV-Vis spectra, zeta potential, and DLS analysis (Figures 2b-d).After functionalization of PMNs with MPA, the surface potential became negative (-39.9 mV), indicating the attachment of numerous negatively charged carboxyl groups. After functionalization with the capture antibody, a significant shift in the ζ-potential toward the positive direction was observed, confirming antibody binding to the PMNs. The increase in the hydrodynamic radius and LSPR peak broadening of the PMNs confirmed successful antibody functionalization. The strong absorption of PMNs at IR wavelengths enabled the use of an IR light source for photothermal conversion (Figure 2d), even after prolonged light exposure. It was confirmed that this prevents photobleaching of a fluorescent dye (SYBR(C) green, SG) by ion beam irradiation.

[0056] 2) Plasmonic photothermal cycling

[0057] The thermal cycling process, which is the core of iPCR, can be accelerated by utilizing the photothermal effect of plasmonic nanoparticles. A homemade IR-LED device was fabricated to study the photothermal conversion of PIMN (Figure 5). The temperature of the solution containing PIMN was measured and recorded using a non-contact IR thermometer. As shown in Figure 2e, we observed a representative temperature profile for one photothermal cycle between 72°C and 95°C at various optical densities (OD) of PIMN. We also observed a linear increase in the heating ramp rate as the OD of PIMN increased from 5 to 15. The shortest photothermal cycling time for one cycle was 6 seconds for the 15 OD sample, with a heating rate of 9.32 ± 0.13°C / s and a cooling rate of 6.45 ± 0.10°C / s (Figure 2f). Considering that the light energy absorbed by gold nanoparticles has no major energy loss channel other than heat generation, we can expect a linear increase in the heating rate. The temperature accuracy was 72.03 ± 0.35 °C and 94.88 ± 0.21 °C at 95 °C and 72 °C, respectively, confirming that the heating rate did not significantly increase above 15.0 OD. These results confirm that the increase in nanoparticle concentration is due to changes in the optical penetration depth or plasmon resonance caused by interparticle coupling. Furthermore, we confirmed that there was no temperature change under IR-LED irradiation without nanoparticles (Figure 6), and that PIMN exhibited excellent thermal stability after thermal cycling (Figure 7).

[0058] (2) Quantitative immunoassay using real-time and end-point fluorescence dual-readout

[0059] Real-time PCR (RT-PCR), which can quantify amplified nucleic acids in real time, is used as the gold standard in molecular diagnostics and is a benchmark technology for fluorescence-based point-of-care biosensors, multiplex PCR, and immuno-PCR. Because gold nanoparticles are known to be efficient fluorescent bleaching agents, we optimized the concentrations of PIMN and SG dye to increase the fluorescence signal. Lambda DNA was amplified by PPT-qiPCR using PIMN, and the optimal conditions for PIMN and SG dye concentrations were determined from the amplification curve and gel electrophoresis analysis results. We confirmed that at a fixed PIMN concentration (7.5 OD), the fluorescence signal was proportional to the SG dye concentration (1x to 7x). However, increasing the dye concentration to 10x SG dye suppressed the amplification reaction (Figure 8). Therefore, we fixed the SG dye concentration at 7x. However, the fastest heat generation rate was observed when the PIMN concentration was 15.0 OD, but at this concentration, the degree of fluorescence quenching was higher than that at 7.5 OD (Figures 9 and 10c), so the nanoparticle concentration was then fixed at 7.5 OD.

