WO2021057513A1 - Phenol recognition sers probe, preparation thereof, use thereof, and sers-based universal ultrasensitive immunoassay method - Google Patents

Phenol recognition sers probe, preparation thereof, use thereof, and sers-based universal ultrasensitive immunoassay method Download PDF

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WO2021057513A1
WO2021057513A1 PCT/CN2020/114769 CN2020114769W WO2021057513A1 WO 2021057513 A1 WO2021057513 A1 WO 2021057513A1 CN 2020114769 W CN2020114769 W CN 2020114769W WO 2021057513 A1 WO2021057513 A1 WO 2021057513A1
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sers
phenol
probe
aqueous solution
ppna
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Chinese (zh)
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李楠
薛巍
陈浩凌
査勇超
牟宗霞
卢杰
崔鑫
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暨南大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34—Purifying; Cleaning
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/38—Diluting, dispersing or mixing samples

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  • the invention belongs to the technical field of biomolecule detection, and specifically relates to a phenol recognition SERS probe, its preparation and application, and a SERS-based general ultra-sensitive immunoassay method.
  • the traditional enzyme-linked immunosorbent assay utilizes the specific bonding characteristics between antigen and antibody, based on the "sandwich” strategy, designing its bonding mechanism, and labeling the second antibody molecule with enzyme. Using the catalytic performance of the enzyme to make the corresponding substrate undergo a color or fluorescence change, it can show whether a specific antigen or antibody is present or not. Quantitative analysis is performed by measuring the corresponding optical intensity change, and the test object is detected.
  • the sensitivity of ELISA immunoassays needs to be improved urgently. Therefore, this technique is difficult to apply to trace or ultra-trace biomolecule detection.
  • SERS Surface Enhanced Raman Scattering
  • the primary purpose of the present invention is to provide a method for preparing a phenol-responsive surface-enhanced Raman scattering (SERS) probe.
  • SERS surface-enhanced Raman scattering
  • Another object of the present invention is to provide a phenol-responsive surface-enhanced Raman scattering (SERS) probe prepared by the above method.
  • Another object of the present invention is to provide an application of the above-mentioned phenol-responsive surface-enhanced Raman scattering (SERS) probe.
  • SERS surface-enhanced Raman scattering
  • Another object of the present invention is to provide a universal ultra-sensitive ELISA immunoassay method based on surface enhanced Raman scattering (SERS).
  • SERS surface enhanced Raman scattering
  • a method for preparing a phenol-responsive surface-enhanced Raman scattering (SERS) probe includes the following steps:
  • DTDBA 2,2'-dithiodiphenylamine
  • DTDBD 2,2'-di Sulfur diphenyl nitrogen tetrafluoroborate
  • Step (1) The mass concentration of the chloroauric acid aqueous solution is 5-25%; the concentration of the hydrochloric acid is 1 mmol/L, and the volume ratio of the chloroauric acid aqueous solution to the hydrochloric acid is 1:125; the hydrochloric acid refers to hydrochloric acid Aqueous solution.
  • the mass ratio of chloroauric acid, polyvinylpyrrolidone and 3-amidino-aniline in the chloroauric acid aqueous solution in step (1) is 1: (1 to 3.2): (2 to 10).
  • step (1) the temperature of the ice-water bath is 0-4°C, and the condition for uniform mixing is shaking and mixing for 10-30 minutes.
  • the conditions for centrifugation and washing in step (1) are: centrifugation at 5000 rpm for 3-10 min, and the centrifugal fluid used for centrifugation is N-methylpyrrolidone (NMP).
  • NMP N-methylpyrrolidone
  • step (1) the gold microparticle CLMPs are dispersed in water and stored at 4°C.
  • the temperature of the ice water bath in step (2) is 0-4°C.
  • Step (2) The molar ratio of 2,2'-disulfide diphenylamine and sodium tetrafluoroborate in the sodium nitrite and 2,2'-disulfide diphenylamine hydrochloric acid aqueous solution is 1:(0.2 ⁇ 1):(20 ⁇ 30).
  • the 2,2'-disulfide diphenylamine hydrochloric acid aqueous solution in step (2) is prepared by the following method: 2,2'-disulfide diphenylamine is dissolved in hydrochloric acid to obtain 2,2'-disulfide diphenylamine hydrochloric acid aqueous solution , Where the concentration of 2,2'-disulfide diphenylamine in hydrochloric acid is 0.03-1.5 mol/L, and the concentration of hydrochloric acid is 0.5-2 mol/L.
  • step (2) the sodium tetrafluoroborate is added in the form of a saturated aqueous solution of sodium tetrafluoroborate.
  • the saturated sodium tetrafluoroborate aqueous solution is first cooled to 0-4°C and then added.
  • step (3) the concentration of the DTDBD in the solvent is 5-30 mmol/L; the content of the gold micro-particle CLMPs in the gold micro-particle CLMPs aqueous solution is 50-200 pcs/mL.
  • the solvent in step (3) is at least one of dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), ethanol, and methanol.
  • DMF dimethyl formamide
  • DMSO dimethyl sulfoxide
  • ethanol ethanol
  • methanol methanol
  • step (3) the ratio of the molar amount of DTDBD to the number of CLMPs of gold microparticles in the gold microparticle CLMPs aqueous solution is (2.5 ⁇ 10 -3 -6 ⁇ 10 -2 ) mmol:1.
  • the washing in step (3) is performed with dimethylformamide (DMF).
  • step (3) the phenol-responsive surface-enhanced Raman scattering (SERS) probe is dispersed in water to obtain a SERS probe aqueous solution, and stored at 4°C.
  • SERS surface-enhanced Raman scattering
  • SERS surface-enhanced Raman scattering
  • a method for detecting phenol based on a phenol-responsive surface-enhanced Raman scattering (SERS) probe including the following steps:
  • the mass ratio of the number of the SERS probes to the phenol in the phenol-containing Na 2 CO 3 aqueous solution is 1: (4.7 ⁇ 10 -11 ⁇ 1.88 ⁇ 10 -6 ) g; the SERS probe in the SERS probe aqueous solution
  • the content of phenol is 50 ⁇ 200/mL; the concentration of phenol in the phenol- containing Na 2 CO 3 aqueous solution is 1 ⁇ 10 -10 ⁇ 1 ⁇ 10 -3 mol/L, and the mass concentration of Na 2 CO 3 is 1 ⁇ 20%; the content of SERS probes in the sample to be tested is 50-200 pieces/mL.
  • the rotation speed of the centrifugation is 1000 rpm and the time is 15 minutes; the washing refers to washing with water.
  • the SERS detection is: take the sample to be tested and dry it for SERS detection, and the conditions are: excitation with a 785nm laser, record the SERS spectrum change on a single SERS probe, the power is 1 mW, and the cumulative time is 5-60 seconds.
  • a method for detecting alkaline phosphatase (ALP) based on phenol-responsive surface-enhanced Raman scattering (SERS) probe including the following steps:
  • the ratio of ALP and SERS probes in the PPNa and ALP solutions is 9.5 ⁇ 10 -4 mmol: (5 ⁇ 10 -6 ⁇ 2.5 ⁇ 10 -3 ) mU: (0.5 ⁇ 2); the PPNa
  • the concentration in the Tris-HCl buffer is 1 mmol/L; the concentration of the ALP solution is 0.1-50mU/L; the SERS probe is added in the form of an aqueous solution, and the content of the SERS probe is 50-200/L mL;
  • the content of SERS probes in the sample to be tested is 50-200 pieces/mL.
  • the pH of the Tris-HCl buffer is 9.8; the washing is washing with water; the drying condition is drying at room temperature for 1 to 3 hours.
  • the conditions of the SERS detection are: excitation with a 785nm laser, record the SERS spectrum change on a single SERS probe, the power is 1 mW, and the cumulative time is 5-60 seconds.
  • the first antibody molecule that specifically recognizes the antigen molecule is fixed on the orifice substrate by incubation. Since the first antibody molecule bound to the orifice substrate still has immunological activity, add the corresponding antigen molecule for immune response, and then add ALP The labeled second antibody molecule reacts with the antigen molecule to form an immunoreactive structure of antigen-antibody molecule bonding;
  • step (1) Mix the antibody antigen molecule with immunoreactive structure in step (1) and PPNa solution uniformly, and incubate at room temperature for 5-30 minutes to make the ALP on the second antibody molecule decompose PPNa to produce phenol, then add SERS probe and continue incubating 10 ⁇ 30min, finally SERS test;
  • the second antibody molecule, the antigen molecule, and the first antibody molecule that specifically recognizes the antigen molecule are all the same biological molecule, and the biological molecule is one of bacteria, cells, viruses, DNA, and RNA;
  • the SERS probe is the above-mentioned phenol-responsive surface-enhanced Raman scattering (SERS) probe.
  • the concentration of the PPNa solution in step (2) is 0.5-3 mmol/L, and the solvent is water.
  • the mass ratio of the PPNa in the PPNa solution to the second antibody molecule in the antibody antigen molecule with an immunoreactive structure is 1: (2-10).
  • the ratio of the number of SERS probes to the molar amount of PPNa in the PPNa solution is 1: (4.5 ⁇ 10 -5 to 1.8 ⁇ 10 -4 ) mmol.
  • step (2) the SERS probe is added in the form of a SERS probe aqueous solution, and the content of the SERS probe in the SERS probe aqueous solution is 50-200 probes/mL.
  • the conditions of the SERS detection in step (2) are: excitation with a 785nm laser, record the SERS spectrum change on a single SERS probe, the power is 1 mW, and the cumulative time is 5-60 seconds.
  • the number of times of washing with PBS buffer is 2 to 3 times.
  • a phenol-responsive surface-enhanced Raman scattering (SERS) probe-based cholera toxin (CT) enzyme-linked immunoassay method includes the following steps:
  • step (2) Mix the CT antigen-antibody molecule with the typical structure of enzyme-linked immunoassay in step (1) and PPNa solution uniformly, and after incubating at room temperature for 5-30 minutes, add the above-mentioned SERS probe, continue incubating for 10-30 minutes, and finally perform SERS detection.
  • Step (1) The concentration of the CT primary antibody molecule in the PBS buffer containing 0.05 mol/L sodium carbonate is 1-50 ⁇ g/mL; the solvent of the CT antigen solution is water, and the concentration is 0.1-100 pg/mL .
  • the mass ratio of the CT first antibody molecule to gelatin in step (1) is 1:100-500.
  • the mass ratio of CT antigen to CT first antibody molecule in the CT antigen solution is 1:1; the mass ratio of the ALP-labeled CT second antibody molecule to CT first antibody molecule is 1:1.
  • the number of times of washing with PBS buffer in step (1) is 2 to 3 times.
  • step (2) the mass ratio of PPNa in the PPNa solution to the second antibody molecule in the CT antigen antibody molecule with the typical structure of enzyme-linked immunoassay is 1: (2-10).
  • the ratio of the number of SERS probes to the molar amount of PPNa in the PPNa solution in step (2) is 1: (4.5 ⁇ 10 -5 ⁇ 1.8 ⁇ 10 -4 ) mmol.
  • the concentration of the PPNa solution is 0.5-3 mmol/L, and the solvent is water.
  • the SERS probe is added in the form of a SERS probe aqueous solution, and the content of the SERS probe in the SERS probe aqueous solution is 50-200 pieces/mL.
  • the conditions of the SERS detection in step (2) are: excitation with a 785nm laser, record the SERS spectrum change on a single SERS probe, the power is 1 mW, and the cumulative time is 5-60 seconds.
  • the number of times of washing with PBS buffer is 2 to 3 times.
  • the present invention has the following advantages and beneficial effects:
  • the present application prepares gold particle self-assembly (SERS substrate) with a "cabbage"-like structure, which has a large number of Raman enhanced "hot spots", which can significantly enhance the SERS signal, and can realize the detection of a single SERS probe, which solves the traditional
  • SERS substrate gold particle self-assembly
  • the problem of uneven measurement signal caused by uneven SERS substrate greatly improves the reproducibility of detection.
  • This application synthesizes a Raman molecule recognized by phenol with high specificity.
  • the molecule contains an azide group that can form an azo compound with phenol.
  • the peak intensity information of the Raman fingerprint at 1141 cm -1 can be accurately reflected Obtain the amount of azobenzene generated, thereby obtaining the concentration of phenol, and the disulfide bonds in the DTDBD molecule can form Au-S bonds with the gold substrate, thereby ensuring the stability of DTDBD and realizing highly sensitive SERS for phenolic substances Detection.
