High-sensitivity quantitative determination method of cardiac troponin I

A quantitative detection method, cardiac troponin technology, applied in the field of biomedical detection, to achieve the effect of reducing the difficulty and cost of experiments, accurate and sensitive quantitative detection, and high sensitivity

Active Publication Date: 2017-08-22
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, the cTnI quantitative detection method based on the specific recognition of nucleic acid aptamer to cTnI, nucleic acid rolling circle amplification and fluorescence resonance energy transfer technology has not been reported.

Method used

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  • High-sensitivity quantitative determination method of cardiac troponin I
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  • High-sensitivity quantitative determination method of cardiac troponin I

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] In the RCA reaction, the success of the cyclization reaction is the basis for the subsequent amplification reaction, and the cyclization reaction can be performed in two ways:

[0053] The first method is in situ circularization, that is, add AP to a transparent 96-well plate immobilized with cTnI, incubate at 37°C for 1h, then add LP, T4DNA ligase and T4DNA ligase Buffer, incubate at 37°C for 1h, and incubate at 96°C Circular ligation on orifice plate. However, the steps of this method are cumbersome and the experiment time is relatively long.

[0054] The second method is to add the pre-prepared aptamer cyclization product to the black 96-well microplate plate on which cTnI has been immobilized, and incubate at 37° C. for 1 h. This method is easy to operate, and a large number of aptamer cyclization products can be prepared at one time for future use, reducing experimental time.

[0055] Through the affinity between biotin-modified AP and streptavidin-modified horse...

Embodiment 2

[0070] Graphene oxide has a series of unique chemical properties and structures, and has a strong adsorption effect on single-stranded DNA. At this time, the energy donor is close to the acceptor, and fluorescence resonance energy transfer occurs, and the fluorescence is quenched; After the complementary sequence is combined, the formed double-stranded DNA has a large amount of negative charges, which repels graphene oxide, weakens the adsorption of graphene oxide, and falls off the surface. At this time, the fluorescent group cannot transfer energy to the energy receiving body. , the fluorescence recovers, so as to achieve the purpose of detection.

[0071] The quenching of ssDNA probes labeled with FAM fluorophores by graphene oxide is a key step in the establishment of graphene oxide sensors. When the target sequence does not exist, the graphene oxide sensor is in the off state. At this time, the quenching efficiency of graphene oxide directly affects the background signal ...

Embodiment 3

[0074] The established quantitative detection method based on nucleic acid rolling circle amplification and fluorescence resonance energy transfer is used in the quantitative detection of cTnI. The detection principle is as follows: figure 1 As shown, the specific detection steps are as follows:

[0075] (1) Coat cTnI antibody diluted in coating buffer onto a black 96-well microtiter plate with 100 μL per well, and then incubate at 37°C for 2 hours;

[0076] (2) Discard the liquid on the black 96-well ELISA plate, add 300 μL of blocking solution to each well, and block at 4°C for 1 hour;

[0077] (3) Discard the liquid on the black 96-well microplate, add 300 μL PBST to each well to wash the black 96-well microplate 3 times, pour off the third washing liquid and pat dry the black 96-well microplate;

[0078] (4) Divide the wells on the black 96-well ELISA plate into 10 experimental groups. cTnI and three groups of samples to be tested were added to 10 different experimental ...

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Abstract

The invention relates to a high-sensitivity quantitative determination method of cardiac troponin I, and belongs to the technical field of biomedical detection. According to the detection method, specific recognition is conducted on the cardiac troponin I through an aptamer, and accurate and sensitive quantitative detection on cTnI is achieved by means of a rolling circle amplification technology of nucleic acid and a graphene oxide-based fluorescence resonance energy transfer technology; the detection method has the advantages of being good in specificity, high in sensitivity, easy and convenient to operate, low in cost and the like, and the clinical test requirements are met.

Description

technical field [0001] The invention relates to a highly sensitive quantitative detection method for cardiac troponin I, belonging to the technical field of biomedical detection. Background technique [0002] Cardiac troponin is a contractile regulatory protein of the myocardium, consisting of three subunits of cardiac troponin I (cTnI), cardiac troponin C (cTnC) and cardiac troponin T (cTnT), among which cTnI has a high degree of Myocardial specificity has become the first choice marker for clinical diagnosis of myocardial infarction. The highly sensitive detection of cTnI is of great significance for improving the early diagnosis rate of acute myocardial infarction, rapid triage, risk stratification and prognosis assessment. [0003] At present, common cTnI quantitative detection methods include radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), chemiluminescence, rapid test strip detection, etc. The detection sensitivity of these methods for cTnI is usual...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 吕雪飞李昱代唯强李永瑞邓玉林
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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