A Raman immunoassay based on a novel SERS probe

A detection method and probe technology, applied in the field of immunological detection, can solve difficult problems such as multi-channel and integrated detection, and achieve the effects of uniform properties, low background noise, and simple modification methods

Active Publication Date: 2022-02-08
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

SERS-immunochromatography detection technology is a fast and highly sensitive immunoassay method that combines SERS detection probes with immunochromatography technology using strip fiber chromatographic membrane as a solid phase substrate, but it is also difficult to achieve multi-channel and integration chemical detection

Method used

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  • A Raman immunoassay based on a novel SERS probe
  • A Raman immunoassay based on a novel SERS probe
  • A Raman immunoassay based on a novel SERS probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] 1.1 Materials

[0076] Reagents: chloroauric acid (Sinopharm Group); silver nitrate (Sinopharm Group); sodium citrate (Sinopharm Group); goat anti-human IgM (Sigma); human IgM (Sigma); goat anti-human IgM-HRP (Sigma); , 5'-Dimercapto bis(2-nitrobenzoic acid) (DTNB) (Sigma); Tyramine (Tyr) (Sigma); DTNB-Tyr; Silver staining reagent (prepared in the laboratory): Solution A ( Silver nitrate aqueous solution), B solution (hydroquinone citric acid buffer solution); single crystal silicon wafer (Zhejiang Lijing).

[0077] Instruments: heating magnetic stirrer (Beijing Century Huake); centrifuge (Eppendorf, Germany); Milli-Q ultrapure water system (Millipore, USA); Hitachi H-7650 transmission electron microscope (Hitachi, Japan); Shimadzu 2600 ultraviolet spectrophotometer (Japan) Shimadzu); i-Raman Plus BWS465-785H Raman spectrometer (B&W Tek, USA); ZEISS EVO LS10 scanning electron microscope (Zeiss, Germany).

[0078] 1.2 Preparation of SERS detection probes

[0079] Firs...

Embodiment 2

[0084] Preparation of immunochip: Add 1 μL of 1 mg / mL capture antibody (goat anti-human IgM) dropwise in the center of each well array of the aldehylation silicon chip, and incubate overnight; then add 5 μL of 1wt% BSA solution, and incubate at 37°C for 2 hours to Block unbound antibody areas; wash with 1×PBST, dry, and set aside.

[0085] SERS immunoassay: Add 5 μL of different concentrations of human IgM (1 μg / mL) to the corresponding wells of the immune chip, PBS as a blank control, react at 37 ° C for 30 min, wash 3 times with 1×PBST, and dry; add to each well 5 μL of detection antibody (goat anti-human IgM-HRP), react at 37°C for 30 minutes, wash 3 times with 1×PBST, and dry; add 5 μL of SERS detection probe to each well, react at 37°C for 30 minutes, wash with 1×PBST 2 times, wash once with water, and dry in the air; add 5 μL of silver staining reagent (mixture A and B in equal proportions) to each well, react in the dark for 2 minutes, wash with water once, and dry in t...

Embodiment 3

[0090] Preparation of immunochip: Add 1 μL of 1 mg / mL capture antibody (goat anti-human IgM) dropwise in the center of each well array of the aldehylation silicon chip, and incubate overnight; then add 5 μL of 1wt% BSA solution, and incubate at 37°C for 2 hours to Block unbound antibody areas; wash with 1×PBST, dry, and set aside.

[0091]SERS immunoassay: Add 5 μL of different concentrations of human IgM (1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, 10 pg / mL) into the corresponding wells of the immune chip, PBS as a blank control, 37 React at ℃ for 30 minutes, wash 3 times with 1×PBST, and dry in the air; add 5 μL of detection antibody (goat anti-human IgM-HRP) to each well, react at 37°C for 30 minutes, wash with 1×PBST 3 times, and dry in the air; Add 5 μL of SERS detection probe to each well, react at 37°C for 30 min, wash twice with 1×PBST, once with water, and dry in the air; add 5 μL of silver staining reagent to each well (mix A and B in equal proportions) , reac...

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Abstract

The invention relates to the field of immunological detection, in particular to a Raman immunological detection method based on a novel SERS probe. A SERS probe, which is a substance coupled with sulfur in gold nanoparticles. The invention provides a new technology of SERS immunodetection, introduces a new type of SERS detection probe Au-DTNB-Tyr NPs, combined with gold-labeled silver staining technology, introduces silver particles around the SERS detection probe to enhance the SERS signal. Through the introduction of the above-mentioned SERS detection probe and gold-labeled silver staining technology, the substance to be detected can be detected quantitatively with high sensitivity, and the detection limit is 10 pg / mL.

Description

technical field [0001] The invention relates to the field of immunological detection, in particular to a Raman immunological detection method based on a novel SERS probe. Background technique [0002] Immunoassays serve as a reliable, simple and inexpensive method for identifying and quantifying the presence of specific antibodies or antigens in complex solution samples. This detection method based on the specific combination of antibody and antigen has been widely used in biochemical research, clinical diagnosis, environmental monitoring and food safety detection and other fields. [0003] Surface-enhanced Raman scattering (SERS) spectroscopy has the advantages of narrow spectral band, high sensitivity, and multi-channel detection. Combined with immunoassay technology, it can be applied to the detection of proteins, viruses, bacteria, etc. Currently, SERS-based immunoassay technologies usually rely on liquid SERS immunomagnetic bead substrates, solid-state SERS immunochip ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/532G01N33/543G01N33/58
Inventor 肖瑞贾小飞荣振王柯莉
Owner ACADEMY OF MILITARY MEDICAL SCI
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