  • This application uses the prepared phenol-responsive SERS probe for the highly sensitive detection of ALP.
  • ALP can efficiently catalyze the hydrolysis of its substrate PPNa to cause the loss of its phosphate group, thereby generating phenol. Therefore, the determination of the concentration of phenol produced by SERS technology can sensitively detect ALP.
  • This application uses the combination of ELISA and SERS for the high-sensitivity detection of CT.
  • ALP is used as the antibody marker
  • the antibody is fixed on the surface of the well plate
  • the antigen in the solution is captured, and then combined
  • the ALP-labeled secondary antibody molecule uses ALP to hydrolyze its substrate PPNa to produce phenol.
  • the change in the signal of the SERS probe caused by phenol can sensitively detect the antigen-cholera toxin CT. Since the clinical determination of ALP is mainly used for the diagnosis of skeletal and hepatobiliary system diseases, and CT detection is an important basis for the diagnosis of the cause of cholera, the present invention has huge clinical application potential.
  • This application uses the prepared SERS probe for detection and immunoassay, which has extremely high universality. Based on the principle of ELISA, by changing the enzyme label, using the highly sensitive SERS technology to detect its corresponding antigen molecules, it can achieve diversification. The nature of this technology makes this technology versatile and can be widely used to detect a variety of biomolecules, such as bacteria, cells, viruses, DNA and RNA, and other clinical applications.
  • the surface-enhanced Raman spectrum of this application has narrow peaks, strong resolution and high sensitivity.
  • the combination of enzyme-linked immunoassay method and SERS detection technology makes the two complementary, which greatly improves ELISA
  • the sensitivity of SERS effectively solves the problems of SERS signal strength and reproducibility, and the development of a general-purpose ultra-sensitive immunoassay technology based on SERS provides new opportunities for the development of both technologies.
  • This application combines SERS detection technology and ELISA detection technology, and has great development space and broad application prospects in the fields of biology, chemical detection, and medical diagnosis.
  • FIG. 1 is a scanning electron micrograph of the gold microparticle CLMP prepared in Example 1.
  • Example 2 is an optical microscope image of the SERS probe particles prepared in Example 1.
  • Figure 3 is the synthesis route and detection reaction mechanism route diagram of DTDBD of the application, in which (a) is the synthesis route diagram of DTDBD which specifically recognizes phenol, (b) is the route diagram of DTDBD and phenol through the azo coupling reaction mechanism, ( c) is a schematic diagram of the self-assembly of DTDBD on the surface of CLMP to form DTDBD-CLMP and its reaction with phenol.
  • Figure 4 shows the SERS spectra of DTDBD-CLMP prepared in Example 1 and the SERS spectra of DTDBD-CLMP and azobenzene-CLAMP produced after reaction with phenol in Example 2.
  • Figure 5 shows the SERS spectra of DTDBD-CLMP in Example 2 after incubation with different concentrations of phenol.
  • Figure 6 shows the relationship between the SERS peak intensity of azobenzene-CLMP at 1141 cm -1 and the phenol concentration in Example 2.
  • Example 7 is a schematic diagram of the mechanism of ALP catalyzing PPNa to generate phenol and its reaction with DTDBD to generate azobenzene in Example 3.
  • Figure 8 shows the SERS spectra of DTDBD-CLMP after incubation with different concentrations of ALP in Example 3.
  • Fig. 9 shows the relationship between the SERS peak intensity of azobenzene-CLMP at 1141 cm -1 and the ALP concentration in Example 3.
  • FIG. 10 is a schematic diagram of the detection principle of the SERS sensor based on phenol recognition used in CT immunoassay in Example 4.
  • FIG. 10 is a schematic diagram of the detection principle of the SERS sensor based on phenol recognition used in CT immunoassay in Example 4.
  • Figure 11 shows the SERS response of DTDBD-CLMP to different CT concentrations in Example 4.
  • Fig. 12 shows the relationship between the SERS peak intensity of azobenzene-CLMP at 1141 cm -1 and the CT concentration in Example 4.
  • the conditions of the SERS test described in the examples of this application are: using 785nm laser excitation, recording the SERS spectrum on a single CLMP, the power is 1mW, and the total cumulative time is 10 seconds. For each sample, 10 CLMP SERS spectra were obtained during measurement for standard deviation calculation.
  • concentrations of the CLMP aqueous solution and the probe aqueous solution in the examples of the application all refer to the number (quantity) of CLMP or probes contained in 1 mL of water.
  • the molecular structure of DTDBD and its synthetic route are shown in Figure 3.
  • the prepared DTDBD has two functional groups: 1) Diazonium ions can react specifically with phenol through an azo coupling reaction to form azobenzene (see Figure 3b); 2) The disulfide group is conducive to the formation of a stable Au-S covalent bond between DTDBD and Au nanosheets during the formation of CLMP.
  • 10 ⁇ L aqueous SERS probe prepared in Example 1 were added to 1mL 2 CO 3 solution containing different concentrations of phenol Na, the concentration of Na 2 CO 3 aqueous solution of phenol were 1 ⁇ 10 -9 mol / L, 5 ⁇ 10 -9 mol/L, 1 ⁇ 10 -8 mol/L, 5 ⁇ 10 -8 mol/L, 1 ⁇ 10 -7 mol/L, 5 ⁇ 10 -7 mol/L, 1 ⁇ 10 -6 mol/L, 1 ⁇ 10 -5 mol/L, 1 ⁇ 10 -3 mol/L, the mass concentration of the Na 2 CO 3 aqueous solution is 5%, and then all are incubated at 4° C.
  • the probe collects the probe and use water After washing, the obtained precipitate is dispersed in water (wherein the content of SERS probe is 100 pcs/mL) to obtain the sample to be tested, a drop of the sample to be tested is drawn and dried at room temperature for 2 hours, and then the SERS measurement is performed.
  • the phenol-responsive SERS sensor proposed in this embodiment can detect phenol with high sensitivity, and its performance is better than the phenol detection method described in the prior art (see Table 1).
  • Carbon nanotubes/polyethyleneimine/electrochemistry A.S.Arribas, E.Bermejo, M.Chicharro, A.Zapardiel, G.L.Luque, N.F.Ferreyra, G.A.Rivas, Anal.Chim.Acta, 2007,596,183.
  • the resulting precipitate is dispersed in water to obtain the sample to be tested (the content of SERS probes is 100/mL), a drop of the sample to be tested is sucked and dropped on the silicon wafer, dried at room temperature for 2 hours, and then SERS measurement is performed .
  • the SERS detection method proposed in this embodiment can detect ALP with high sensitivity, and its performance is better than the ALP detection method described in the prior art (see Table 2).
  • CT primary antibody molecule PBS buffer containing 0.05mol/L sodium carbonate
  • CT primary antibody molecule Anti-Cholera Toxin antibody (ab123129), purchased from Abcam (Shanghai) Trading company
  • CT primary antibody molecule Anti-Cholera Toxin antibody (ab123129)
  • Abcam Abcam (Shanghai) Trading company
  • CT primary antibody molecule Anti-Cholera Toxin antibody (ab123129)
  • PBS buffer 3 times
  • excess gelatin the mass ratio of CT primary antibody molecule to gelatin is 1: 200
  • CT antigen Cholera Toxin from Vibrio cholera (c8052) purchased from Sigma-Aldrich
  • concentrations are 0.1pg/mL, 0.5pg/mL, 1pg/mL, 5pg/mL, 10pg/mL, 50pg/mL, 100pg/mL, and after react
  • the results of CT detection are shown in Figures 11 and 12.
  • the results show that the SERS probe can sensitively respond to the presence of CT (see Figure 11), and the intensity of the SERS peak at 1141 cm -1 gradually increases with the increase of CT concentration.
  • the linear response of CT concentration is from 8.3 ⁇ 10 -14 mol/L ( 0.1pg/mL) to 8.3 ⁇ 10 -12 mol/L (10pg/mL) (see Figure 12).
  • the standard addition method was used to determine the CT recovery rate in the serum sample to evaluate the reliability of the CT determination method provided in this embodiment.
  • Standard CT samples of different concentrations were added to the blank serum samples (the final concentrations in the serum samples were 0.5 pg/mL, 1 pg/mL, and 5 pg/mL, respectively, and the solvent was water), and the SERS test was performed.
  • RSD relative standard deviation

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Abstract

A phenol recognition SERS probe and preparation thereof, use thereof, and a SERS-based universal ultrasensitive immunoassay method. First, a phenol-responsive SERS probe is prepared by reducing chloroauric acid using DTDBA, and then ELISA is used in combination with SERS, a biomolecule is labelled with ALP, a substrate PPNa thereof is hydrolysed using ALP to produce phenol, and a biomolecule is detected sensitively by means of a SERS probe signal caused by phenol. The present invention can overcome the defect of low sensitivity of conventional enzyme-linked immunoassay; can solve the problem of enhancement and poor reproducibility of an SERS detection signal; has significant universality; can be widely used in immunoassay using ALP as enzyme marker to detect a wide variety of biomolecules; provides a solid foundation for the development of immunological technology on the bass of SERS detection; and has great development space and broad application prospects in the fields of biology, chemical detection, and medical diagnosis.

Description

一种苯酚识别SERS探针及其制备、应用和基于SERS通用超灵敏免疫分析方法A phenol-recognizing SERS probe, its preparation, application, and a general-purpose ultra-sensitive immunoassay method based on SERS 技术领域Technical field
本发明属于生物分子检测技术领域，具体涉及一种苯酚识别SERS探针及其制备、应用和基于SERS通用超灵敏免疫分析方法。The invention belongs to the technical field of biomolecule detection, and specifically relates to a phenol recognition SERS probe, its preparation and application, and a SERS-based general ultra-sensitive immunoassay method.
背景技术Background technique
传统的酶联免疫吸附测定(ELISA)是利用抗原抗体之间专一性键结特性，基于“三明治”策略，设计其键结机制，以酶标记第二抗体分子。利用酶的催化性能使其相应的底物发生颜色或荧光变化，即可显示特定抗原或抗体是否存在，通过测定相应的光学强度变化进行定量分析，对待测物进行检测。但是受限于这些光谱方法的灵敏度，ELISA免疫分析的灵敏度亟待提高，因此该技术较难应用于痕量或超痕量生物分子检测。表面增强拉曼散射(Surface Enhanced Raman Scattering，SERS)是将激光拉曼光谱应用于表面化学研究中发现的一种新的表面光学现象。SERS凭借其超高的灵敏度、独一无二的指纹谱、窄峰宽及无水干扰等特点，成为一种广泛采用的检测手段，可用于痕量分析乃至单分子检测。SERS探针是指单个纳米粒子上修饰拉曼分子，具有灵敏度高、抗光漂白、指纹识别能力强、稳定性好及多重检测等优点，为高灵敏生物分析检测提供了可能。然而，SERS光谱检测仍存在不足，譬如绝大多数采用的基于简单纳米结构的金银或其合金形成的基底，由于缺乏丰富的“热点”区域使得拉曼信号增强不够；此外，SERS基底具有较随机的结构，即便是同批制备的基底，彼此之间的SERS增强因子差别仍可能较大，使得SERS信号的均一性不佳；再者，溶液相中游离态的被检测物在SERS基底上的自发吸附较弱，也将造成拉曼信号较 弱。此外，SERS检测时信号无规律波动的问题亟待解决，SERS信号重复性与可靠性有待于提高。The traditional enzyme-linked immunosorbent assay (ELISA) utilizes the specific bonding characteristics between antigen and antibody, based on the "sandwich" strategy, designing its bonding mechanism, and labeling the second antibody molecule with enzyme. Using the catalytic performance of the enzyme to make the corresponding substrate undergo a color or fluorescence change, it can show whether a specific antigen or antibody is present or not. Quantitative analysis is performed by measuring the corresponding optical intensity change, and the test object is detected. However, limited by the sensitivity of these spectroscopic methods, the sensitivity of ELISA immunoassays needs to be improved urgently. Therefore, this technique is difficult to apply to trace or ultra-trace biomolecule detection. Surface Enhanced Raman Scattering (SERS) is a new surface optical phenomenon discovered in the application of laser Raman spectroscopy to surface chemistry research. SERS has become a widely used detection method due to its ultra-high sensitivity, unique fingerprint spectrum, narrow peak width and no water interference. It can be used for trace analysis and even single molecule detection. SERS probe refers to the modified Raman molecule on a single nanoparticle. It has the advantages of high sensitivity, resistance to photobleaching, strong fingerprint recognition ability, good stability and multiple detection, which provides the possibility for highly sensitive biological analysis and detection. However, SERS spectroscopy detection still has shortcomings. For example, most of the substrates formed of gold and silver or its alloys based on simple nanostructures have insufficient Raman signal enhancement due to the lack of abundant "hot spots"; in addition, SERS substrates have relatively high Random structure, even if the substrates prepared in the same batch, the difference in SERS enhancement factor between each other may still be large, making the uniformity of the SERS signal poor; in addition, the free state of the test substance in the solution phase is on the SERS substrate. The weaker spontaneous adsorption will also result in a weaker Raman signal. In addition, the problem of irregular signal fluctuations during SERS detection needs to be solved urgently, and the repeatability and reliability of SERS signals need to be improved.
