A kit for early diagnosis of lung cancer based on signal amplification of nafion-induced atom transfer radical polymerization

A technology of polymerization reaction and signal amplification, applied in the field of biological analysis, to achieve the effect of increasing polymerization rate and stability, improving stability and reproducibility, and improving polymerization efficiency

Active Publication Date: 2022-07-08
HENAN UNIV OF CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the current problem of detecting lung cancer DNA, the purpose of the present invention is to provide a lung cancer early diagnosis kit based on Nafion-induced atom transfer radical polymerization signal amplification

Method used

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  • A kit for early diagnosis of lung cancer based on signal amplification of nafion-induced atom transfer radical polymerization
  • A kit for early diagnosis of lung cancer based on signal amplification of nafion-induced atom transfer radical polymerization
  • A kit for early diagnosis of lung cancer based on signal amplification of nafion-induced atom transfer radical polymerization

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1. Construction of diagnostic kit

[0055] The kit includes amino Fe 3 O 4 Magnetic bead solution (10 mg / mL), Sulfo-SMCC solution (100 μM), PNA solution (0.5 μM), ZrClO 2 solution (5mM), Nafion solution ( 117 solution), Fluorescein O-acrylate (FA) solution (10 mM), AA solution (2 mM), Cu II Br / Me 6 TREN solution (10 mM), PBS buffer, DMF (N,N-dimethylformamide).

[0056] The preparation method of PBS buffer is: weigh 5.8g Na 2 HPO 4 .12H 2 O, 0.592g NaH 2 PO 4 .2H 2 O, 2.34g NaCl and 200μL ultrapure water were mixed to obtain; the concentration was 0.1M, and the pH was 7.4.

[0057] The preparation method of the ascorbic acid (AA) solution is as follows: AA is mixed with an ethanol solution with a volume fraction of 70% to prepare an AA solution with a concentration of 2 mM, which is stored at 4° C. for later use.

[0058] The preparation method of the fluorescein FA solution is as follows: FA and DMF are mixed to prepare a fluorescein FA solution wi...

Embodiment 2

[0060] Embodiment 2, the detection method of test kit

[0061] (1) Treatment and modification of amino magnetic beads:

[0062] ①Measure a certain amount of amino Fe 3 O 4 Magnetic bead solution (10mg / mL), magnetic separation, remove supernatant, wash with PBS buffer, magnetic separation, remove supernatant, add PBS buffer to resuspend and restore to the measured volume of magnetic bead solution;

[0063] ②Measure 20μL of magnetic bead resuspension, add 20μL of Sulfo-SMCC solution and 160μL of PBS buffer, and after mixing, react in the dark for 2h under the condition of constant temperature shaker at 37°C and 200r / s, so that one end of the cross-linking agent SMCC is mixed with Amino magnetic beads are bound by amide bonds;

[0064] 3. Magnetically separate the mixture obtained in step (1)-2, remove the supernatant, wash with PBS buffer, magnetically separate, and remove the supernatant;

[0065] ④Add 195μL of PBS buffer and 5μL of PNA solution, mix well, react overnight i...

Embodiment 3

[0077] Example 3. Feasibility verification

[0078] First, the fluorescence intensity of aminomagnetic beads with different modification states was detected by using a fluorescence spectrometer. like figure 2 As shown, no fluorescence intensity was detected with the addition of only PBS buffer (0.1 M, pH 7.4). Instead, add SMCC / PNA / tDNA / Zr 4+ / Nafion / Cu II Br / Me 6 Obvious fluorescence emission peaks could still be observed after multiple washings after TREN, which is because a large amount of FA was introduced to the magnetic beads through ATRP, which produced a great fluorescence signal. At the same time, without adding Zr 4+ , Cu II Br / Me 6 Under the conditions of TREN, Nafion, PNA, and tDNA, only relatively weak fluorescent signals were detected, which indicated that the experiment had high sensitivity. Through comparative experiments, it is fully proved that the fluorescence signal amplification strategy based on ATRP polymerization is feasible for the detection o...

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Abstract

The invention discloses an early diagnosis kit for lung cancer based on signal amplification of atom transfer radical polymerization reaction initiated by Nafion, comprising amino Fe 3 O 4 Magnetic Bead Solution, Sulfo‑SMCC Solution, PNA Solution, ZrClO 2 Solution, Nafion solution, Fluorescein O-acrylate solution, AA solution, CuBr 2 / Me 6 TREN solution, PBS buffer, DMF. The present invention utilizes the polymerization characteristics of Nafion to apply it in the ATRP reaction, as an initiator to improve the polymerization ability of the monomers, a large number of fluorescent monomers are connected and polymerized through the ATRP reaction, and finally, the fluorescence signal intensity determined by a fluorescence spectrometer is related to the target DNA (tDNA). ) concentration was positively correlated, and a detection kit for lung cancer biomarker CYFRA21-1 based on signal amplification of Nafion-induced atom transfer radical polymerization reaction was successfully constructed. The kit of the invention has the characteristics of high sensitivity, high stability, strong anti-interference, low detection limit and the like, and opens up a new way for the clinical detection of lung cancer DNA.

Description

technical field [0001] The invention relates to an early diagnosis kit for lung cancer based on signal amplification of atom transfer radical polymerization reaction initiated by Nafion, and belongs to the technical field of biological analysis. Background technique [0002] Perfluorosulfonic acid (Nafion) is a synthetic polymer with ionic properties, known as an ionomer. It opens up a whole new class of polymers, which are mostly used in membrane materials, fuel cells, and electrode modification. Since the ionic properties of Nafion are formed by adding sulfonic acid groups to the polymer matrix, the sulfonic acid groups have very strong ionic chemical components, and some of the C-F bonds in Nafion have low energy and can generate free radicals. Under the polymerization reaction Easy free radical polymerization. The perfluorosulfonic acid polymer has excellent ionic conductivity, excellent chemical stability and good thermal stability. [0003] Atom Transfer Radical Pol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/682
CPCC12Q1/6886C12Q1/682C12Q2563/107C12Q2563/143C12Q2563/149C12Q2527/125Y02E60/50
Inventor 杨怀霞张京玉刘艳菊巴妍妍赵利营郭壮壮
Owner HENAN UNIV OF CHINESE MEDICINE
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