[0060] Subsequently, rapid and sensitive real-time PPT-qiPCR using PIMN was performed with various concentrations of human IL-6 under optimized conditions (Figure 10a). Human IL-6 was detected through a sandwich immunoassay using a PIMN-conjugated anti-human IL-6 monoclonal capture antibody and a biotinylated anti-human IL-6 monoclonal detection antibody. Serial dilutions of human IL-6 were incubated with the capture and detection antibodies to form a sandwich structure. After washing, the biotinylated detection antibody was assembled with a pre-conjugated streptavidin-DNA template and used as a signal probe in a later PCR reaction. After three washes, the PCR reaction mixture containing primers and polymerase was added to a PCR tube and subjected to ultrafast real-time PCR using an IR-LED device. Real-time PPT-qiPCR was performed. The initial denaturation temperature (95°C for 5 seconds) and two-step photothermal cycling between denaturation (1 second at 95°C) and annealing / extension (8 seconds at 72°C) were controlled 35 times using a non-contact IR thermometer. The fluorescence signal indicating probe DNA amplification was then monitored in real time using a blue LED and spectrophotometer. The amplification reaction was completed in less than 10 minutes based on the temperature profile (Figure 10b). Compared to commercial thermal cyclers that transfer heat to the reaction solution via PCR tubes in a heating block, our system with uniformly dispersed nanoparticles allows heat to be distributed directly and evenly throughout the reaction mixture, enabling exceptionally fast thermal cycling. A graph plotting the real-time amplification signal of PPT-qiPCR versus IL-6 concentration shows a typical qPCR amplification curve and was used to determine the quantification cycle (Cq) number (Figure 10c). Cq values ​​ranged from 5 to 1000 pg mL. -1 We confirmed that the assay showed a consistent linear relationship with IL-6 concentrations (Figure 3D->10d). The achieved sensitivity and dynamic range of real-time PPT-qiPCR were compared with those of a commercial ELISA kit and are shown in Table 1 below (Table 1).

[0061] [Table 1]

[0062] After confirming real-time quantitative detection as described above, we applied in situ endpoint fluorescence measurement to the PPT-qiPCR system to achieve even higher sensitivity. PIMN can be removed from the solution by simple magnetic separation, significantly reducing fluorescence quenching. After the PPT-qiPCR reaction, PIMN was collected at the bottom of the reaction tube with a magnet, and the in situ endpoint fluorescence signal of the amplicon was measured at 530 nm and plotted against the IL-6 concentration (Figure 10e). Measurement of the in situ endpoint fluorescence signal, including magnetic separation, was confirmed to be completed within 15 seconds (Figures 10b and 10e). Furthermore, as can be seen from the results, eliminating fluorescence quenching enabled in situ endpoint PPT-qiPCR to achieve a detection range of 0.05–1000 pg mL. -1 ) and improved sensitivity. The quantification plot also showed correct linearity. The fluorescence images before and after magnetic separation shown in Figure 10f and the nanoparticle concentration-dependent fluorescence signal shown in Figure 11 confirmed that the decrease in fluorescence quenching was due to the removal of nanoparticles. [FL]=32208+4269log[C](R 2 = 0.989), the analytical LOD calculated from the correlation equation (FL: fluorescence signal of SG at 530 nm, C: concentration of IL-6) was 21.3 fg mL -1 (3σ Blank ) was confirmed. Furthermore, as shown in Table 1, when compared with the LOD of a commercial ELISA analysis kit, PPT-qiPCR was confirmed to have a sensitivity approximately 100 times lower. Compared to commercial ELISA kits using microtiter plates, the immunomagnetic process using PIMN allows for washing after antigen capture, enabling flexible sampling of the analysis solution, shortening washing time, and reducing nonspecific binding and background signals. Furthermore, the selectivity of PPT-qiPCR for nonspecific target proteins was investigated, and as shown in Figure 10g, there was no fluorescent signal distinguishable from the blank and nonspecific target, demonstrating excellent selectivity. confirmed.

[0063] (3) Colorimetric detection

[0064] Colorimetric biosensors are one of the most suitable platforms for POC diagnostics and resource-limited countries because they are easy to operate and eliminate expensive and complex equipment for signal detection. While maintaining the exponential amplification power of PCR, we extended the system to colorimetric detection using a chromogenic substrate commonly used in ELISA (Figure 12a). To demonstrate that the PPT-qiPCR method is applicable to visually distinguishable colorimetric detection, we performed a colorimetric assay using reactive oxygen species (ROS) (reactive oxygen species). They utilized the photocatalytic properties of the DNA-SG complex to generate singlet oxygen species. In particular, the oxidation of TMB can be induced by singlet oxygen generated in the DNA-SG complex when emitted from underneath a blue LED. The generated singlet oxygen catalyzes the oxidation of TMB, resulting in a color change, and the amount of singlet oxygen is directly related to the amount of amplified target DNA.