发明内容Summary of the invention
为解决现有技术的缺点和不足，本发明的首要目的在于提供一种苯酚响应型表面增强拉曼散射(SERS)探针的制备方法。In order to solve the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a method for preparing a phenol-responsive surface-enhanced Raman scattering (SERS) probe.
本发明的另一目的在于提供上述方法制得的一种苯酚响应型表面增强拉曼散射(SERS)探针。Another object of the present invention is to provide a phenol-responsive surface-enhanced Raman scattering (SERS) probe prepared by the above method.
本发明的再一目的在于提供上述一种苯酚响应型表面增强拉曼散射(SERS)探针的应用。Another object of the present invention is to provide an application of the above-mentioned phenol-responsive surface-enhanced Raman scattering (SERS) probe.
本发明的再一目的在于提供一种基于表面增强拉曼散射(SERS)的通用超灵敏ELISA免疫分析方法。Another object of the present invention is to provide a universal ultra-sensitive ELISA immunoassay method based on surface enhanced Raman scattering (SERS).
本发明目的通过以下技术方案实现：The purpose of the present invention is achieved through the following technical solutions:
一种苯酚响应型表面增强拉曼散射(SERS)探针的制备方法，包括以下步骤：A method for preparing a phenol-responsive surface-enhanced Raman scattering (SERS) probe includes the following steps:
(1)将氯金酸水溶液溶于盐酸中，然后加入聚乙烯吡咯烷酮(PVP)，冰水浴条件下反应5～20min，加入3-脒基-苯胺(NAAN)，混合均匀，2～8℃下静置反应12～48h，离心洗涤，得到金微米粒子CLMPs；(1) Dissolve the chloroauric acid aqueous solution in hydrochloric acid, then add polyvinylpyrrolidone (PVP), react for 5-20min under ice-water bath conditions, add 3-amidino-aniline (NAAN), mix well, 2-8℃ Let stand and react for 12～48h, centrifuge and wash to obtain CLMPs of gold microparticles;
(2)在冰水浴条件下，将亚硝酸钠加入到2,2'-二硫二苯胺(DTDBA)盐酸水溶液中，反应0.5～2h后，加入四氟硼酸钠，得到2,2'-二硫二苯氮四氟硼酸(DTDBD)，即为特异性识别苯酚的拉曼分子；(2) Under ice-water bath conditions, add sodium nitrite to 2,2'-dithiodiphenylamine (DTDBA) hydrochloric acid aqueous solution, after reaction for 0.5-2h, add sodium tetrafluoroborate to obtain 2,2'-di Sulfur diphenyl nitrogen tetrafluoroborate (DTDBD) is a Raman molecule that specifically recognizes phenol;
(3)将DTDBD溶于溶剂中，加入金微米粒子CLMPs水溶液，混合均匀，在2～8℃孵育0.5～4h后，洗涤，得到DTDBD-CLMPs，即为苯酚响应型表面增强拉曼散射(SERS)探针。(3) Dissolve DTDBD in a solvent, add gold microparticle CLMPs aqueous solution, mix well, incubate at 2～8℃ for 0.5～4h, wash, and obtain DTDBD-CLMPs, namely phenol-responsive surface enhanced Raman scattering (SERS) ) Probe.
步骤(1)所述氯金酸水溶液的质量浓度为5～25％；所述盐酸的浓度为1mmol/L，所述氯金酸水溶液与盐酸的体积比为1：125；所述盐酸指盐酸水溶液。Step (1) The mass concentration of the chloroauric acid aqueous solution is 5-25%; the concentration of the hydrochloric acid is 1 mmol/L, and the volume ratio of the chloroauric acid aqueous solution to the hydrochloric acid is 1:125; the hydrochloric acid refers to hydrochloric acid Aqueous solution.
步骤(1)所述氯金酸水溶液中的氯金酸、聚乙烯吡咯烷酮和3-脒基-苯胺的质量比为1：(1～3.2)：(2～10)。The mass ratio of chloroauric acid, polyvinylpyrrolidone and 3-amidino-aniline in the chloroauric acid aqueous solution in step (1) is 1: (1 to 3.2): (2 to 10).
步骤(1)所述冰水浴的温度为0～4℃，所述混合均匀的条件为震荡混合10～30min。In step (1), the temperature of the ice-water bath is 0-4°C, and the condition for uniform mixing is shaking and mixing for 10-30 minutes.
步骤(1)所述离心洗涤的条件为：在5000rpm下离心3～10min，离心所用的离心液为N-甲基吡咯烷酮(NMP)。The conditions for centrifugation and washing in step (1) are: centrifugation at 5000 rpm for 3-10 min, and the centrifugal fluid used for centrifugation is N-methylpyrrolidone (NMP).
步骤(1)所述金微米粒子CLMPs分散于水中，并于4℃保存。In step (1), the gold microparticle CLMPs are dispersed in water and stored at 4°C.
步骤(2)所述冰水浴的温度为0～4℃。The temperature of the ice water bath in step (2) is 0-4°C.
步骤(2)所述亚硝酸钠、2,2'-二硫二苯胺盐酸水溶液中2,2'-二硫二苯胺和四氟硼酸钠的摩尔比为1：(0.2～1)：(20～30)。Step (2) The molar ratio of 2,2'-disulfide diphenylamine and sodium tetrafluoroborate in the sodium nitrite and 2,2'-disulfide diphenylamine hydrochloric acid aqueous solution is 1:(0.2～1):(20 ~30).
步骤(2)所述2,2'-二硫二苯胺盐酸水溶液由以下方法制得：将2,2'-二硫二苯胺溶于盐酸中，得到2,2'-二硫二苯胺盐酸水溶液，其中2,2'-二硫二苯胺在盐酸中的浓度为0.03～1.5mol/L，盐酸的浓度为0.5～2mol/L。The 2,2'-disulfide diphenylamine hydrochloric acid aqueous solution in step (2) is prepared by the following method: 2,2'-disulfide diphenylamine is dissolved in hydrochloric acid to obtain 2,2'-disulfide diphenylamine hydrochloric acid aqueous solution , Where the concentration of 2,2'-disulfide diphenylamine in hydrochloric acid is 0.03-1.5 mol/L, and the concentration of hydrochloric acid is 0.5-2 mol/L.
步骤(2)所述四氟硼酸钠以饱和四氟硼酸钠水溶液的形式加入。所述饱和四氟硼酸钠水溶液先降温至0～4℃后再加入。In step (2), the sodium tetrafluoroborate is added in the form of a saturated aqueous solution of sodium tetrafluoroborate. The saturated sodium tetrafluoroborate aqueous solution is first cooled to 0-4°C and then added.
步骤(3)所述DTDBD在溶剂中的浓度为5～30mmol/L；所述金微米粒子CLMPs水溶液中金微米粒子CLMPs的含量为50～200个/mL。In step (3), the concentration of the DTDBD in the solvent is 5-30 mmol/L; the content of the gold micro-particle CLMPs in the gold micro-particle CLMPs aqueous solution is 50-200 pcs/mL.
步骤(3)所述溶剂为二甲基甲酰胺(DMF)、二甲基亚砜(DMSO)、乙醇、甲醇中的至少一种。The solvent in step (3) is at least one of dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), ethanol, and methanol.
步骤(3)所述DTDBD摩尔量和金微米粒子CLMPs水溶液中金微米粒子CLMPs的个数比为(2.5×10 -3～6×10 -2)mmol：1个。 In step (3), the ratio of the molar amount of DTDBD to the number of CLMPs of gold microparticles in the gold microparticle CLMPs aqueous solution is (2.5×10 -3 -6×10 -2 ) mmol:1.
步骤(3)所述洗涤采用二甲基甲酰胺(DMF)进行。The washing in step (3) is performed with dimethylformamide (DMF).
步骤(3)所述苯酚响应型表面增强拉曼散射(SERS)探针分散于水中得到SERS探针水溶液，并于4℃保存。In step (3), the phenol-responsive surface-enhanced Raman scattering (SERS) probe is dispersed in water to obtain a SERS probe aqueous solution, and stored at 4°C.
上述方法制得的一种苯酚响应型表面增强拉曼散射(SERS)探针。A phenol-responsive surface-enhanced Raman scattering (SERS) probe prepared by the above method.
上述一种苯酚响应型表面增强拉曼散射(SERS)探针在生物分析与传感检 测领域中的应用。The application of the above-mentioned phenol-responsive surface-enhanced Raman scattering (SERS) probe in the field of biological analysis and sensing detection.
一种基于苯酚响应型表面增强拉曼散射(SERS)探针检测苯酚的方法，包括以下步骤：A method for detecting phenol based on a phenol-responsive surface-enhanced Raman scattering (SERS) probe, including the following steps:
将上述SERS探针分散于水溶液中得到SERS探针水溶液，再加入到含苯酚的Na 2CO 3溶液中，混合均匀，2～8℃静置反应5～30min，离心，洗涤，得到沉淀物，分散于水中，得到待测样品，然后进行SERS检测。 Disperse the above-mentioned SERS probe in the aqueous solution to obtain the SERS probe aqueous solution, then add it to the phenol-containing Na 2 CO 3 solution, mix well, let stand for 5-30 min at 2-8°C, centrifuge and wash to obtain a precipitate. Disperse in water to obtain the sample to be tested, and then perform SERS detection.
所述SERS探针个数与含苯酚的Na 2CO 3水溶液中苯酚的质量比为1个：(4.7×10 -11～1.88×10 -6)g；所述SERS探针水溶液中SERS探针的含量为50～200个/mL；所述含苯酚的Na 2CO 3水溶液中苯酚的浓度为1×10 -10～1×10 -3mol/L，Na 2CO 3的质量浓度为1～20％；所述待测样品中SERS探针的含量为50～200个/mL。 The mass ratio of the number of the SERS probes to the phenol in the phenol-containing Na 2 CO 3 aqueous solution is 1: (4.7×10 -11 ～1.88×10 -6 ) g; the SERS probe in the SERS probe aqueous solution The content of phenol is 50～200/mL; the concentration of phenol in the phenol- containing Na 2 CO 3 aqueous solution is 1×10 -10 ～1×10 -3 mol/L, and the mass concentration of Na 2 CO 3 is 1～ 20%; the content of SERS probes in the sample to be tested is 50-200 pieces/mL.
所述离心的转速为1000rpm，时间为15min；所述洗涤指用水洗涤。The rotation speed of the centrifugation is 1000 rpm and the time is 15 minutes; the washing refers to washing with water.
所述SERS检测为：取待测样品干燥后进行SERS检测，其条件为：用785nm激光激发，记录单个SERS探针上SERS光谱变化，功率为1mW，累计时间为5～60秒。The SERS detection is: take the sample to be tested and dry it for SERS detection, and the conditions are: excitation with a 785nm laser, record the SERS spectrum change on a single SERS probe, the power is 1 mW, and the cumulative time is 5-60 seconds.
