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Nafion-based atom transfer radical polymerization signal amplification triggering lung cancer early diagnosis kit

A technology of atom transfer and polymerization, which is applied in the field of biological analysis to achieve the effects of improving detection sensitivity, polymerization efficiency and stability.

Active Publication Date: 2019-09-20
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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  • Nafion-based atom transfer radical polymerization signal amplification triggering lung cancer early diagnosis kit
  • Nafion-based atom transfer radical polymerization signal amplification triggering lung cancer early diagnosis kit
  • Nafion-based atom transfer radical polymerization signal amplification triggering lung cancer early diagnosis kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1, the construction of diagnostic kit

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

[0056] The preparation method of PBS buffer solution is: weigh 5.8g Na 2 HPO 4 .12H 2 O, 0.592g NaH 2 PO 4 .2H 2 O, 2.34gNaCl and 200μL of ultrapure water are mixed to obtain the product; the concentration is 0.1M and the pH is 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, and stored at 4° C. for future use.

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

Embodiment 2

[0060] Embodiment 2, the detection method of kit

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

[0062] ① Take a certain amount of amino Fe 3 o 4 Magnetic bead solution (10mg / mL), magnetically separated, remove supernatant, wash with PBS buffer, magnetically separate, remove supernatant, add PBS buffer to resuspend and return to the measured volume of magnetic bead solution;

[0063] ② Measure 20 μL of magnetic bead resuspension, add 20 μL of Sulfo-SMCC solution and 160 μL of PBS buffer, mix well, and react in the dark for 2 hours under the conditions of 37°C and 200 r / s on a constant temperature shaker, so that one end of the cross-linking agent SMCC and Amino magnetic beads are bound by amide bonds;

[0064] ③Magnetically separating the mixture obtained in steps (1)-②, removing the supernatant, washing with PBS buffer, magnetically separating, removing the supernatant;

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

Embodiment 3

[0077] Embodiment 3, feasibility verification

[0078] First, the fluorescence intensity of amino magnetic beads in different modification states was detected by using a fluorescence spectrometer. Such as figure 2 As shown, only adding PBS buffer (0.1 M, pH 7.4) did not detect any fluorescence intensity. Instead, adding SMCC / PNA / tDNA / Zr 4+ / Nafion / Cu II Br / Me 6 After multiple washes after TREN, obvious fluorescence emission peaks can still be observed, because a large amount of FA was introduced to the magnetic beads through ATRP, resulting in a huge 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 ATRP polymerization-based fluorescence signal amplification strategy is feasible for the detection of tDNA.

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Abstract

The invention discloses a Nafion-based atom transfer radical polymerization signal amplification triggering lung cancer early diagnosis kit, which comprises amino Fe3O4 magnetic bead solution, Sulfo-SMCC solution, PNA solution, ZrClO2 solution, Nafion solution, fluorescein O-acrylate solution, AA solution, CuBr2 / Me6TREN solution, PBS solution and DMF. Due to the polymerization characteristic of Nafion, Nafion is applied as an initiator to ATRP reaction to improve monomer polymerization performance, a great quantity of fluorescent monomers are polymerized through ATRP reaction, and finally, the Nafion-based atom transfer radical polymerization signal amplification triggering lung cancer biomarker CYFRA21-1 detection kit is constructed successfully through positive correlation of fluorescence signal intensity determined by a fluorescence spectrophotometer and target DNA (tDNA) concentration. The kit has advantages of high sensitivity, high stability, great anti-interference performance, low detection limit and the like and creates a new approach for lung cancer DNA clinical detection.

Description

technical field [0001] The invention relates to a kit for early diagnosis of lung cancer based on signal amplification of atom transfer free radical polymerization induced by Nafion, which belongs to the technical field of bioanalysis. Background technique [0002] Perfluorosulfonic acid (Nafion) is a synthetic polymer with ionic properties, known as ionomer. It opens up a new type of polymer, which is 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 a very strong ionic chemical composition. Some C-F bonds in Nafion have low energy and can generate free radicals. Under the polymerization reaction Easy free radical polymerization. Perfluorosulfonic acid polymers have excellent ion conductivity, excellent chemical stability and good thermal stability. [0003] Atom Transfer Radical Polymerization (ATRP) usually uses ini...

Claims

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Application Information

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Patent Type & Authority Applications(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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