SERS probe and substrate labeled by multiple antibodies as well as preparation method and application of SERS probe and substrate

A technology of antibody labeling and probe, applied in the field of molecular biology, to achieve the effect of simple and convenient labeling method, small sample size and high coincidence degree

Active Publication Date: 2018-02-27
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no reports in the literature on the use of SERS probe-labeled immunoassay and exosome-associated migration inhibitory factor (MIF) to detect pancreatic cancer exosomes and realize stage judgment.

Method used

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  • SERS probe and substrate labeled by multiple antibodies as well as preparation method and application of SERS probe and substrate
  • SERS probe and substrate labeled by multiple antibodies as well as preparation method and application of SERS probe and substrate
  • SERS probe and substrate labeled by multiple antibodies as well as preparation method and application of SERS probe and substrate

Examples

Experimental program
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Effect test

Embodiment 1

[0043] (1) Preparation of various antibody-labeled SERS probes and substrates

[0044] Add 50μL of 10μg / mL anti-MIF, anti-GPC1, anti-EGFR, anti-EpCAM, anti-CD9, anti-CD63 six antibodies to the 5mM gold-silver nanoparticle solution with core-shell structure, and 60μL 10mg / mL dopamine solution, react at room temperature for 1 hour. The reacted solution was centrifuged and then dispersed with deionized water. In addition, soak ordinary glass slides in 12M NaOH for 5 hours, then add 8ml of 20mg / ml dopamine solution to the solution, shake the above petri dish on a special shaker for 1 hour, and then rinse it with deionized water. Place the modified and cleaned slides in 1% BSA containing 5mg / mL anti-MIF, anti-GPC1, anti-EGFR, anti-EpCAM, anti-CD9, anti-CD63 for 2h, and deionized water After rinsing 3 times, let it dry naturally.

[0045] (2) Cell culture and exosome extraction

[0046] Pancreatic cancer cell line PANC-01 was cultured in RPMI 1640 medium, which was supplemented wi...

Embodiment 2

[0056] (1) Preparation of various antibody-labeled SERS probes and substrates

[0057] Add 80μL of 20μg / mL anti-MIF, anti-GPC1, anti-EGFR, anti-EpCAM, anti-CD9, anti-CD63 and other antibodies to the 8mM gold-silver nanoparticle solution with core-shell structure and 60μL of 10mg / mL mL dopamine solution, react at room temperature for 1 hour. The reacted solution was centrifuged and then dispersed with deionized water. In addition, soak ordinary glass slides in 12M NaOH for 5 hours, then add 8ml of 20mg / ml dopamine solution to the solution, shake the above petri dish on a special shaker for 1 hour, and then rinse it with deionized water. Place the modified and cleaned slides in 1% BSA containing 10mg / mL anti-MIF, anti-GPC1, anti-EGFR, anti-EpCAM, anti-CD9, anti-CD63 for 2h, and deionized water After rinsing 3 times, let it dry naturally.

[0058] (2) Cell culture and exosome extraction

[0059] Pancreatic cancer cell line PANC-01 was cultured in RPMI 1640 medium, which was s...

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Abstract

The invention belongs to the technical field of molecular biology and in particular relates to an SERS probe and an SERS substrate labeled by multiple antibodies as well as a preparation method and application of the SERS probe and the SERS substrate. Dopamine is embedded and combined with multiple antibodies under auto-polymerization conditions so as to obtain the SERS substrate and the SERS labeled probe modified by the polydopamine and the antibodies. By virtue of immune binding force, high-sensitivity detection and diagnosis of exosome extracted from pancreatic cancer cells and serum samples of clinical pancreatic cancer patients can be realized by utilizing Raman spectroscopy. Statistical data analysis discovers that an SERS platform based on an MIF antibody is capable of realizing phased tumor judgment of the pancreatic cancer. The SERS probe disclosed by the invention is simple and convenient in labeling method, is used for detection of exosome of the pancreatic cancer, is rapid, sensitive, high in specificity and small in amount of needed samples, does not need a high-speed centrifugal separation process and can be applicable to clinical diagnosis and phased tumor metastasis judgment.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to various antibody-labeled SERS probes and substrates, their preparation methods and applications. Background technique [0002] Pancreatic cancer is one of the malignant tumors of the digestive tract with the worst prognosis. The National Cancer Center analyzed and estimated the 2012 malignant tumor registration data collected by the National Cancer Registry Center in 2015, and the incidence of pancreatic cancer was 86,000, the incidence rate is 6.35 / 10 5 , ranking 10th in the incidence of all malignant tumors, the number of deaths reached 77,000, and the mortality rate was 5.72 / 10 5 , ranking 6th. Pancreatic cancer lacks specific clinical manifestations and sensitive early diagnostic indicators. The commonly used serum marker for diagnosis and follow-up is CA19-9, but its diagnostic specificity is poor. At present, the preoperative diagnosis and staging o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/65G01N33/574
CPCG01N21/658G01N33/57438G01N33/57484
Inventor 陈惠张人李腾达黄志鹏孔继烈
Owner FUDAN UNIV
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