一种基于苯酚响应型表面增强拉曼散射(SERS)探针检测碱性磷酸酶(ALP)的方法，包括以下步骤：A method for detecting alkaline phosphatase (ALP) based on phenol-responsive surface-enhanced Raman scattering (SERS) probe, including the following steps:
将苯基磷酸二钠(PPNa)溶于Tris-HCl缓冲液中，加入ALP溶液，室温下孵育10～30min，加入上述SERS探针，再孵育10～30min，洗涤，得到沉淀物，将其分散于水中得到待测样品，将待测样品滴加在硅片上，干燥，进行SERS检测；Dissolve phenyl disodium phosphate (PPNa) in Tris-HCl buffer, add ALP solution, incubate at room temperature for 10-30 minutes, add the above SERS probe, incubate for another 10-30 minutes, wash to obtain a precipitate, and disperse it Obtain the sample to be tested in water, drop the sample to be tested on the silicon wafer, dry, and perform SERS detection;
其中，所述PPNa、ALP溶液中的ALP和SERS探针的比例为9.5×10 -4mmol：(5×10 -6～2.5×10 -3)mU：(0.5～2)个；所述PPNa在Tris-HCl缓冲液中的浓度为1mmol/L；所述ALP溶液的浓度为0.1～50mU/L；所述SERS探针以水溶液的形式加入，其中SERS探针的含量为50～200个/mL；所述待测样品中SERS探针的含量为50～200个/mL。 Wherein, the ratio of ALP and SERS probes in the PPNa and ALP solutions is 9.5×10 -4 mmol: (5×10 -6 ～2.5×10 -3 ) mU: (0.5～2); the PPNa The concentration in the Tris-HCl buffer is 1 mmol/L; the concentration of the ALP solution is 0.1-50mU/L; the SERS probe is added in the form of an aqueous solution, and the content of the SERS probe is 50-200/L mL; The content of SERS probes in the sample to be tested is 50-200 pieces/mL.
所述Tris-HCl缓冲液的pH为9.8；所述洗涤为用水洗涤；所述干燥的条件为室温下干燥1～3h。The pH of the Tris-HCl buffer is 9.8; the washing is washing with water; the drying condition is drying at room temperature for 1 to 3 hours.
所述SERS检测的条件为：用785nm激光激发，记录单个SERS探针上SERS光谱变化，功率为1mW，累计时间为5～60秒。The conditions of the SERS detection are: excitation with a 785nm laser, record the SERS spectrum change on a single SERS probe, the power is 1 mW, and the cumulative time is 5-60 seconds.
一种基于表面增强拉曼散射(SERS)的通用超灵敏ELISA免疫分析方法，包括以下步骤：A universal ultra-sensitive ELISA immunoassay method based on surface-enhanced Raman scattering (SERS), including the following steps:
(1)将特异性识别抗原分子的第一抗体分子通过孵育固定于孔板基底，由于结合孔板基底的第一抗体分子仍具有免疫活性，加入相应的抗原分子进行免疫反应，然后再加入ALP标记的第二抗体分子，与抗原分子通过免疫反应形成抗原抗体分子键结的免疫反应结构；(1) The first antibody molecule that specifically recognizes the antigen molecule is fixed on the orifice substrate by incubation. Since the first antibody molecule bound to the orifice substrate still has immunological activity, add the corresponding antigen molecule for immune response, and then add ALP The labeled second antibody molecule reacts with the antigen molecule to form an immunoreactive structure of antigen-antibody molecule bonding;
(2)将步骤(1)具有免疫反应结构的抗体抗原分子与PPNa溶液混合均匀，室温下孵育5～30min，使第二抗体分子上的ALP分解PPNa产生苯酚，然后加入SERS探针，继续孵育10～30min，最后进行SERS检测；(2) Mix the antibody antigen molecule with immunoreactive structure in step (1) and PPNa solution uniformly, and incubate at room temperature for 5-30 minutes to make the ALP on the second antibody molecule decompose PPNa to produce phenol, then add SERS probe and continue incubating 10～30min, finally SERS test;
其中第二抗体分子、抗原分子和特异性识别抗原分子的第一抗体分子均属同一种生物分子，所述生物分子为细菌、细胞、病毒、DNA和RNA中的一种；The second antibody molecule, the antigen molecule, and the first antibody molecule that specifically recognizes the antigen molecule are all the same biological molecule, and the biological molecule is one of bacteria, cells, viruses, DNA, and RNA;
所述SERS探针为上述一种苯酚响应型表面增强拉曼散射(SERS)探针。The SERS probe is the above-mentioned phenol-responsive surface-enhanced Raman scattering (SERS) probe.
步骤(2)所述PPNa溶液的浓度为0.5～3mmol/L，溶剂为水。所述PPNa溶液中的PPNa与具有免疫反应结构的抗体抗原分子中的第二抗体分子的质量比为1：(2～10)。所述SERS探针数量和PPNa溶液中PPNa的摩尔量比为1个：(4.5×10 -5～1.8×10 -4)mmol。 The concentration of the PPNa solution in step (2) is 0.5-3 mmol/L, and the solvent is water. The mass ratio of the PPNa in the PPNa solution to the second antibody molecule in the antibody antigen molecule with an immunoreactive structure is 1: (2-10). The ratio of the number of SERS probes to the molar amount of PPNa in the PPNa solution is 1: (4.5×10 -5 to 1.8×10 -4 ) mmol.
步骤(2)所述SERS探针以SERS探针水溶液的形式加入，所述SERS探针水溶液中SERS探针的含量为50～200个/mL。In step (2), the SERS probe is added in the form of a SERS probe aqueous solution, and the content of the SERS probe in the SERS probe aqueous solution is 50-200 probes/mL.
步骤(2)所述SERS检测的条件为：用785nm激光激发，记录单个SERS探针上SERS光谱变化，功率为1mW，累计时间为5～60秒。所述用PBS缓冲液洗涤的次数为2～3次。The conditions of the SERS detection in step (2) are: excitation with a 785nm laser, record the SERS spectrum change on a single SERS probe, the power is 1 mW, and the cumulative time is 5-60 seconds. The number of times of washing with PBS buffer is 2 to 3 times.
一种基于苯酚响应型表面增强拉曼散射(SERS)探针的霍乱毒素(CT)酶 联免疫检测方法，包括以下步骤：A phenol-responsive surface-enhanced Raman scattering (SERS) probe-based cholera toxin (CT) enzyme-linked immunoassay method includes the following steps:
(1)将CT第一抗体分子加入到孔板中，加入含有0.05mol/L碳酸钠的PBS缓冲液，在37℃孵育1～3h后，用PBS缓冲液洗涤，然后加入明胶封闭CT第一抗体分子未覆盖的位点，用PBS缓冲液洗涤，再加入CT抗原溶液，在37℃孵育0.5～2小时，除去未反应的CT抗原溶液并用PBS缓冲液洗涤，再加入ALP标记的CT第二抗体分子，在37℃孵育1～3h后，用PBS缓冲液洗涤，得到具有酶联免疫典型结构的CT抗原抗体键结结构；(1) Add CT primary antibody molecules to the well plate, add PBS buffer containing 0.05 mol/L sodium carbonate, incubate at 37°C for 1 to 3 hours, wash with PBS buffer, and then add gelatin to block CT primary The uncovered sites of antibody molecules are washed with PBS buffer, then CT antigen solution is added, and incubated at 37°C for 0.5 to 2 hours. Unreacted CT antigen solution is removed and washed with PBS buffer, and then ALP-labeled CT second The antibody molecule is incubated at 37°C for 1 to 3 hours, and then washed with PBS buffer to obtain a CT antigen-antibody bonding structure with a typical structure of enzyme-linked immunoassay;
(2)将步骤(1)具有酶联免疫典型结构的CT抗原抗体分子和PPNa溶液混合均匀，室温下孵育5～30min后，加入上述SERS探针，继续孵育10～30min，最后进行SERS检测。(2) Mix the CT antigen-antibody molecule with the typical structure of enzyme-linked immunoassay in step (1) and PPNa solution uniformly, and after incubating at room temperature for 5-30 minutes, add the above-mentioned SERS probe, continue incubating for 10-30 minutes, and finally perform SERS detection.
步骤(1)所述CT第一抗体分子在含有0.05mol/L碳酸钠的PBS缓冲液中的浓度为1～50μg/mL；所述CT抗原溶液的溶剂为水，浓度为0.1～100pg/mL。Step (1) The concentration of the CT primary antibody molecule in the PBS buffer containing 0.05 mol/L sodium carbonate is 1-50 μg/mL; the solvent of the CT antigen solution is water, and the concentration is 0.1-100 pg/mL .
步骤(1)所述CT第一抗体分子与明胶的质量比1：100～500。所述CT抗原溶液中的CT抗原与CT第一抗体分子的质量比为1：1；所述ALP标记的CT第二抗体分子与CT第一抗体分子的质量比为1：1。The mass ratio of the CT first antibody molecule to gelatin in step (1) is 1:100-500. The mass ratio of CT antigen to CT first antibody molecule in the CT antigen solution is 1:1; the mass ratio of the ALP-labeled CT second antibody molecule to CT first antibody molecule is 1:1.
步骤(1)所述用PBS缓冲液洗涤的次数均为2～3次。The number of times of washing with PBS buffer in step (1) is 2 to 3 times.
步骤(2)所述PPNa溶液中的PPNa与具有酶联免疫典型结构的CT抗原抗体分子中的第二抗体分子的质量比为1：(2～10)。In step (2), the mass ratio of PPNa in the PPNa solution to the second antibody molecule in the CT antigen antibody molecule with the typical structure of enzyme-linked immunoassay is 1: (2-10).
步骤(2)所述SERS探针个数和PPNa溶液中PPNa的摩尔量比为1个：(4.5×10 -5～1.8×10 -4)mmol。所述PPNa溶液的浓度为0.5～3mmol/L，溶剂为水。所述SERS探针以SERS探针水溶液的形式加入，所述SERS探针水溶液中SERS探针的含量为50～200个/mL。 The ratio of the number of SERS probes to the molar amount of PPNa in the PPNa solution in step (2) is 1: (4.5×10 -5 ～1.8×10 -4 ) mmol. The concentration of the PPNa solution is 0.5-3 mmol/L, and the solvent is water. The SERS probe is added in the form of a SERS probe aqueous solution, and the content of the SERS probe in the SERS probe aqueous solution is 50-200 pieces/mL.
步骤(2)所述SERS检测的条件为：用785nm激光激发，记录单个SERS探针上SERS光谱变化，功率为1mW，累计时间为5～60秒。所述用PBS缓冲液洗涤的次数为2～3次。The conditions of the SERS detection in step (2) are: excitation with a 785nm laser, record the SERS spectrum change on a single SERS probe, the power is 1 mW, and the cumulative time is 5-60 seconds. The number of times of washing with PBS buffer is 2 to 3 times.
与现有技术相比，本发明具有以下优点及有益效果：Compared with the prior art, the present invention has the following advantages and beneficial effects:
1、本申请制备具有“卷心菜”状结构的金颗粒自组装体(SERS基底)，其拥有大量拉曼增强“热点”，可显著增强SERS信号，又可实现单个SERS探针检测，解决了传统SERS基底不均匀所造成的测量信号不均一的问题，极大提高检测重现性。1. The present application prepares gold particle self-assembly (SERS substrate) with a "cabbage"-like structure, which has a large number of Raman enhanced "hot spots", which can significantly enhance the SERS signal, and can realize the detection of a single SERS probe, which solves the traditional The problem of uneven measurement signal caused by uneven SERS substrate greatly improves the reproducibility of detection.
2、本申请合成高特异性苯酚识别的拉曼分子，该分子中含有叠氮基团，可与苯酚形成偶氮类化合物，通过拉曼指纹在1141cm -1处的峰强度信息，能体现准确地获得生成偶氮苯的含量数量，从而获得苯酚的浓度，且DTDBD分子中的二硫键可和金基底形成Au-S键，从而保证DTDBD的稳定性，可实现苯酚类物质的高灵敏SERS检测。 2. This application synthesizes a Raman molecule recognized by phenol with high specificity. The molecule contains an azide group that can form an azo compound with phenol. The peak intensity information of the Raman fingerprint at 1141 cm -1 can be accurately reflected Obtain the amount of azobenzene generated, thereby obtaining the concentration of phenol, and the disulfide bonds in the DTDBD molecule can form Au-S bonds with the gold substrate, thereby ensuring the stability of DTDBD and realizing highly sensitive SERS for phenolic substances Detection.
