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A mass spectrometry immunoassay method and application for disease marker detection

A technology of immunoassay and mass spectrometry, which is applied in biological testing, material analysis, material analysis by electromagnetic means, etc. It can solve the problems of not meeting the requirements of detection sensitivity, affecting detection sensitivity, and complicated preparation.

Active Publication Date: 2021-07-09
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Literature (R.Du, L.Zhu, J.Gan, Y.Wang, L.Qiao and B.Liu.Anal.Chem., 2016, 88, 6767-6772 and J.R.Lee, J.Lee, S.K.Kim, K.P. Kim, H.S.Park and W.Yeo.Angew.Chem.Int.Ed., 2008, 47, 9518–9521) reported the current typical labeled probes and mass spectrometry immunoassays that can be applied to mass spectrometry immunoassays. However, due to Probes need to be dissociated by laser, which greatly increases the analysis cost and instrument requirements of mass spectrometry. At the same time, laser dissociation produces various forms of signals, which disperses the amplification effect of labeling signals and affects detection sensitivity.
In the literature (S.Chen, Q.Wan and A.K.Badu-Tawiah.J.Am.Chem.Soc., 2016,138,6356-6359), labeled probes and low-cost papers that can be dissociated by chemical means have been reported. Spray immunoassay, but the probe has a single structure, complex preparation, poor scalability, and poor signal amplification, making this mass spectrometry immunoassay unable to meet the detection sensitivity requirements in current clinical diagnosis

Method used

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  • A mass spectrometry immunoassay method and application for disease marker detection
  • A mass spectrometry immunoassay method and application for disease marker detection
  • A mass spectrometry immunoassay method and application for disease marker detection

Examples

Experimental program
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Effect test

Embodiment 1

[0070] Example 1: Using the mass spectrometry immunoassay method of the present invention to measure thrombin in plasma.

[0071] Specific steps are as follows:

[0072] (1) Use an indium tin oxide glass sheet with a length of 20 mm and a width of 5 mm as a protein capture chip. One end of the chip is in the shape of an isosceles triangle with a tip angle of 45°. A 20-nm-thick gold layer was deposited by ion sputtering in a waist-tangential circular region with a diameter of 1.5 mm as a protein capture region.

[0073] Soak the gold layer area in 2 μL of 5’ end HS-(CH 2 ) 6 - In the thiol-modified thrombin deoxyribonucleic acid thrombin aptamer, the aptamer is dissolved in tris-hydrochloric acid buffer solution (25mmol / L, pH=7.4, containing 100mmol / L sodium chloride and 5mmol / L L magnesium chloride), the aptamer sequence is: 5'-SH-(CH 2 ) 6 - GTG GTTGGT GTG GTT GG-3' (SEQ ID No: 1). Incubate at 4°C in the dark for 16 h in a humid environment, and wash twice with tris-hyd...

Embodiment 2

[0079] Example 2: Determination of carcinoembryonic antigen 125 (CA125) in serum using the mass spectrometry immunoassay of the present invention.

[0080] Specific steps are as follows:

[0081] (1) The material of the protein capture chip is the same as that in Example 1. Prepare antibody-immobilized CA125 detection chip on its surface.

[0082] Soak the rest of the gold layer in 200 μL ethanol solution of 11-mercaptoundecanoic acid with a concentration of 1 mg / mL, place it in the dark for 12 hours, wash it twice with ethanol and deionized water twice, and soak the area in 200 μL Concentration 10mg / mL NHS and concentration 50mg / mL EDC aqueous solution, react at room temperature for 30min, wash with deionized water once, immerse the area in 2μL anti-CA125 phosphate buffer solution with a concentration of 0.1mg / mL, avoid at 4℃ Incubate with light for 12 hours, and then wash twice with phosphate buffer solution to obtain antibody-immobilized chips, which are set aside.

[00...

Embodiment 3

[0089] Prepare a variety of mass spectrometry-labeled probes and try to detect multiple mass spectrometry-labeled probes at the same time. The specific steps are as follows:

[0090] (1) Add 100 μL of acetonitrile solutions with a concentration of 100 μmol / L of formula VIII, formula IX, and formula X to 2 mL of a gold nanoparticle dispersion with a diameter of 25 nm at a concentration of 1 nmol / L, and react for 12 hours at room temperature in the dark. Centrifuge at 6000rpm for 15min, discard the supernatant, add water to disperse, centrifuge and wash twice, and finally disperse in 2mL of water. The gold nanoparticle dispersions modified with Formula VIII, Formula IX, and Formula X were mixed in a volume ratio of 1:1:1.

[0091]

[0092] (2) Add 2 μL of the mixed dispersion solution dropwise on the chip and let it dry.

[0093] (3) The chip was placed on the sample track in front of the entrance of the mass spectrometer, and the dissociation of the three mass spectrometry ...

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Abstract

The invention discloses a mass spectrometry immunoassay method that can be used for detection of disease markers and its application, including: capture of target protein on the chip surface, labeling of target protein by mass spectrometry labeling probe, chip electrospray ionization mass spectrometry detection label Mass spectrometry signal molecule on target protein, quantitative calculation of protein concentration. The method has strong universality, strong expandability, simple device and high sensitivity. Among them, the chip participates in the ionization process of mass spectrometry in the form of an array to achieve fast, efficient, high-throughput, and low-cost sample analysis. Mass spectrometry-labeled probes mark a large number of mass spectrometry-labeled molecules on the target protein through the specific recognition of aptamers to achieve multi-step amplification of the target protein signal and achieve high-sensitivity detection. A variety of mass spectrometry marker molecules can realize the simultaneous detection of multiple target proteins, further improving the analysis throughput and analysis efficiency. The method has broad application prospects in the fields of early clinical diagnosis, tumor marker screening and prognosis treatment.

Description

technical field [0001] The invention relates to the construction and application of a protein immune analysis method, in particular to the preparation method of a universal protein capture and detection chip and a mass spectrometry labeling probe, an immunodetection method based on mass spectrometry signals and its application in the detection of protein markers Applications. Background technique [0002] Protein molecules are widely involved in important life activities in the living body, including body defense, material transportation, signal transduction, etc. The protein expression level can usually reflect the life state of the body. A variety of protein molecules have been used as biomarkers for various diseases mainly including tumors, such as alpha-fetoprotein, carcinoembryonic antigen, prostate specific antigen, etc., for clinical diagnosis , Pathological analysis and prognosis evaluation provide important reference indicators. The detection of protein molecules ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N27/62G01N33/536
CPCG01N33/6848G01N27/624G01N33/536
Inventor 白玉徐姝婷刘虎威
Owner PEKING UNIV