Raman immune detection method based on novel SERS probe
A detection method and probe technology, applied in the field of immunological detection, can solve the problems of difficult multi-channel and integrated detection, and achieve the effects of uniform nature, simple modification method and low background noise.
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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; add 5 μL of silver staining reagent to each well (mix A and B in equal proportions) , react in the da...
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