3、本申请将制得的苯酚响应性SERS探针用于ALP的高灵敏检测，ALP能高效地催化其底物PPNa水解导致其磷酸基团的失去，从而生成苯酚。因此通过SERS技术对所产生的苯酚的浓度测定可灵敏检测ALP。3. This application uses the prepared phenol-responsive SERS probe for the highly sensitive detection of ALP. ALP can efficiently catalyze the hydrolysis of its substrate PPNa to cause the loss of its phosphate group, thereby generating phenol. Therefore, the determination of the concentration of phenol produced by SERS technology can sensitively detect ALP.
4、本申请将ELISA与SERS联用用于CT的高灵敏检测，基于ELISA中的“三明治”检测策略，采用ALP作为抗体标记物，将抗体固定于孔板表面，捕获溶液中抗原，再结合ALP标记的第二抗体分子，利用ALP水解其底物PPNa产生苯酚，通过苯酚引起的SERS探针信号的改变，可灵敏检测抗原-霍乱毒素CT。由于临床上测定ALP主要用于骨骼、肝胆系统疾病的诊断，且CT检测是霍乱病因诊断的重要依据，本发明具有巨大的临床应用潜力。4. This application uses the combination of ELISA and SERS for the high-sensitivity detection of CT. Based on the "sandwich" detection strategy in ELISA, ALP is used as the antibody marker, the antibody is fixed on the surface of the well plate, the antigen in the solution is captured, and then combined The ALP-labeled secondary antibody molecule uses ALP to hydrolyze its substrate PPNa to produce phenol. The change in the signal of the SERS probe caused by phenol can sensitively detect the antigen-cholera toxin CT. Since the clinical determination of ALP is mainly used for the diagnosis of skeletal and hepatobiliary system diseases, and CT detection is an important basis for the diagnosis of the cause of cholera, the present invention has huge clinical application potential.
5、本申请将制得的SERS探针用于检测免疫分析，具有极高的普适性，基于ELISA原理，通过改变酶标记物，利用高灵敏的SERS技术检测其相应的抗原分子，实现多样性，使得该技术具有通用性，可广泛用于检测多种生物分子，如细菌、细胞、病毒、DNA和RNA等临床应用潜力。5. This application uses the prepared SERS probe for detection and immunoassay, which has extremely high universality. Based on the principle of ELISA, by changing the enzyme label, using the highly sensitive SERS technology to detect its corresponding antigen molecules, it can achieve diversification. The nature of this technology makes this technology versatile and can be widely used to detect a variety of biomolecules, such as bacteria, cells, viruses, DNA and RNA, and other clinical applications.
6、本申请所述表面增强拉曼光谱谱峰较窄，具有较强的分辨率及高灵敏度，将酶联免疫分析方法与SERS检测技术相结合，使得二者互补，既极大地提高了ELISA的灵敏度，又有效解决了SERS信号强度和重现性问题，开发出一种基于SERS的通用超灵敏的免疫分析技术，为两种技术的发展都提供了新的机遇。6. The surface-enhanced Raman spectrum of this application has narrow peaks, strong resolution and high sensitivity. The combination of enzyme-linked immunoassay method and SERS detection technology makes the two complementary, which greatly improves ELISA The sensitivity of SERS effectively solves the problems of SERS signal strength and reproducibility, and the development of a general-purpose ultra-sensitive immunoassay technology based on SERS provides new opportunities for the development of both technologies.
7、本申请将SERS检测技术和ELISA检测技术相结合，在生物、化学检测，医学诊断等领域都有很大的发展空间和广阔的应用前景。7. This application combines SERS detection technology and ELISA detection technology, and has great development space and broad application prospects in the fields of biology, chemical detection, and medical diagnosis.
附图说明Description of the drawings
图1为实施例1制得的金微米粒子CLMP的扫描电镜图。FIG. 1 is a scanning electron micrograph of the gold microparticle CLMP prepared in Example 1.
图2为实施例1制得的SERS探针颗粒的光学显微镜图。2 is an optical microscope image of the SERS probe particles prepared in Example 1.
图3为本申请DTDBD的合成路线以及检测反应机理路线图，其中(a)为特异性识别苯酚的DTDBD的合成路线图，(b)为DTDBD与苯酚通过偶氮偶联反应机理线路图，(c)为DTDBD在CLMP表面上自组装形成DTDBD-CLMP以及其与苯酚反应示意图。Figure 3 is the synthesis route and detection reaction mechanism route diagram of DTDBD of the application, in which (a) is the synthesis route diagram of DTDBD which specifically recognizes phenol, (b) is the route diagram of DTDBD and phenol through the azo coupling reaction mechanism, ( c) is a schematic diagram of the self-assembly of DTDBD on the surface of CLMP to form DTDBD-CLMP and its reaction with phenol.
图4为实施例1制得的DTDBD-CLMP的SERS光谱以及实施例2中DTDBD-CLMP和与苯酚反应后生成的偶氮苯-CLAMP的SERS光谱。Figure 4 shows the SERS spectra of DTDBD-CLMP prepared in Example 1 and the SERS spectra of DTDBD-CLMP and azobenzene-CLAMP produced after reaction with phenol in Example 2.
图5为实施例2中DTDBD-CLMP与不同浓度的苯酚孵育后的SERS光谱。Figure 5 shows the SERS spectra of DTDBD-CLMP in Example 2 after incubation with different concentrations of phenol.
图6为实施例2中偶氮苯-CLMP在1141cm -1处的SERS峰强度与苯酚浓度的关系。 Figure 6 shows the relationship between the SERS peak intensity of azobenzene-CLMP at 1141 cm -1 and the phenol concentration in Example 2.
图7为实施例3中ALP催化PPNa生成苯酚及其与DTDBD反应生成偶氮苯的机理示意图。7 is a schematic diagram of the mechanism of ALP catalyzing PPNa to generate phenol and its reaction with DTDBD to generate azobenzene in Example 3.
图8为实施例3中DTDBD-CLMP与不同浓度的ALP孵育后的SERS光谱。Figure 8 shows the SERS spectra of DTDBD-CLMP after incubation with different concentrations of ALP in Example 3.
图9为实施例3中在1141cm -1处的偶氮苯-CLMP的SERS峰强度与ALP浓度的关系。 Fig. 9 shows the relationship between the SERS peak intensity of azobenzene-CLMP at 1141 cm -1 and the ALP concentration in Example 3.
图10为实施例4中基于苯酚识别的SERS传感用于CT免疫分析的检测原理示意图。FIG. 10 is a schematic diagram of the detection principle of the SERS sensor based on phenol recognition used in CT immunoassay in Example 4. FIG.
图11为实施例4中DTDBD-CLMP对不同CT浓度的SERS响应。Figure 11 shows the SERS response of DTDBD-CLMP to different CT concentrations in Example 4.
图12为实施例4中在1141cm -1处的偶氮苯-CLMP的SERS峰强度与CT浓度的关系。 Fig. 12 shows the relationship between the SERS peak intensity of azobenzene-CLMP at 1141 cm -1 and the CT concentration in Example 4.
具体实施方式detailed description
下面结合实施例和附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。The present invention will be further described in detail below in conjunction with the examples and drawings, but the implementation of the present invention is not limited thereto.
本申请实施例中未注明的工艺参数，均可见常规方法进行，所用原料均可通过商业渠道获得。The process parameters not specified in the examples of this application can be carried out by conventional methods, and all the raw materials used can be obtained through commercial channels.
本申请实施例所述SERS测试的条件为：使用785nm激光激发，记录单个CLMP上的SERS光谱，功率为1mW，总累计时间为10秒。每个样品，测量时分别获得10个CLMP的SERS光谱用于标准偏差计算。The conditions of the SERS test described in the examples of this application are: using 785nm laser excitation, recording the SERS spectrum on a single CLMP, the power is 1mW, and the total cumulative time is 10 seconds. For each sample, 10 CLMP SERS spectra were obtained during measurement for standard deviation calculation.
本申请实施例中所述CLMP水溶液、探针水溶液的浓度均指1mL水中含有CLMP或探针的个数(数量)。The concentrations of the CLMP aqueous solution and the probe aqueous solution in the examples of the application all refer to the number (quantity) of CLMP or probes contained in 1 mL of water.
实施例1Example 1
(1)“卷心菜”结构金微米颗粒的合成(1) Synthesis of "Cabbage" structure gold micron particles
将8μL质量浓度为10％的氯金酸水溶液溶于1mL浓度为1mmol/L的盐酸中，加入1.6mg PVP，放置在冰水中10分钟，再加入40μL 100mg/mL NAAN盐酸水溶液，震荡20分钟后在4℃下静置反应24小时；在5000rpm下离心10min，离心后用NMP洗涤，得到金微米粒子CLMP，然后分散于水中得到浓度为100个/mL的CLMP水溶液，4℃保存备用。所制备的“卷心菜”结构金微米颗粒CLMP的形貌如图1所示。Dissolve 8μL of chloroauric acid aqueous solution with a mass concentration of 10% in 1mL of hydrochloric acid with a concentration of 1mmol/L, add 1.6mg PVP, place in ice water for 10 minutes, then add 40μL of 100mg/mL NAAN hydrochloric acid aqueous solution, shake for 20 minutes The reaction was allowed to stand at 4°C for 24 hours; centrifuged at 5000 rpm for 10 minutes, washed with NMP after centrifugation to obtain gold microparticle CLMP, then dispersed in water to obtain a CLMP aqueous solution with a concentration of 100/mL, and stored at 4°C for later use. The morphology of the prepared "Cabbage" structure gold micron particles CLMP is shown in Figure 1.
(2)特异性识别苯酚的拉曼分子的合成(2) Synthesis of Raman molecules that specifically recognize phenol
将2.82mmol(700mg)DTDBA溶于40mL浓度为1mol/L的HCl中，在冰水(0℃)浴中冷却，然后边搅拌边加入2mL浓度为3.1mmol/mL的亚硝酸钠水溶液，冰水浴中持续搅拌1小时后，在搅拌状态下向上述混合液中加入15mL冷却的饱和四氟硼酸钠水溶液(即浓度为108g/100mL)，反应10分钟得到的沉淀即为DTDBD；过滤后用乙腈/二乙基乙基重结晶，将最终产物在50℃真空干燥24h并储存在冰箱中备用。Dissolve 2.82mmol (700mg) DTDBA in 40mL of 1mol/L HCl, cool in an ice water (0℃) bath, and then add 2mL of 3.1mmol/mL sodium nitrite aqueous solution while stirring, ice water bath After stirring continuously for 1 hour, add 15mL of cooled saturated sodium tetrafluoroborate aqueous solution (i.e. the concentration of 108g/100mL) to the above mixed solution under stirring. The precipitate obtained after reaction for 10 minutes is DTDBD; after filtration, use acetonitrile/ Diethyl ethyl was recrystallized, and the final product was vacuum dried at 50°C for 24 hours and stored in a refrigerator for later use.
DTDBD的分子结构及其合成路线如图3所示，制得的DTDBD具有两个官能团：1)重氮离子可通过偶氮偶合反应与苯酚特异反应形成偶氮苯(见图3b)；2)二硫化物基团有利于在CLMP中形成过程中DTDBD与Au纳米片形成稳固的Au-S共价键。The molecular structure of DTDBD and its synthetic route are shown in Figure 3. The prepared DTDBD has two functional groups: 1) Diazonium ions can react specifically with phenol through an azo coupling reaction to form azobenzene (see Figure 3b); 2) The disulfide group is conducive to the formation of a stable Au-S covalent bond between DTDBD and Au nanosheets during the formation of CLMP.
(3)苯酚响应的SERS探针的合成(3) Synthesis of phenol-responsive SERS probe
将3.7mg步骤(2)中生成的DTDBD溶于1mL DMF中，加入10μL步骤(1)的CLMP水溶液，混合均匀得到混合液，将混合溶液在冰箱(4℃)中静置反应2小时，反应产物用DMF洗涤后分散于水中形成DTDBD-CLMP水溶液，该水溶液中DTDBD-CLMP的含量为100个/mL，即为SERS探针水溶液。DTDBD-CLMP在二氧化硅晶片上的光学显微镜图如图2所示，DTDBD-CLMP的SERS光谱(见图4中曲线1)显示分别位于1078cm -1和1590cm -1的苯基的两个强拉曼峰，表明CLMP携带大量DTDBD分子。 Dissolve 3.7 mg of the DTDBD produced in step (2) in 1 mL DMF, add 10 μL of the CLMP aqueous solution from step (1), and mix well to obtain a mixed solution. The mixed solution is allowed to stand in a refrigerator (4°C) for 2 hours to react. The product was washed with DMF and dispersed in water to form an aqueous solution of DTDBD-CLMP. The content of DTDBD-CLMP in the aqueous solution was 100 pcs/mL, which was the SERS probe aqueous solution. DTDBD-CLMP optical microscope on silica wafer shown in FIG, DTDBD-CLMP SERS spectrum of FIG. 2 (see FIG. 4, curve 1) display at two strong 1078cm -1 1590cm -1 and phenyl, respectively, The Raman peak indicates that CLMP carries a large number of DTDBD molecules.
