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A rapid detection method for bacteria based on surface-enhanced Raman scattering-laser-induced breakdown spectroscopy

A technology of bacteria and spectroscopy, applied in Raman scattering, measuring devices, thermal excitation analysis, etc., can solve the problems of poor reproducibility and stability, great difficulty, and the inability to ensure uniform distribution of nanoparticles, etc., to achieve good accuracy , the effect of simple operation

Active Publication Date: 2021-11-19
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Researchers usually mix bacteria with silver or gold sol, and then induce aggregation with inorganic salts to tightly combine nanoparticles with bacterial cells. However, this method cannot ensure uniform distribution of nanoparticles on bacterial cells, so the reproducibility and stability are poor.
In addition, because SERS is a near-field phenomenon, the degree of Raman enhancement is affected by many factors, so it is still difficult to use SERS for quantitative analysis.
[0004] Laser-induced breakdown spectroscopy (Laser Induced Breakdown Spectroscopy, LIBS) is based on the interaction between high-power pulsed laser and matter to generate transient plasma. A spectroscopic technique for quantitative analysis, which has not yet been combined with SER for qualitative and quantitative detection of bacteria

Method used

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  • A rapid detection method for bacteria based on surface-enhanced Raman scattering-laser-induced breakdown spectroscopy
  • A rapid detection method for bacteria based on surface-enhanced Raman scattering-laser-induced breakdown spectroscopy
  • A rapid detection method for bacteria based on surface-enhanced Raman scattering-laser-induced breakdown spectroscopy

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1 Detection method of the present invention

[0041] Parameters of the LIBS-Raman combined device: the continuous laser used to excite Raman scattering is a semiconductor-pumped all-solid-state laser with an output wavelength of 532nm and a power of 50-400mW. The pulsed laser used for LIBS is a Q-switched Nd:YAG laser with an output wavelength of 1064nm, a pulse repetition frequency of 1-20Hz, and a single pulse with a maximum energy of 100mJ.

[0042] When performing Raman testing, the laser power is attenuated to 15mW through the attenuation sheet, the objective lens is 10 times larger, the slit of the spectrometer is 25um, and the Raman scanning range is 500-2000cm -1 , the resolution is about 4-7cm -1 ; When performing LIBS experiments, the pulse energy of the pulsed laser is 30mJ, and the repetition frequency of the pulsed laser is 10Hz.

[0043]1) Bacterial culture: Inoculate the known strains in 25 mL of trypticase soy broth (TSB), shake and culture ...

Embodiment 2

[0047] Embodiment 2 The present invention qualitatively detects bacteria

[0048] 1) Bacterial culture: inoculate unknown strains in 25 mL of tryptone soy broth (TSB), shake and culture at 37°C for 10 hours, take 5 mL of cultured bacterial liquid, centrifuge at 6000 rpm for 5 min, and wash with ultrapure water Three times, finally dispersed in ultrapure water, measure OD 600 And determine the bacterial concentration by plate count.

[0049] 2) Hydrophobic substrate preparation: In the present invention, a silicon wafer is used as a substrate-loaded sample for SERS and LIBS detection. In order to prevent the signal strength from weakening due to sample diffusion, the silicon wafer needs to be hydrophobized in advance, that is, the silicon wafer is ultrasonicated in acetone and water ( 40KHz) for 10 minutes, then soak in concentrated H 2 SO 4 (98%) and H 2 o 2 (30%) in a mixed solution with a volume ratio of 3:1 and heated at 90°C for 30 minutes, washed with deionized water...

Embodiment 3

[0052] Embodiment 3 The present invention quantitatively detects bacteria

[0053] (1) Take a known bacterial solution with a certain volume concentration and disperse it in ultrapure water to make a concentration of 5×10~5×10 7 CFU / mL bacterial solution, as a standard;

[0054] (2) Take a 0.8cm*0.8cm silicon wafer, wash it ultrasonically with acetone and water for 10 minutes, and then soak it in concentrated H 2 SO 4 (98%) and H 2 o 2 (30%) in a mixed solution with a volume ratio of 3:1 and heated at 90°C for 30 minutes, washed with deionized water, soaked in 5% HF solution for 30 minutes to form Si-H bonds on the surface, and finally nitrogen Blow dry to get hydrophobic silicon wafer.

[0055] (3) Bacterial in situ synthesis of AgNPs: Take 1mL of the standard product in a 1.5mL centrifuge tube, add 10μL of 0.1mol / L silver nitrate, vortex and mix for 10 minutes, then add 10μL of 0.2mol / L sodium borohydride, turn over and centrifuge Tubes generate AgNPs in situ in the ba...

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Abstract

The invention discloses a sample processing method for detecting bacteria by using SERS. The steps are as follows: 1) take a sample to be tested, collect bacteria, and disperse with water to obtain a bacteria liquid; 2) add nitric acid to the bacteria liquid obtained in step 1) Silver, after mixing, then add sodium borohydride to generate silver nanoparticles to obtain a co-existing sol of bacteria and silver nanoparticles; 3) take the sol obtained in step 2), drop it on a hydrophobic silicon chip, and evaporate naturally. . The invention also discloses a method for detecting bacteria by using SERS-LIBS combined technology. The sample processing method of the invention can improve the reproducibility and stability of the Raman spectrum, and has excellent application prospects.

Description

technical field [0001] The invention belongs to the field of bacteria detection, and relates to a rapid detection method for bacteria based on surface-enhanced Raman scattering-laser-induced breakdown spectroscopy Background technique [0002] Rapid and accurate detection of bacteria in food and water plays a vital role in food safety and human health. At present, a large number of bacterial detection methods have been developed, including culture methods, immunoassays, molecular detection methods, and spectroscopic methods. As a classic method for bacterial detection, the culture method mainly relies on the growth of microorganisms, which has the problem of long detection cycle. Although immunoassays such as enzyme-linked immunosorbent assay (Enzyme-Linked Immunosorbnent Assay, ELISA) have the advantage of strong specificity, the required monoclonal antibodies are difficult to prepare and inactivated, and the cost is relatively high. Molecular detection methods such as pol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/65G01N21/71
CPCG01N21/658G01N21/718
Inventor 段忆翔廖文龙林庆宇
Owner SICHUAN UNIV
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