Method for detecting brain natriuretic peptide through composite probe fluorescence "turning on-turning off-turning on" strategy on basis of cobalt nanomaterial/nucleic acid aptamer

A nucleic acid aptamer and composite probe technology, applied in the field of fluorescence analysis, can solve the problems of cumbersome operation, high cost, and time-consuming, and achieve the effect of good selectivity and strong practicability

Inactive Publication Date: 2019-08-06
CHONGQING MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to provide a new homogeneous fluorescence method based on cobalt oxyhydroxide nanorings for highly sensitive detection of brain natriuretic peptide, to solve the problems of complicated operation, time-consuming, high cost and low sensitivity in the prior art. Simple, fast, high sensitivity and good selectivity, it is expected to be used for timely diagnosis and prognosis of heart failure, etc.

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  • Method for detecting brain natriuretic peptide through composite probe fluorescence "turning on-turning off-turning on" strategy on basis of cobalt nanomaterial/nucleic acid aptamer
  • Method for detecting brain natriuretic peptide through composite probe fluorescence "turning on-turning off-turning on" strategy on basis of cobalt nanomaterial/nucleic acid aptamer
  • Method for detecting brain natriuretic peptide through composite probe fluorescence "turning on-turning off-turning on" strategy on basis of cobalt nanomaterial/nucleic acid aptamer

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Experimental program
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Effect test

Embodiment 1

[0044] Preparation and characterization of embodiment 1 CoOOH nanomaterial

[0045] The preparation method of CoOOH nanometer material comprises the following steps:

[0046] (1) 10mL NaOH solution (1mol / L) was quickly added to 40mL CoCl under magnetic stirring 2 ·6H 2O (10mmol / L) solution, after stirring and mixing, sonicate for 1min; (2) add 2mL of NaClO solution with a concentration of 0.9mol / L dropwise during sonication, and continue sonicating for 10min; (3) gradually add to the mixed solution Add 1mol / L HCl dropwise to adjust the pH of the solution to 7.3; (4) Transfer the above mixed solution to a centrifuge tube, centrifuge at 10,000rpm for 10min, discard the supernatant, and ultrasonically disperse the lower precipitate in 25mL ultrapure water, Repeat the centrifugal washing operation at least 3 times; (5) place the last precipitate in an oven, and dry it at 60° C. to obtain the final product (brown black powder), which is ready for use.

[0047] Characterization o...

Embodiment 2

[0052] The preparation and detection method of embodiment 2 solution

[0053] Preparation of FAM-aptamer stock solution

[0054] The freeze-dried powder of FAM-aptamer was centrifuged at 6000 rpm for 5 min, and 800 μL of ultrapure water was added to prepare a stock solution of 2 μmol / L, which was stored in a refrigerator at 4°C. Dilute the concentration to 100nmol / L before use.

[0055] Preparation of BNP standard stock solution

[0056] Add 2mL of ultrapure water to the BNP freeze-dried powder, and equilibrate at room temperature (20-30°C) for 15-20min to completely dissolve the freeze-dried powder, then gently rotate / invert the reagent until uniform, and the concentration of the prepared stock solution is 1273pg / mL, stored in -20°C refrigerator, diluted before use.

[0057] Preparation of CoOOH Nanoring Dispersion

[0058] Weigh 0.0063 g of CoOOH nanorings, add 250 mL of ultrapure water, and sonicate until the CoOOH nanomaterials are evenly dispersed.

[0059] Detection...

Embodiment 3

[0064] Embodiment 3 influence factor investigation

[0065] The inventors studied the influence of the concentration of CoOOH nanorings and the pH of the system on the detection results, as follows.

[0066] Effect of CoOOH nanoring concentration: Add different concentrations of CoOOH nanoring dispersions to the mixed solution of Tris-HCl buffer (pH 7.4) and FAM-aptamer (3.75nmol / L) in turn, and dilute to 400 μL, vortex mixed, let stand at room temperature for 30 min, and measure according to the fluorescence parameters in Example 2.

[0067] When the CoOOH nanoring concentration is 0-18.75 μg / mL, the fluorescence emission intensity of FAM-aptamer is as follows Figure 4 shown. It can be seen that with the increase of the concentration of CoOOH nanorings, the fluorescence intensity of FAM-aptamer gradually decreases. When the concentration of CoOOH nanorings is 18.75 μg / mL, the fluorescence quenching efficiency reaches 80%. conditions for subsequent experiments.

[0068] I...

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Abstract

The invention discloses a method for detecting brain natriuretic peptide through a composite probe fluorescence "turning on-turning off-turning on" strategy on the basis of cobalt nanomaterial / nucleicacid aptamer. According to the method, a CoOOH nano-ring with a high-efficiency fluorescence quenching effect is synthesized; a CoOOH nano-ring / FAM-aptamer composite probe is constructed; backgroundsignals are reduced by means of the fluorescence quenching effect of the CoOOH nano-ring for a dye; the fluorescent dye can be made to move away from the CoOOH nano-ring by means of the recognition effect of the BNP (brain natriuretic peptide) for the specific molecules of FAM-aptamer, so that fluorescent signals can be recovered; and fluorescence recovery efficiency and BNP concentration are in agood linear relationship in a range of 0.030 to 0.80pg / mL. The method has the advantages of simplicity, rapidness, high sensitiveness, high specificity and the like, and can be used for the quantitative detection of BNP in clinical specimens. ROC curve analysis shows that the method has high accuracy in diagnosing heart failure (AUC=0.87, 95% CI: 0.71-1.00, and P<0.01), and the method has good specificity (diagnostic specificity is 87. 9%); and the use cost of the method is greatly reduced. The method is suitable for being widely promoted and used in the diagnosis and prognosis of the heart failure of children and adults in primary-level organizations.

Description

[0001] The invention belongs to the technical field of medical examination, and in particular relates to a new fluorescent analysis method for detecting brain natriuretic peptide with high sensitivity, high specificity and low cost by using a cobalt nanomaterial / nucleic acid aptamer composite probe. Background technique [0002] Brain natriuretic peptide (BNP) has become the "gold standard" for the diagnosis of heart failure (HF). release to keep blood pressure and plasma volume stable. HF-related guidelines also list its application value as a grade IA recommendation. The pathophysiological effects of BNP released into the blood are to antagonize the renin-angiotensin-aldosterone system and the sympathetic nervous system. Blood pressure, reduce cardiac load and anti-myocardial fibrosis. Compared with heart failure diagnostic methods such as echocardiography, electrocardiogram, and chest X-ray, which are time-consuming, expensive, and easily affected by subjective factors suc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/74G01N33/68G01N33/577G01N33/543G01N33/533
CPCG01N33/533G01N33/54326G01N33/577G01N33/68G01N33/74G01N2333/58
Inventor 张普涂爱萍尚京川尹一兵袁川吉
Owner CHONGQING MEDICAL UNIVERSITY
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