实施例2Example 2
一种苯酚的SERS检测方法。A SERS detection method for phenol.
将10μL实施例1制得的SERS探针水溶液分别加入到1mL含有不同浓度苯酚的Na 2CO 3水溶液中，所述Na 2CO 3水溶液中苯酚的浓度分别为1×10 -9mol/L、5×10 -9mol/L、1×10 -8mol/L、5×10 -8mol/L、1×10 -7mol/L、5×10 -7mol/L、1×10 -6mol/L、1×10 -5mol/L、1×10 -3mol/L，所述Na 2CO 3水溶液的质量浓度为5％，然后均在4℃下孵育15min，收集探针并用水洗涤，得到沉淀物再分散于水中(其中SERS探针的含量为100个/mL)，得到待测样品，吸取一滴待测样品在室温下干燥2h后进行SERS测量。 10μL aqueous SERS probe prepared in Example 1 were added to 1mL 2 CO 3 solution containing different concentrations of phenol Na, the concentration of Na 2 CO 3 aqueous solution of phenol were 1 × 10 -9 mol / L, 5×10 -9 mol/L, 1×10 -8 mol/L, 5×10 -8 mol/L, 1×10 -7 mol/L, 5×10 -7 mol/L, 1×10 -6 mol/L, 1×10 -5 mol/L, 1×10 -3 mol/L, the mass concentration of the Na 2 CO 3 aqueous solution is 5%, and then all are incubated at 4° C. for 15 min, collect the probe and use water After washing, the obtained precipitate is dispersed in water (wherein the content of SERS probe is 100 pcs/mL) to obtain the sample to be tested, a drop of the sample to be tested is drawn and dried at room temperature for 2 hours, and then the SERS measurement is performed.
DTDBD-CLMP与Na 2CO 3水溶液中的苯酚(浓度为1mmol/L)孵育后，DTDBD与苯酚反应生成偶氮苯，在1141cm -1、1391cm -1和1438cm -1处均产生新拉曼峰(见图4中的曲线2)。不同浓度的苯酚Na 2CO 3水溶液与DTDBD-CLMP反应后所得SERS光谱显示，与1078cm -1处基本不变的SERS峰强度相比，位于1141cm -1处的峰强度随着苯酚浓度的增加而增强，表现出特征的被测物浓度 依赖性特征(见图5)。结果表明该SERS探针对于苯酚浓度线性响应从1×10 -9mol/L增加到5×10 -7mol/L，基于LOD＝3×δ/m，理论检测限(LOD)计算为0.4×10 -9mol/L，其中δ是最低测试浓度(此处为1nmol/L)的响应的标准偏差，m为浓度依赖性响应的斜率(见图6)。 After 2 CO 3 aqueous solution and phenol DTDBD-CLMP of Na (concentration of 1mmol / L) were incubated, DTDBD phenol is reacted with azobenzene generated in 1141cm -1, 1391cm -1 and 1438cm -1 at a new Raman peaks are (See curve 2 in Figure 4). Different concentrations of Na 2 CO 3 aqueous phenol and the resulting reaction DTDBD-CLMP SERS spectra show substantially unchanged compared with the SERS peak intensity at 1078 cm -1, a peak intensity at 1141cm -1 positioned with increasing phenol concentration Enhancement, showing characteristic analyte concentration-dependent characteristics (see Figure 5). The results showed that the linear response of the SERS probe to phenol concentration increased from 1×10 -9 mol/L to 5×10 -7 mol/L, based on LOD=3×δ/m, the theoretical limit of detection (LOD) was calculated as 0.4× 10 -9 mol/L, where δ is the standard deviation of the response at the lowest tested concentration (here 1nmol/L), and m is the slope of the concentration-dependent response (see Figure 6).
本实施例所提出的苯酚响应型SERS传感器可高灵敏度检测苯酚，其性能优于现有技术所述苯酚的检测方法(见表1)。The phenol-responsive SERS sensor proposed in this embodiment can detect phenol with high sensitivity, and its performance is better than the phenol detection method described in the prior art (see Table 1).
表1本实施例所述方法与其他苯酚检测方法的对比Table 1 Comparison of the method described in this example with other phenol detection methods
Figure PCTCN2020114769-appb-000001
Figure PCTCN2020114769-appb-000001
电化学/酪氨酸酶N.Li,M.H.Xue,H.Yao,J.J.Zhu,Anal.Bioanaly.Chem.,2005,383,1127.Electrochemistry/Tyrosinase N.Li, M.H. Xue, H. Yao, J.J. Zhu, Anal.Bioanaly.Chem., 2005, 383, 1127.
聚丙烯酰胺微凝胶/电化学J.P.Hervás Pérez,M.Sánchez-Paniagua López,E.López-Cabarcos,B.López-Ruiz,Biosens.Bioelectron.,2006,22,429.Polyacrylamide microgel/electrochemistry J.P. Hervás Pérez, M. Sánchez-Paniagua López, E. López-Cabarcos, B. López-Ruiz, Biosens. Bioelectron., 2006, 22, 429.
氧化石墨烯–氧化锌/电化学T.Arfin,S.N.Rangari,Anal.Methods,2018,10,347.Graphene Oxide-Zinc Oxide/Electrochemistry T.Arfin, S.N.Rangari, Anal.Methods, 2018, 10, 347.
碳纳米管/聚乙烯亚胺/电化学A.S.Arribas,E.Bermejo,M.Chicharro,A.Zapardiel,G.L.Luque,N.F.Ferreyra,G.A.Rivas,Anal.Chim.Acta,2007,596,183.Carbon nanotubes/polyethyleneimine/electrochemistry A.S.Arribas, E.Bermejo, M.Chicharro, A.Zapardiel, G.L.Luque, N.F.Ferreyra, G.A.Rivas, Anal.Chim.Acta, 2007,596,183.
光学/酪氨酸酶J.Abdullah,M.Ahmad,N.Karuppiah,L.Y.Heng,H.Sidek,Sensor.Actuat.B:Chem.,2006,114,604.Optical/tyrosinase J. Abdullah, M. Ahmad, N. Karuppiah, L. Y. Heng, H. Sidek, Sensor. Actuat. B: Chem., 2006, 114, 604.
实施例3Example 3
一种ALP的SERS检测方法A SERS detection method for ALP
分别向950μL含浓度为1mmol/L PPNa的Tris-HCl缓冲液(pH 9.8)中，加入50μL不同浓度(分别为0.1mU/L、0.5mU/L、1mU/L、5mU/L、10mU/L、50mU/L)的ALP水溶液。室温下孵育20分钟，分别加入10μL实施例1制得的SERS探针水溶液，在室温下孵育15min。用水洗涤后，所得沉淀物分散于水中，得到待测样品(其中SERS探针的含量为100个/mL)，吸取一滴待测样品滴加在硅晶片上，在室温下干燥2h后进行SERS测量。Add 50μL of different concentrations (0.1mU/L, 0.5mU/L, 1mU/L, 5mU/L, 10mU/L, respectively, to 950μL Tris-HCl buffer (pH 9.8) containing 1mmol/L PPNa. , 50mU/L) ALP water solution. Incubate for 20 minutes at room temperature, add 10 μL of the SERS probe aqueous solution prepared in Example 1, and incubate for 15 minutes at room temperature. After washing with water, the resulting precipitate is dispersed in water to obtain the sample to be tested (the content of SERS probes is 100/mL), a drop of the sample to be tested is sucked and dropped on the silicon wafer, dried at room temperature for 2 hours, and then SERS measurement is performed .
本实施例反应机理及检测结果如图7、8、9所示。1141cm -1处SERS峰强度明显随ALP浓度的增加而持续增强(见图8)。根据本实施例的方法，ALP的检出限为0.04mU/L，线性范围为0.1～50mU/L(见图9)。 The reaction mechanism and test results of this embodiment are shown in Figures 7, 8, and 9. The intensity of the SERS peak at 1141 cm -1 obviously continued to increase with the increase of the ALP concentration (see Figure 8). According to the method of this embodiment, the detection limit of ALP is 0.04 mU/L, and the linear range is 0.1-50 mU/L (see Figure 9).
本实施例所提出的SERS检测方法可高灵敏度检测ALP，其性能优于现有技术所述ALP的检测方法(见表2)。The SERS detection method proposed in this embodiment can detect ALP with high sensitivity, and its performance is better than the ALP detection method described in the prior art (see Table 2).
表2本实施例所述方法与其他ALP检测方法的比较Table 2 Comparison of the method described in this embodiment with other ALP detection methods
Figure PCTCN2020114769-appb-000002
Figure PCTCN2020114769-appb-000002
电化学发光H.Jiang,X.Wang,Anal.Chem.,2012,84,6986.Electrochemiluminescence H. Jiang, X. Wang, Anal. Chem., 2012, 84, 6986.
比色/有机金属骨架C.Wang,J.Gao,Y.Cao,H.Tan,Anal.Chim.Acta,2018,1004,74.Colorimetric/organometallic framework C. Wang, J. Gao, Y. Cao, H. Tan, Anal. Chim. Acta, 2018, 1004, 74.
SERS J.Zhang,L.He,X.Zhang,J.Wang,L.Yang,B.Liu,C.Jiang,Z.Zhang,Sensor Actuat.B Chem.,2017,253,839.SERS J. Zhang, L. He, X. Zhang, J. Wang, L. Yang, B. Liu, C. Jiang, Z. Zhang, Sensor Actuat. B Chem., 2017, 253, 839.
实施例4Example 4
一种CT的SERS检测方法A CT SERS Detection Method
先将100μL含有0.05mol/L碳酸钠的CT第一抗体分子PBS缓冲液加入到96孔板中，其中CT第一抗体分子(Anti-Cholera Toxin antibody(ab123129)，购买于艾博抗(上海)贸易公司)的浓度为10μg/mL，在37℃孵育2小时，随后用PBS缓冲液洗涤3次，并加入过量明胶封闭抗体未覆盖的位点(CT第一抗体分子与明胶的质量比1：200)，然后再用PBS缓冲液洗涤除去过量的明胶，随后分别在不同的孔板中注入不同浓度的CT抗原(Cholera Toxin from Vibrio cholera(c8052)购买自西格玛奥德里奇公司)水溶液各100μL，浓度分别为0.1pg/mL、0.5pg/mL、1pg/mL、5pg/mL、10pg/mL、50pg/mL、100pg/mL，37℃反应1小时后，用PBS缓冲液洗涤3次移去未反应的CT抗原溶液；再加入100μL浓度为1mg/mL ALP标记的CT第二抗体分子(Anti-Cholera Toxin antibody(ALP)，购买于艾博抗(上海)贸易公司)水溶液，并在37℃温育1小时后用PBS缓冲液洗涤以清除过量的CT第二抗体分子；然后分别将90μL浓度为1mmol/L的PPNa水溶液注入上述含CT第一抗体分子、抗原和第二抗体分子的孔板中，室温下孵育20分钟后分别加入实施例1制得的SERS探针水溶液10μL，混合均匀得到混合液，室温下孵育15分钟后，取反应产物进行测量SERS光谱，通过测定单个SERS光谱上的拉曼信号变化计算检测线性范围和检出限。ELISA CT传感检测机制如图10所示。First add 100μL of CT primary antibody molecule PBS buffer containing 0.05mol/L sodium carbonate to the 96-well plate, in which CT primary antibody molecule (Anti-Cholera Toxin antibody (ab123129), purchased from Abcam (Shanghai) Trading company) with a concentration of 10μg/mL, incubate at 37°C for 2 hours, then wash with PBS buffer 3 times, and add excess gelatin to block the uncovered sites of the antibody (the mass ratio of CT primary antibody molecule to gelatin is 1: 200), and then washed with PBS buffer to remove excess gelatin, and then inject different concentrations of CT antigen (Cholera Toxin from Vibrio cholera (c8052) purchased from Sigma-Aldrich) into different well plates with 100 μL each, The concentrations are 0.1pg/mL, 0.5pg/mL, 1pg/mL, 5pg/mL, 10pg/mL, 50pg/mL, 100pg/mL, and after reacting at 37°C for 1 hour, wash with PBS buffer three times to remove Reactive CT antigen solution; then add 100μL of 1mg/mL ALP-labeled CT secondary antibody molecule (Anti-Cholera Toxin Antibody (ALP), purchased from Abcam (Shanghai) Trading Company) aqueous solution, and warm it at 37℃ After incubating for 1 hour, wash with PBS buffer to remove excess CT secondary antibody molecules; then respectively inject 90 μL of 1mmol/L PPNa aqueous solution into the above-mentioned well plates containing CT primary antibody molecules, antigen and secondary antibody molecules After incubating for 20 minutes at room temperature, 10 μL of the SERS probe aqueous solution prepared in Example 1 was added, and mixed uniformly to obtain a mixed solution. After incubating for 15 minutes at room temperature, the reaction product was taken to measure the SERS spectrum. Mann signal changes to calculate the detection linear range and detection limit. The ELISA CT sensing detection mechanism is shown in Figure 10.
