Method for detecting biomacromolecule based on magnetic separation-quantum dot immunofluorescence sensing and reagent preparation method

A biomacromolecule and immunofluorescence technology, applied in the field of detection of biomacromolecules, can solve the problems of fluorescence background interference, loss of fluorescence signal, and the inability of fluorescence to be effectively excited.

Inactive Publication Date: 2014-01-29
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are problems to be solved in the traditional magnetic separation-quantum dot sensor. The fluorescent signal of the traditional magnetic separation-quantum dot immunosensor comes from the quantum dot immunoprobe bound by the immunomagnetic beads through the antigen-antibody specific reaction. This analysis The mode has two disadvantages: first, the immunomagnetic beads themselves have fluorescence, which will cause fluorescence background interference; second, the diameter of the quantum dots is an order of magnitude smaller than that of the immunomagnetic beads. When the laser is irradiated, the quantum dots on the other surface of the magnetic beads The fluorescence of the dots cannot be effectively excited, this phenomenon is like a "day and night" phenomenon, thus resulting in the loss of effective fluorescent signal
But for the solution to the second problem, there is no related report

Method used

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  • Method for detecting biomacromolecule based on magnetic separation-quantum dot immunofluorescence sensing and reagent preparation method
  • Method for detecting biomacromolecule based on magnetic separation-quantum dot immunofluorescence sensing and reagent preparation method
  • Method for detecting biomacromolecule based on magnetic separation-quantum dot immunofluorescence sensing and reagent preparation method

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Experimental program
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Embodiment 1

[0031] Embodiment 1: the preparation of simulated sample

[0032] Take the detection of the content of Fusarium wilt in the sample as an example.

[0033] Take 15 corn seed samples in a 50mL centrifuge tube, soak and disinfect with 30mL 3-5% NaCl0 for 5min-15min, and rinse with 10mL sterile water for 3 times. Soak with 40mL PBS buffer solution (0.01mol / L, pH 7.4), overnight at 4°C. Transfer the soaking liquid into a centrifuge tube, centrifuge at 1000r / min for 10min, take the supernatant and transfer it to another centrifuge tube, centrifuge at 10000r / mmin for 15min, discard the supernatant, suspend and precipitate with 100mL of the above PBS buffer solution, and prepare corn seed matrix liquid. Use this matrix solution as diluent to prepare different content corn blight bacteria (10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 and 0 cfu / mL) of serial positive samples. Negative samples were prepared according to the above steps without adding corn wilt bacteria.

Embodiment 2

[0034] Embodiment 2: Preparation of immunomagnetic bead reagent

[0035] Take the detection of the content of Fusarium wilt in the sample as an example.

[0036] 1. Preparation of phosphate buffer solution: disodium hydrogen phosphate (0.2mol L -1 ) and sodium dihydrogen phosphate (0.2mol L -1 ) according to (Na 2 HPO 4 / NaH 2 PO 4 =4:1 ratio) mixed to prepare 0.01mol L -1 Phosphate buffer solution, pH=7.4.

[0037] 2. Preparation of blocking solution: use a molar concentration of 0.01mol L -1 Bovine serum albumin (BSA) was dissolved in PBS of PH=7.4 so that the concentration by weight was 0.5%.

[0038] 3. Activation of supercisive nano-magnetic beads: Take 2mg of supercisive nano-magnetic beads with a particle diameter of 1000nm carboxyl group in a 1.5mL centrifuge tube, place it in an ultrasonic cleaner for 30s, put it into a vortex oscillator and vibrate for 10s, and use Separation of magnetic separation rack to obtain solid superparasitic nano-magnetic beads, the...

Embodiment 3

[0040] Embodiment 3: quantum dot fluorescent probe (QDs-Ab 2 )(2) Preparation

[0041] Take the detection of the content of Fusarium wilt in the sample as an example.

[0042] 1. Activation of carboxyl groups on the surface of QDs: Take 100 μL of QDs solution in a 1.5 mL centrifuge tube, place it in an ultrasonic cleaner for 30 s, place it in a vortex shaker for 10 s, then add 10 μL of EDC with a mass concentration of 10 mg / mL and 5 μL of NHS with a mass concentration of 10mg / mL was activated by shaking for 15 minutes;

[0043] 2. QDs-Ab 2 Preparation of fluorescent probe (2): Add 0.3 mg of Fusarium wilt monoclonal antibody (protein content 5 mg / mL) to the above-mentioned activated QDs solution, react at room temperature for 2 h, and then add 50 μL of BSA at a concentration of 1 % PBS buffer solution, blocking reaction for 0.5h, then transferred to an ultrafiltration centrifuge tube with a molecular weight of 100kd, centrifuged at a centrifugal force of 8000g for 15min, rem...

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Abstract

The invention provides a method for detecting a biomacromolecule based on magnetic separation-quantum dot immunofluorescence sensing and a reagent preparation method. The method is characterized by comprising the following steps: with superparamagnetic nano beads MBs as carriers, coupling the carriers with an antibody Ab1 of a biomacromolecule to be detected to prepare a capturing antibody MB-Ab1; coupling a quantum dot with an antibody Ab2 of the biomacromolecule to be detected to prepare a quantum dot fluorescence probe QDs-Ab2; enriching the biomacromolecule Ag to be detected in a test sample by using the MB-Ab1 to prepare a serial compound MB-Ab1-Ag; reacting the MB-Ab1-Ag with the QDs-Ab2 to generate an MB-Ab1-Ag-Ab2-QDs and carrying out magnetic separation to separate the MB-Ab1-Ag-Ab2-QDs from a liquid-phase QDs-Ab2 immunofluorescence probe; and measuring QDs-Ab2 fluorescence intensity X in a liquid phase and indirectly measuring C by utilizing an inverse relation represented between the concentration C of the biomacromolecule to be detected and the X. The method is simple and convenient to operate, good in stability and high in detection sensitivity.

Description

technical field [0001] The invention relates to a method for detecting biological macromolecules, in particular to a magnetic separation-quantum dot immunofluorescence sensing detection method based on an indirect signal analysis mode and a method for preparing and using related reagents. Background technique [0002] Infectious animal and plant pathogenic bacteria and viruses are the key monitoring objects in the entry-exit inspection and quarantine work. Rapid, accurate and convenient detection of these infectious harmful organisms is essential for protecting human health and life safety, ensuring national security, Maintaining social stability is of great significance. [0003] The current detection methods for detecting the pathogenic bacteria and viruses mainly include methods such as isolation and culture detection, immunological detection, and molecular biology. The isolation and culture method is simple and easy to implement, but due to its low detection sensitivity...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/54326G01N33/542G01N33/56911G01N33/56983G01N33/588G01N2446/80
Inventor 邹明强陈翊平李莉刘峰刘彩虹
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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