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Ultra-sensitive biochip based on surface enhanced Raman scattering and preparation method thereof

A surface-enhanced Raman and biochip technology, applied in Raman scattering, material excitation analysis, instruments, etc., can solve the problems of poor repeatability and low detection sensitivity of SERS immune substrates, achieve uniform distribution, improve detection uniformity, and enhance The effect of sensitivity

Inactive Publication Date: 2021-07-09
JILIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The object of the present invention is to solve the problems of low detection sensitivity and poor repeatability of the SERS immune substrate modified by metal nanoparticles, and provide a SARS-CoV-2 ultrasensitive biochip based on surface-enhanced Raman scattering (SERS) and its preparation method

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  • Ultra-sensitive biochip based on surface enhanced Raman scattering and preparation method thereof
  • Ultra-sensitive biochip based on surface enhanced Raman scattering and preparation method thereof
  • Ultra-sensitive biochip based on surface enhanced Raman scattering and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1: Preparation of SERS immune substrate

[0036] (1) Preparation of Au NPs solution:

[0037] 0.985mg chloroauric acid (HAuCl 4 ) was mixed with 0.365g cetyltrimethylammonium bromide (CTAB), and then the volume was adjusted to 10mL with deionized water, and then 0.6mL, 10×10 -3 M sodium borohydride solution (NaBH 4 ), stirred for 2 minutes, and placed at 27° C. for 3 hours to ensure that the reaction was complete to obtain a CTAB-coated gold nanocluster solution. 2mL, 200×10 - 3 M cetyltrimethylammonium chloride solution (CTAC) with 1.5mL, 100×10 -3 Mix M ascorbic acid solution (AA) and 50 μL CTAB-coated gold nanocluster solution, and then add 2 mL, 0.5×10 -3 M HAuCl 4 solution, reacted at 27°C for 15 minutes, and then centrifuged at 14500rpm for 30 minutes to resuspend in 1mL, 20×10 -3 In the MCTAC solution, a gold nanoparticle seed solution with a particle size of 10 nm was obtained. 2mL, 100×10 -3 MCTAC solution with 130 μL, 10×10 -3 The MAA soluti...

Embodiment 2

[0042] Embodiment 2: Preparation of SERS immunoprobe solution

[0043] (1) Preparation of Ag NPs solution:

[0044] Dissolve 0.02536g of ascorbic acid and 0.21175g of trisodium citrate in 240mL of deionized water, then add 2.4mL of 0.1M silver nitrate solution (AgNO 3 ), stirred at 30°C for 15 minutes, and aged at 100°C for 2 hours to obtain the Ag NPs solution.

[0045] (2) Preparation of Ag@4MBA solution:

[0046] Add 200 μL, 0.1 mM ethanol solution of p-mercaptobenzoic acid (4MBA) to 1 mL of the AgNPs solution obtained in step (1), stir at 30 °C for 12 h, centrifuge at 8500 rpm for 15 minutes and disperse in 1 mL of PBS to obtain 4MBA-modified AgNPs NPs solution (Ag@4MBA);

[0047] (3) Preparation of SERS immunoprobe solution:

[0048] Add 30 μL, 10 μg / mL SARS-CoV-2 spike antibody solution in PBS to 1 mL of the Ag@4MBA solution in step (2), incubate at 4°C for 6 h, then centrifuge at 6500 rpm for 15 minutes and disperse in 1 mL of PBS. Finally, 50 μL of 5% by mass BSA ...

Embodiment 3

[0049] Embodiment 3: Preparation and detection of SARS-CoV-2 ultrasensitive biochip

[0050] (1) Incubation of the target antigen:

[0051] To the surface of the SERS immune substrate obtained in Example 1, 10 μL of the SARS-CoV-2 spike antigen with a concentration of 1fg / mL, 10fg / mL, 100fg / mL, 1pg / mL, 10pg / mL, 100pg / mL and 1ng / mL was added dropwise. The PBS solution of protein and the PBS solution of BSA in the control group were incubated at 37°C for 2.5h, and then washed 3 times with PBS and a large amount of deionized water.

[0052] (2) Preparation of SARS-CoV-2 ultrasensitive biochip

[0053] Add 15 μL of the SERS immunoprobe solution obtained in Example 2 dropwise to the substrate obtained in step (1), incubate at 4°C for 2.5 hours, and then wash with PBS and deionized water 3 times to obtain a SARS-CoV-2 ultrasensitive biological chip.

[0054] (3) Detection of SARS-CoV-2 ultrasensitive biochip

[0055] Place the SARS-CoV-2 ultra-sensitive biochip obtained in step ...

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Abstract

The invention discloses an SARS-CoV-2 ultra-sensitive biochip based on surface enhanced Raman scattering and a preparation method thereof, and belongs to the technical field of functional materials and biosensing detection. The preparation method comprises the following steps: assembling gold nanoparticles by taking a silicon wafer as a substrate, and modifying a novel coronavirus spike protein antibody (SARS-CoV-2spike antibody) and bovine serum albumin to obtain an SERS immune substrate; sequentially modifying the surface of Ag NPs with a Raman molecule p-mercaptobenzoic acid and an SARS-CoV-2spike antibody so as to prepare an SERS immune probe; then, using the SERS immune substrate for adsorbing novel coronavirus spike protein antigens (SAR-CoV-2spike antigen protein) with different concentrations and an SERS immune probe so that the SAR-CoV-2 ultra-sensitive biochip based on surface enhanced Raman scattering, which is of a sandwich immune sandwich structure, is obtained. According to the invention, the problems of low sensitivity and poor uniformity of the enhanced substrate prepared by an electrostatic adsorption method are solved, and the purpose of further improving the SERS detection sensitivity is achieved.

Description

technical field [0001] The invention belongs to the technical field of functional materials and biosensing detection, in particular to a SARS-CoV-2 ultrasensitive biochip based on Surface Enhanced Raman Scattering (SERS) and a preparation method thereof. Background technique [0002] The rapid spread of the COVID-19 outbreak caused by the novel coronavirus (SARS-CoV-2) has created a worrying situation around the world. It has been confirmed that the virus can be transmitted through respiratory droplets, aerosols and contact with non-biological surfaces, and asymptomatic infection and transmission have become a major public health problem. Therefore, there is an urgent need to develop sensitive, rapid and low-cost diagnostic tools for early screening of infected individuals so that appropriate isolation and treatment can be facilitated. [0003] In clinical practice, there are three main diagnostic methods for COVID-19: (1) chest CT scan; (2) ribonucleic acid (RNA) detection...

Claims

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Application Information

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IPC IPC(8): G01N21/65G01N33/531
CPCG01N21/658G01N33/531G01N2021/656
Inventor 卢革宇刘晓敏张美玲潘嘉林张玲包浩强
Owner JILIN UNIV
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