CT检测结果如图11、12所示。结果表明SERS探针可灵敏响应CT的存在(见图11)，且1141cm -1处的SERS峰强度随着CT浓度的增加而逐渐增加，CT浓度线性响应从8.3×10 -14mol/L(0.1pg/mL)增加到8.3×10 -12mol/L(10pg/mL)(见图12)。此外，采用标准加入法对血清样品中CT回收率进行测定，以评估本实施例提供的CT测定方法的可靠性。向空白血清样品中分别加入不同浓度的标准CT样品(使其在血清样品中的最终浓度分别为0.5pg/mL、1pg/mL和5pg/mL，其溶剂为水)，进行SERS测试。SERS测量结果表明CT回收率在98.8～103.3％范围内，相对标准偏差(RSD)小于5.6％(n＝3)(见表3)。这表明本实施例提供的基于SERS的免疫分析技术可高灵敏检测CT，且将ALP 标记物替换为其他抗原，该技术均可应用于多种生物待测物的检测，在生物分析和疾病诊断中具有广阔的应用前景。 The results of CT detection are shown in Figures 11 and 12. The results show that the SERS probe can sensitively respond to the presence of CT (see Figure 11), and the intensity of the SERS peak at 1141 cm -1 gradually increases with the increase of CT concentration. The linear response of CT concentration is from 8.3×10 -14 mol/L ( 0.1pg/mL) to 8.3×10 -12 mol/L (10pg/mL) (see Figure 12). In addition, the standard addition method was used to determine the CT recovery rate in the serum sample to evaluate the reliability of the CT determination method provided in this embodiment. Standard CT samples of different concentrations were added to the blank serum samples (the final concentrations in the serum samples were 0.5 pg/mL, 1 pg/mL, and 5 pg/mL, respectively, and the solvent was water), and the SERS test was performed. SERS measurement results show that the CT recovery rate is in the range of 98.8-103.3%, and the relative standard deviation (RSD) is less than 5.6% (n=3) (see Table 3). This shows that the SERS-based immunoassay technology provided in this example can detect CT with high sensitivity, and replace the ALP marker with other antigens. This technology can be applied to the detection of a variety of biological analytes, in biological analysis and disease diagnosis. China has broad application prospects.
表3基于SERS的免疫分析技术检测血清样本中CT含量Table 3 Detection of CT content in serum samples based on SERS immunoassay technology
Figure PCTCN2020114769-appb-000003
Figure PCTCN2020114769-appb-000003
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, etc. made without departing from the spirit and principle of the present invention Simplified, all should be equivalent replacement methods, and they are all included in the protection scope of the present invention.

Claims (10)

  1. 一种苯酚响应型表面增强拉曼散射探针的制备方法，其特征在于，包括以下步骤：A method for preparing a phenol-responsive surface-enhanced Raman scattering probe is characterized in that it comprises the following steps:
    (1)将氯金酸水溶液溶于盐酸中，然后加入聚乙烯吡咯烷酮，冰水浴条件下反应5～20min，加入3-脒基-苯胺，混合均匀，2～8℃下静置反应12～48h，离心洗涤，得到金微米粒子CLMPs；(1) Dissolve the chloroauric acid aqueous solution in hydrochloric acid, then add polyvinylpyrrolidone, react for 5-20 minutes under ice-water bath conditions, add 3-amidino-aniline, mix well, and let stand at 2-8℃ for 12～48h , Centrifugal washing to obtain CLMPs of gold microparticles;
    (2)在冰水浴条件下，将亚硝酸钠加入到2,2'-二硫二苯胺盐酸水溶液中，反应0.5～2h后，加入四氟硼酸钠，得到2,2'-二硫二苯氮四氟硼酸，即为特异性识别苯酚的拉曼分子；(2) Under ice-water bath conditions, add sodium nitrite to 2,2'-disulfide diphenylamine hydrochloric acid aqueous solution, after reaction for 0.5 to 2 hours, add sodium tetrafluoroborate to obtain 2,2'-disulfide diphenyl Nitrogentetrafluoroboric acid is a Raman molecule that specifically recognizes phenol;
    (3)将DTDBD溶于溶剂中，加入金微米粒子CLMPs水溶液，混合均匀，在2～8℃孵育0.5～4h后，洗涤，得到DTDBD-CLMPs，即为苯酚响应型表面增强拉曼散射探针。(3) Dissolve DTDBD in solvent, add gold microparticle CLMPs aqueous solution, mix well, incubate at 2～8℃ for 0.5～4h, wash, and obtain DTDBD-CLMPs, which are phenol-responsive surface-enhanced Raman scattering probes .
  2. 根据权利要求1所述一种苯酚响应型表面增强拉曼散射探针的制备方法，其特征在于，步骤(1)所述氯金酸水溶液的质量浓度为5～25％；所述盐酸的浓度为1mmol/L，所述氯金酸水溶液与盐酸的体积比为1：125；The method for preparing a phenol-responsive surface-enhanced Raman scattering probe according to claim 1, wherein the mass concentration of the aqueous solution of chloroauric acid in step (1) is 5-25%; the concentration of the hydrochloric acid It is 1 mmol/L, and the volume ratio of the chloroauric acid aqueous solution to hydrochloric acid is 1:125;
    步骤(1)所述氯金酸水溶液中的氯金酸、聚乙烯吡咯烷酮和3-脒基-苯胺的质量比为1：(1～3.2)：(2～10)；The mass ratio of chloroauric acid, polyvinylpyrrolidone and 3-amidino-aniline in the chloroauric acid aqueous solution in step (1) is 1:(1～3.2):(2～10);
    步骤(2)所述亚硝酸钠、2,2'-二硫二苯胺盐酸水溶液中2,2'-二硫二苯胺和四氟硼酸钠的摩尔比为1：(0.2～1)：(20～30)；Step (2) The molar ratio of 2,2'-disulfide diphenylamine and sodium tetrafluoroborate in the sodium nitrite and 2,2'-disulfide diphenylamine hydrochloric acid aqueous solution is 1:(0.2～1):(20 ～30);
    步骤(3)所述DTDBD摩尔量和金微米粒子CLMPs水溶液中金微米粒子CLMPs的个数比为(2.5×10 -3～6×10 -2)mmol：1个。 In step (3), the ratio of the molar amount of DTDBD to the number of CLMPs of gold microparticles in the gold microparticle CLMPs aqueous solution is (2.5×10 -3 -6×10 -2 ) mmol:1.
  3. 根据权利要求2所述一种苯酚响应型表面增强拉曼散射探针的制备方法，其特征在于，步骤(2)所述2,2'-二硫二苯胺盐酸水溶液由以下方法制得：将2,2'-二硫二苯胺溶于盐酸中，得到2,2'-二硫二苯胺盐酸水溶液，其中2,2'-二硫二苯胺在盐酸中的浓度为0.03～1.5mol/L，盐酸的浓度为0.5～2mol/L；The method for preparing a phenol-responsive surface-enhanced Raman scattering probe according to claim 2, wherein the 2,2'-dithiodiphenylamine hydrochloric acid aqueous solution in step (2) is prepared by the following method: 2,2'-disulfide diphenylamine is dissolved in hydrochloric acid to obtain 2,2'-disulfide diphenylamine hydrochloric acid aqueous solution, wherein the concentration of 2,2'-disulfide diphenylamine in hydrochloric acid is 0.03～1.5mol/L, The concentration of hydrochloric acid is 0.5～2mol/L;
    步骤(2)所述四氟硼酸钠以饱和四氟硼酸钠水溶液的形式加入；Step (2) The sodium tetrafluoroborate is added in the form of a saturated aqueous solution of sodium tetrafluoroborate;
    步骤(3)所述DTDBD在溶剂中的浓度为5～30mmol/L；所述金微米粒子CLMPs水溶液中金微米粒子CLMPs的含量为50～200个/mL；Step (3) The concentration of the DTDBD in the solvent is 5-30 mmol/L; the content of the gold micro-particle CLMPs in the gold micro-particle CLMPs aqueous solution is 50-200 pcs/mL;
    步骤(3)所述溶剂为二甲基甲酰胺、二甲基亚砜、乙醇、甲醇中的至少一种。The solvent in step (3) is at least one of dimethylformamide, dimethylsulfoxide, ethanol, and methanol.
  4. 权利要求1～3任一项所述方法制得的一种苯酚响应型表面增强拉曼散射探针。A phenol-responsive surface-enhanced Raman scattering probe prepared by the method of any one of claims 1 to 3.
  5. 权利要求4所述一种苯酚响应型表面增强拉曼散射探针在生物分析与传感检测领域中的应用。The application of a phenol-responsive surface-enhanced Raman scattering probe of claim 4 in the field of biological analysis and sensing detection.
  6. 一种基于表面增强拉曼散射的通用超灵敏ELISA免疫分析方法，其特征在于，包括以下步骤：A universal ultra-sensitive ELISA immunoassay method based on surface-enhanced Raman scattering, which is characterized in that it comprises the following steps:
    (1)将特异性识别抗原分子的第一抗体分子通过孵育固定于孔板基底，由于结合孔板基底的第一抗体分子仍具有免疫活性，加入相应的抗原分子进行免疫反应，然后再加入ALP标记的第二抗体分子，与抗原分子通过免疫反应形成抗原抗体分子键结的免疫反应结构；(1) The first antibody molecule that specifically recognizes the antigen molecule is fixed on the orifice substrate by incubation. Since the first antibody molecule bound to the orifice substrate still has immunological activity, add the corresponding antigen molecule for immune response, and then add ALP The labeled second antibody molecule reacts with the antigen molecule to form an immunoreactive structure of antigen-antibody molecule bonding;
    (2)将步骤(1)具有免疫反应结构的抗体抗原分子与PPNa溶液混合均匀，室温下孵育5～30min，使第二抗体分子上的ALP分解PPNa产生苯酚，然后加入SERS探针，继续孵育10～30min，最后进行SERS检测；(2) Mix the antibody antigen molecule with immunoreactive structure in step (1) and PPNa solution uniformly, and incubate at room temperature for 5-30 minutes to make the ALP on the second antibody molecule decompose PPNa to produce phenol, then add SERS probe and continue incubating 10～30min, finally SERS test;
    其中第二抗体分子、抗原分子和特异性识别抗原分子的第一抗体分子均属同一种生物分子，所述生物分子为细菌、细胞、病毒、DNA和RNA中的一种；The second antibody molecule, the antigen molecule, and the first antibody molecule that specifically recognizes the antigen molecule are all the same biological molecule, and the biological molecule is one of bacteria, cells, viruses, DNA, and RNA;
    所述SERS探针为权利要求4所述的苯酚响应型表面增强拉曼散射探针。The SERS probe is the phenol-responsive surface-enhanced Raman scattering probe of claim 4.