[0065] First, we investigated the photocatalytic activity of DNA-SG, which generates reactive oxygen species, as a function of pH, TMB concentration, and light exposure time, by irradiating the SG at a fixed concentration of 7x. The absorbance intensity at 650 nm, which indicates TMB oxidation, was monitored, and MES buffer (pH 4.7) was added after PPT-qiPCR to adjust the solution pH. The optimal pH for TMB oxidation using DNA-SG was confirmed to be 5.0, compared with horseradish peroxidase (HRP) (Figure 13A). The change in absorbance with the presence or absence of target DNA was greatest at 3 mM TMB (Figure 13B). Because the absorbance did not increase significantly with light exposure times longer than 10 minutes (Figure 14), further experiments were performed with a 10-minute exposure time and a TMB concentration of 5.0 and 3 mM, respectively. After immunomagnetic analysis and PPT-qiPCR using serial dilutions of IL-6, a TMB solution in MES buffer was added to a PCR tube to confirm colorimetric detection. After 10 minutes of irradiation with a blue LED, the DNA-SG induced TMB oxidation, and the PMNs were magnetically separated for 15 seconds to confirm the hue change. Because the degree of photocatalytic activity of the DNA-SG is directly related to the amount of amplified amplicon, we confirmed that the target could be quantified using the hue change and saturation associated with the amount of amplified PCR product. As shown in Figure 12b, the colorless-to-blue transition served as a reliable indicator of the presence or absence of the target, while the intensity of the blue color served as evidence of target concentration. The hue readings were more accurately quantified using a UV-Vis spectrophotometer (Figure 12c). The relationship between absorbance at 650 nm and IL-6 concentration was plotted, and the curve showed [Abs] = 1.46 + 0.67 log[C](R 2 = 0.99) and showed a strong linear correlation with the equation (Abs: absorbance of TMB oxidation, C: IL-6 concentration). The colorimetric method, like the fluorometric end-point analysis, demonstrated a linear correlation of 0.05 pg mL -1 to 1 ng mL -1 Wide detection range up to 39.5 fg mL -1 Therefore, the LOD was 0.05 pg mL -1The amplified PPT-qiPCR was further characterized by agarose gel electrophoresis (Figure 10f), which confirmed a clear change in band intensity as the IL-6 concentration increased.

Claims

1. (a) forming plasmonic immunomagnetic nanoparticles (PIMNs) by binding capture antibodies to plasmonic nanoparticles; (b) mixing the plasmonic immunomagnetic nanoparticles, a detection antibody, a streptavidin-nucleic acid complex, and a target antigen to form a sandwich-structured immune complex; (c) isolating the sandwich immune complexes using magnetic separation; (d) amplifying the nucleic acid in the sandwich immunocomplex by photothermal cycling amplification using the sandwich immunocomplex, a primer, and a nucleic acid polymerase; and (e) detecting the target antigen; The method, wherein the capture antibody and the detection antibody bind to the same target antigen.

2. The method of claim 1 , wherein the plasmonic immunomagnetic nanoparticles in step (a) have magnetic and photothermal properties.

3. The method of claim 1 , wherein in step (a), the capture antibody is covalently bound to the surface of the nanoparticle.

4. The method of claim 1, wherein the detection antibody in step (b) is biotinylated.

5. 2. The method of claim 1, wherein the polymerase in step (d) is one or more selected from the group consisting of Taq polymerase, VENT polymerase, DEEPVENT polymerase, PWO polymerase, and Pfu polymerase.

6. 2. The method of claim 1, wherein in step (e), the detection is performed using one or more selected from the group consisting of colorimetric analysis, fluorescent analysis, Raman analysis, and gel electrophoresis.

Images