Cell sensor based on surface enhanced Raman scattering and application thereof

A surface-enhanced Raman and cell sensor technology, applied in Raman scattering, instruments, scientific instruments, etc., can solve the problems of cumbersome operations, large differences in human cells, and long cycles, so as to reduce the false negative rate or false positive rate , Reduce the complexity of the process and improve the evaluation speed

Active Publication Date: 2022-06-28
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] (1) The amount of impurities used in animal experiments is large, the cost is high, and the cycle is long;
[0005] (2) The test based on prokaryotic cells is quite different from human cells, and it is not suitable for the evaluation of GTI that requires metabolic activation;
[0006] (3) Computer-based virtual screening methods are prone to false positives / false negatives;
[0007] (4) Evaluation methods such as molecular hybridization or adduct detection for a single gene mutation site or damage type are difficult to achieve rapid evaluation of various gene damage;
[0008] (5) Other methods that require separation or staining are cumbersome and cannot be detected in situ in real time, which may miss or interfere with the body's response to gene damage

Method used

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  • Cell sensor based on surface enhanced Raman scattering and application thereof
  • Cell sensor based on surface enhanced Raman scattering and application thereof
  • Cell sensor based on surface enhanced Raman scattering and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] 1. Instruments and Reagents

[0074] 1.1 Instruments

[0075] Confocal inverted microscope fluorescence bright and dark field imaging Raman spectrometer (Beijing Zhuoli Hanguang, equipped with Olympus® LUCPLFLN40X telephoto achromatic objective, Lumenera® INFINITY 3-1 camera, 3 gratings, 3-channel laser light source, fluorescence Light source and filters, etc., can perform brightfield, darkfield imaging and Raman spectroscopy detection and mapping imaging), constant temperature incubator, microplate reader, UV spectrophotometer, transmission electron microscope, ultrapure water meter, etc.

[0076] reagent

[0077] Chloroauric acid tetrahydrate (HAuCl 4 ·4H 2 O), trisodium citrate, SH-PEG-NH 2 (MW2000), 4-mercaptobenzonitrile (4-MBN), glutaraldehyde (25%), PBS, bovine serum albumin (BSA), TAT, Anti-γH2AX (phospho S139) antibody, methyl methanesulfonate (MMS), cisplatin (cis-Pt), 5-fluorouracil (5-Fu), N-nitrosodiethylamine (NDEA).

[0078] Probe Construction

[0...

Embodiment 2

[0110] 1. Instruments and Reagents

[0111] 1.1 Instruments

[0112] Confocal inverted microscope fluorescence bright and dark field imaging Raman spectrometer (Beijing Zhuoli Hanguang, equipped with Olympus® LUCPLFLN40X telephoto achromatic objective, Lumenera® INFINITY 3-1 camera, 3 gratings, 3-channel laser light source, fluorescence Light source and filters, etc., can perform brightfield, darkfield imaging and Raman spectroscopy detection and mapping imaging), constant temperature incubator, ultrapure water meter, etc.

[0113] reagent

[0114] Chloroauric acid tetrahydrate (HAuCl 4 ·4H 2 O), trisodium citrate, SH-PEG-NH 2 (MW2000), 4-mercaptobenzonitrile (4-MBN), 4-mercaptobenzoic acid (4-MBA), 4-mercaptophenylboronic acid (4-MBN), glutaraldehyde (25%), PBS, bovine serum Albumin (BSA), TAT, NLS, Anti-γH2AX (phospho S139) antibody.

[0115] Raman molecule selection optimization

[0116] Since the structures of 4-MBN, 4-MBA and 4-MPBA are similar ( Figure 13 ), and...

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PUM

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Abstract

The invention discloses a cell sensor based on surface enhanced Raman scattering and application of the cell sensor. The cell sensor is constructed by introducing a surface enhanced Raman scattering probe into a human hepatocyte line. The surface enhanced Raman scattering probe is prepared by taking gold nanoparticles as a detection substrate, a gene damage effect molecular antibody as a recognition unit, a Raman molecule as a report unit, SH-PEG-NH2 as a stable chain and cell-penetrating peptide as an auxiliary penetration unit. The cell sensor is exposed to various drug impurities, Raman signals are detected, and the type and level of genotoxic impurities are evaluated. The method has the advantages of being good in detection universality, high in reliability, small in impurity dosage and the like, and evaluation of genotoxic impurities in the drug research and development process is promoted.

Description

technical field [0001] The invention belongs to the field of drug analysis and detection, and relates to a cell sensor based on surface-enhanced Raman scattering (SERS) and its application in the evaluation of genotoxic impurities. Background technique [0002] Toxicity evaluation and limit control of various impurities that may be generated in various links of drug development and clinical use are important requirements to ensure drug quality and safety. Genotoxic Impurities (GTI) are a class of impurities that can cause genetic damage at low concentrations and have a risk of carcinogenicity. Drug regulatory agencies in various countries have set strict limits on the content of GTI in drugs. 1-2 . With the development of modern analytical technology, effective detection and control of impurities with known genotoxicity (such as N-nitrosodimethylamine, methyl methanesulfonate) have been implemented. However, for impurities with unknown genotoxicity, how to quickly and effe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/65B22F9/24B82Y40/00
CPCG01N21/658B22F9/24B82Y40/00
Inventor 柳文媛韩凌飞冯锋
Owner CHINA PHARM UNIV
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