  7. 根据权利要求6所述一种基于表面增强拉曼散射的通用超灵敏免疫分析技术，其特征在于，步骤(2)所述PPNa溶液的浓度为0.5～3mmol/L，溶剂为水；所述PPNa溶液中的PPNa与具有免疫反应结构的抗体抗原分子中的第二抗体分子的质量比为1：(2～10)；所述SERS探针数量和PPNa溶液中PPNa的摩尔量比为1个：(4.5×10 -5～1.8×10 -4)mmol； The universal ultra-sensitive immunoassay technique based on surface-enhanced Raman scattering according to claim 6, wherein the concentration of the PPNa solution in step (2) is 0.5-3 mmol/L, and the solvent is water; the PPNa The mass ratio of PPNa in the solution to the second antibody molecule in the antibody antigen molecule with immunoreactive structure is 1: (2-10); the ratio of the number of SERS probes to the molar amount of PPNa in the PPNa solution is 1: (4.5×10 -5 ～1.8×10 -4 )mmol;
    步骤(2)所述SERS探针以SERS探针水溶液的形式加入，所述SERS探针水溶液中SERS探针的含量为50～200个/mL。In step (2), the SERS probe is added in the form of a SERS probe aqueous solution, and the content of the SERS probe in the SERS probe aqueous solution is 50-200 probes/mL.
  8. 一种基于苯酚响应型表面增强拉曼散射探针检测苯酚的方法，其特征在于，包括以下步骤：A method for detecting phenol based on a phenol-responsive surface-enhanced Raman scattering probe is characterized in that it comprises the following steps:
    将权利要求4所述SERS探针分散于水溶液中得到SERS探针水溶液，再加入到含苯酚的Na 2CO 3溶液中，混合均匀，2～8℃静置反应5～30min，离心，洗涤，得到沉淀物，分散于水中，得到待测样品，然后进行SERS检测； Disperse the SERS probe of claim 4 in the aqueous solution to obtain the SERS probe aqueous solution, then add it to the phenol-containing Na 2 CO 3 solution, mix well, stand at 2-8°C for reaction for 5-30 min, centrifuge, wash, Obtain the precipitate, disperse it in water, obtain the sample to be tested, and then perform SERS detection;
    所述SERS探针个数与含苯酚的Na 2CO 3水溶液中苯酚的质量比为1个：(4.7×10 -11～1.88×10 -6)g；所述SERS探针水溶液中SERS探针的含量为50～200个/mL；所述含苯酚的Na 2CO 3水溶液中苯酚的浓度为1×10 -10～1×10 -3mol/L，Na 2CO 3的质量浓度为1～20％；所述待测样品中SERS探针的含量为50～200个/mL。 The mass ratio of the number of the SERS probes to the phenol in the phenol-containing Na 2 CO 3 aqueous solution is 1: (4.7×10 -11 ～1.88×10 -6 ) g; the SERS probe in the SERS probe aqueous solution The content of phenol is 50～200/mL; the concentration of phenol in the phenol- containing Na 2 CO 3 aqueous solution is 1×10 -10 ～1×10 -3 mol/L, and the mass concentration of Na 2 CO 3 is 1～ 20%; the content of SERS probes in the sample to be tested is 50-200 pieces/mL.
  9. 一种基于苯酚响应型表面增强拉曼散射探针检测碱性磷酸酶的方法，其特征在于，包括以下步骤：A method for detecting alkaline phosphatase based on a phenol-responsive surface-enhanced Raman scattering probe is characterized in that it comprises the following steps:
    将苯基磷酸二钠溶于Tris-HCl缓冲液中，加入ALP溶液，室温下孵育10～30min，加入权利要求4所述SERS探针，再孵育10～30min，洗涤，得到沉淀物，将其分散于水中得到待测样品，将待测样品滴加在硅片上，干燥，进行SERS检测；Dissolve phenyl phosphate disodium in Tris-HCl buffer, add ALP solution, incubate at room temperature for 10-30 minutes, add the SERS probe of claim 4, incubate for 10-30 minutes, and wash to obtain a precipitate. Disperse in water to obtain the sample to be tested, drop the sample to be tested on the silicon wafer, dry, and perform SERS detection;
    所述PPNa、ALP溶液中的ALP和SERS探针的比例为9.5×10 -4mmol：(5×10 -6～2.5×10 -3)mU：(0.5～2)个；所述PPNa在Tris-HCl缓冲液中的浓度为1mmol/L；所述ALP溶液的浓度为0.1～50mU/L；所述SERS探针以水溶液的形式加入，其中SERS探针的含量为50～200个/mL；所述待测样品中SERS探针的含量为50～200个/mL。 The ratio of ALP and SERS probes in the PPNa and ALP solutions is 9.5×10 -4 mmol: (5×10 -6 ～2.5×10 -3 ) mU: (0.5～2); the PPNa is in Tris -The concentration in the HCl buffer is 1 mmol/L; the concentration of the ALP solution is 0.1-50mU/L; the SERS probe is added in the form of an aqueous solution, and the content of the SERS probe is 50-200/mL; The content of SERS probes in the sample to be tested is 50-200 probes/mL.
  10. 一种基于苯酚响应型表面增强拉曼散射探针的霍乱毒素酶联免疫检测方法，其特征在于，包括以下步骤：A cholera toxin enzyme-linked immunoassay method based on a phenol-responsive surface-enhanced Raman scattering probe, which is characterized in that it comprises the following steps:
    (1)将CT第一抗体分子加入到孔板中，加入含有0.05mol/L碳酸钠的PBS 缓冲液，在37℃孵育1～3h后，用PBS缓冲液洗涤，然后加入明胶封闭CT第一抗体分子未覆盖的位点，用PBS缓冲液洗涤，再加入CT抗原溶液，在37℃孵育0.5～2小时，除去未反应的CT抗原溶液并用PBS缓冲液洗涤，再加入ALP标记的CT第二抗体分子，在37℃孵育1～3h后，用PBS缓冲液洗涤，得到具有酶联免疫典型结构的CT抗原抗体键结结构；(1) Add CT primary antibody molecules to the well plate, add PBS buffer containing 0.05 mol/L sodium carbonate, incubate at 37°C for 1 to 3 hours, wash with PBS buffer, and then add gelatin to block CT primary The uncovered sites of antibody molecules are washed with PBS buffer, then CT antigen solution is added, and incubated at 37°C for 0.5 to 2 hours. Unreacted CT antigen solution is removed and washed with PBS buffer, and then ALP-labeled CT second The antibody molecule is incubated at 37°C for 1 to 3 hours, and then washed with PBS buffer to obtain a CT antigen-antibody bonding structure with a typical structure of enzyme-linked immunoassay;
    (2)将步骤(1)具有酶联免疫典型结构的CT抗原抗体分子和PPNa溶液混合均匀，室温下孵育5～30min后，加入权利要求4所述SERS探针，继续孵育10～30min，最后进行SERS检测；(2) Mix the CT antigen antibody molecule with the typical structure of enzyme-linked immunoassay in step (1) and PPNa solution uniformly, and after incubating at room temperature for 5-30 minutes, add the SERS probe of claim 4, and continue to incubate for 10-30 minutes, and finally Perform SERS testing;
    步骤(1)所述CT第一抗体分子在含有0.05mol/L碳酸钠的PBS缓冲液中的浓度为1～50μg/mL；所述CT抗原溶液的溶剂为水，浓度为0.1～100pg/mL；Step (1) The concentration of the CT primary antibody molecule in the PBS buffer containing 0.05 mol/L sodium carbonate is 1-50 μg/mL; the solvent of the CT antigen solution is water, and the concentration is 0.1-100 pg/mL ；
    步骤(1)所述CT第一抗体分子与明胶的质量比1：100～500；所述CT抗原溶液中的CT抗原与CT第一抗体分子的质量比为1：1；所述ALP标记的CT第二抗体分子与CT第一抗体分子的质量比为1：1；Step (1) The mass ratio of CT first antibody molecule to gelatin is 1:100-500; the mass ratio of CT antigen to CT first antibody molecule in the CT antigen solution is 1:1; the ALP labeled The mass ratio of CT second antibody molecule to CT first antibody molecule is 1:1;
    步骤(2)所述PPNa溶液中的PPNa与具有酶联免疫典型结构的CT抗原抗体分子中的第二抗体分子的质量比为1：(2～10)；所述SERS探针个数和PPNa溶液中PPNa的摩尔量比为1个：(4.5×10 -5～1.8×10 -4)mmol；所述PPNa溶液的浓度为0.5～3mmol/L，溶剂为水；所述SERS探针以SERS探针水溶液的形式加入，所述SERS探针水溶液中SERS探针的含量为50～200个/mL。 Step (2) The mass ratio of PPNa in the PPNa solution to the second antibody molecule in the CT antigen antibody molecule with the typical structure of enzyme-linked immunosorbent is 1: (2-10); the number of SERS probes and PPNa The molar ratio of PPNa in the solution is 1: (4.5×10 -5 ～1.8×10 -4 ) mmol; the concentration of the PPNa solution is 0.5～3 mmol/L, the solvent is water; the SERS probe is SERS The probe is added in the form of an aqueous solution, and the content of the SERS probe in the SERS probe aqueous solution is 50-200 probes/mL.
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113092442A (en) * 2021-04-09 2021-07-09 上海海洋大学 Method for rapidly detecting histamine
CN113295682A (en) * 2021-05-21 2021-08-24 吉林大学 Phenolic compound analysis method based on polyphenol oxidase activity nanoenzyme
CN113624739A (en) * 2021-08-10 2021-11-09 中国人民解放军军事科学院军事医学研究院 Detection method and kit for rapidly detecting quaternary ammonium oxime heavy activator in biological sample
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Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110658177B (en) * 2019-09-25 2021-11-23 暨南大学 Phenol recognition SERS probe, preparation and application thereof, and SERS-based general ultrasensitive immunoassay method
CN111230138B (en) * 2020-01-16 2022-06-21 暨南大学 Pi-Pi directional self-assembly-based three-dimensional gold super particle, preparation and application thereof in SERS substrate
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110027901A1 (en) * 2009-04-13 2011-02-03 Richard Samuel Gaster Methods and devices for detecting the presence of an analyte in a sample
US20120212733A1 (en) * 2010-10-27 2012-08-23 The Board Of Trustees Of The University Of Illinois Tailored raman spectrocopic probes for ultrasensitive and highly multiplexed assays
CN103411945A (en) * 2013-06-24 2013-11-27 吉林大学 Method for performing surface-enhanced Raman detection on phenolic and polycylic aromatic hydrocarbon compounds by generating azo molecule
CN110658177A (en) * 2019-09-25 2020-01-07 暨南大学 Phenol recognition SERS probe, preparation and application thereof, and SERS-based general ultrasensitive immunoassay method

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005508495A (en) * 2001-09-06 2005-03-31 ジェノミック プロファイリング システムズ インコーポレイティッド Rapid and sensitive detection of molecules
CN103969436B (en) * 2014-05-08 2015-09-16 东南大学 The new method that a kind of hypersensitive alkaline phosphatase detects

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110027901A1 (en) * 2009-04-13 2011-02-03 Richard Samuel Gaster Methods and devices for detecting the presence of an analyte in a sample
US20120212733A1 (en) * 2010-10-27 2012-08-23 The Board Of Trustees Of The University Of Illinois Tailored raman spectrocopic probes for ultrasensitive and highly multiplexed assays
CN103411945A (en) * 2013-06-24 2013-11-27 吉林大学 Method for performing surface-enhanced Raman detection on phenolic and polycylic aromatic hydrocarbon compounds by generating azo molecule
CN110658177A (en) * 2019-09-25 2020-01-07 暨南大学 Phenol recognition SERS probe, preparation and application thereof, and SERS-based general ultrasensitive immunoassay method

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LI NAN, CHEN HAOLING, ZHANG MEIYING, ZHA YONGCHAO, MU ZONGXIA, MA YING, CHEN PENG: "A universal ultrasensitive platform for enzyme-linked immunoassay based on responsive surface-enhanced Raman scattering", SENSORS AND ACTUATORS B: CHEMICAL, ELSEVIER BV, NL, vol. 315, 1 July 2020 (2020-07-01), NL, pages 128135, XP055795984, ISSN: 0925-4005, DOI: 10.1016/j.snb.2020.128135 *
MA YING, PROMTHAVEEPONG KITTITHAT, LI NAN: "Chemical Sensing on a Single SERS Particle", ACS SENSORS, vol. 2, no. 1, 27 January 2017 (2017-01-27), pages 135 - 139, XP055795999, ISSN: 2379-3694, DOI: 10.1021/acssensors.6b00617 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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