Ultra-high sensitive colorimetric method for detecting thrombin

A technology of sensitive detection and thrombin, applied in the field of biochemical analysis, can solve the problems of cumbersome, waste of resources, etc., achieve ultra-high sensitive detection, improve detection sensitivity, and improve the effect of signal-to-noise ratio

Active Publication Date: 2015-06-24
QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The occurrence of rolling circle replication in this invention requires the connection of capture antibodies, thrombin, detection antibodies, primer probes and circular templates one by one, which is cumbersome and wastes resources, and it is cumbersome to prepare a nano-gold solution with uniform particle size in advance.

Method used

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  • Ultra-high sensitive colorimetric method for detecting thrombin
  • Ultra-high sensitive colorimetric method for detecting thrombin
  • Ultra-high sensitive colorimetric method for detecting thrombin

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Experimental program
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Embodiment 1

[0032] Experimental part of embodiment 1

[0033] 1. Modification of 96-well plate:

[0034] (1) Add 200 μL of 5 mM glutaraldehyde to 6 microwells of a 96-well plate, shake in a water bath at 37° C. in a wet box for 4 hours, and coat the bottom of the microwells with glutaraldehyde.

[0035] (2) The reaction solution was discarded, the 6 microwells were washed 5 times with PBS of pH 5 and water respectively, and 200 μL of 1×10 -6 M primer (5′-GAC GGC GAA GGA TTG ATA CT-3′), in a water bath at 37°C for 4 hours, and the primer was modified onto the microwell plate.

[0036] (3) Discard the reaction solution, wash 6 microwells once with 200 μL water, add 25 μL 1×10 -6M circle template (5′-CTT CGC CGT CCC CAA CCC GCC CTA CCC GGT TGG TGT GGT TGG CCC AAC CCG CCC TAC CCA GTA TCA ATC-3′), 3 μL 10×T4 ligase buffer, 90°C for 10 min, room temperature After annealing for 1 hour, connect the circle template to the primer on the surface of the microwell plate.

[0037] (4) Add 2 μL of T...

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Abstract

The invention provides an ultra-high sensitive colorimetric method for detecting thrombin; a lot of DNA (Deoxyribonucleic Acid) enzymes can be formed by adopting a rolling-circle replicating and amplifying technology; detection signals are amplified; therefore, the detection sensitivity of thrombin is increased; influence of unreacted hemin to reaction can be eliminated by adopting a 96-pore plate connection primer; the signal to noise ratio is increased; and therefore, the detection sensitivity is further increased. By adopting a sensitive colorimetric system, gold ions are reduced to be red or blue generated in a dispersed or united state by utilizing hydrogen peroxide; whether a target molecule exists can be judged directly; a nano gold solution is unnecessary to prepare in advance; the ultra-high sensitive colorimetric method is simple and feasible; complex apparatuses and equipment are unnecessary; by means of the ultra-high sensitive colorimetric method, ultra-high sensitive detection of thrombin can be realized; and the detection limit is up to 10-17 M (about six thrombin molecules).

Description

Technical field: [0001] The invention belongs to the technical field of biochemical analysis, in particular to a colorimetric method for detecting thrombin with ultrahigh sensitivity. Background technique: [0002] Thrombin is a proteolytic enzyme consisting of two peptide chains connected by disulfide bonds. Thrombin converts fibrinogen in the blood into fibrin, accelerates the aggregation of platelets, and achieves the purpose of rapid hemostasis. The metastasis and spread of tumor tissue and tumor cells in cancer patients will change the blood coagulation mechanism of patients, and cancer and other diseases can be evaluated by detecting the concentration of thrombin. At present, the methods for detecting thrombin mainly include fluorescence method, chromogenic substrate method and electrochemical method, but the sensitivity of these three methods is not good, the detection limit is high, and it cannot meet the trace detection required by modern medicine; Raman Scatterin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/56
CPCC12Q1/56C12Q1/6844C12Q2531/125
Inventor 王宗花王赛毕赛夏建飞张菲菲杨敏桂日军李延辉夏延致
Owner QINGDAO UNIV
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