Fluorescence method for detecting brain natriuretic peptide based on graphene oxide/nucleic acid aptamer

A technology of nucleic acid aptamer and brain natriuretic peptide, which is applied in the field of medical testing, can solve the problems of detection susceptibility, susceptibility to interference, high detection cost, etc., to overcome cross-reaction interference, improve method selectivity, and high specificity detection Effect

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

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Problems solved by technology

Among them, the most commonly used chemiluminescent immunoassay in clinical practice has the advantages of high sensitivity and wide linear range, but the detection is susceptible to interference, and requires the introduction of magnetic beads for separation, which results in high detection costs; ELISA has good specificity , has been used to develop various commercial detection kits, but low sensitivity and precision limit its application; immunochromatography is simple, fast, and portable, and is suitable for point of care testing (POCT), However, its sensitivity is not enough and it is susceptible to interference, which limits the accurate determination
In addition, the above-mentioned methods are all immunoassays, and these methods will also detect proBNP and BNP metabolites while detecting BNP, because they have the same antibody epitope, which leads to the concentration of BNP is often overestimated

Method used

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  • Fluorescence method for detecting brain natriuretic peptide based on graphene oxide/nucleic acid aptamer
  • Fluorescence method for detecting brain natriuretic peptide based on graphene oxide/nucleic acid aptamer
  • Fluorescence method for detecting brain natriuretic peptide based on graphene oxide/nucleic acid aptamer

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The preparation and detection method of embodiment 1 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 boiled ultrapure water was added to prepare a 2 μmol / L stock solution, which was stored in a refrigerator at 4°C. Dilute to a concentration of 100nmol / L before use.

[0055] Preparation of BNP standard stock solution

[0056] Use a pipette gun to add 2 mL of boiled ultrapure water to the lyophilized powder, and equilibrate at room temperature (20-30°C) for 15-20 minutes to completely dissolve the lyophilized powder, then gently rotate / invert the reagent until uniform, and configure The stock solution has a concentration of 1482pg / mL, stored in a -20°C refrigerator, and diluted before use.

[0057] Detection method

[0058] Blank group 1: Add 40 μL of PB buffer (pH7.4) and 15 μL of FAM-aptamer (3.75 nmol / L) in sequence to a 1.5 mL centrifuge tube, add boile...

Embodiment 2

[0062] The investigation of embodiment 2 influencing factors

[0063] The inventors studied the effect of GO concentration on the fluorescence of the FAM-aptamer probe as follows.

[0064] A series of GO solutions with different concentrations were added to the mixed solution of PB buffer (pH7.4) and FAM-aptamer (3.75nmol / L), and then the volume was adjusted to 400 μL with ultrapure water, vortexed and kept at room temperature. Stand still for 30 minutes, and measure the fluorescence parameters according to Example 1.

[0065] When the GO concentration is 0-40 μg / mL, the fluorescence intensity of FAM-aptamer in the system changes as follows figure 2 shown. It can be seen from the figure that when GO was added, the fluorescence of FAM-aptamer was rapidly quenched, and with the increase of GO concentration, the fluorescence intensity gradually decreased until it was completely quenched. In the present invention, 20 μg / mL GO was selected as the subsequent detection condition....

Embodiment 3

[0066] The drawing of embodiment 3 standard curve

[0067] A series of BNP standard solutions with different concentrations were added to the mixed solution consisting of 20 μL GO (400 μg / mL) and 40 μL PB buffer (pH 7.4), vortexed, allowed to stand at room temperature for 30 min, and then added to each 15 μL of FAM-aptamer (3.75 nmol / L) was added to the mixed solution, and the volume was adjusted to 400 μL with ultrapure water, vortexed to mix, and left to stand at room temperature for 30 min, and measured according to the fluorescence parameters set in Example 1. Repeat the experiment three times.

[0068] Depend on image 3 It can be seen that the fluorescence intensity of GO / FAM-aptamer composite probe gradually recovers with the increase of BNP concentration. Fluorescence recovery efficiency ((F 2 -F 1 ) / (F 0 -F 1 )) is the ordinate, the BNP concentration is the abscissa, and the fitting linearity has a good linear relationship ( Figure 4 ), the standard curve equa...

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Abstract

The invention provides a homogeneous fluorescence detection novel method of brain natriuretic peptide (BNP) with high sensitivity, high selectivity and low cost. The method comprises the following steps of through constructing a graphene oxide / fluorescent dye-labeled nucleic acid aptamer composite probe, using a fluorescence quenching effect of the graphene oxide to the dye to reduce a detected fluorescent background signal; when BNP is added and a nucleic acid aptamer make the fluorescent dye be far away from the graphene oxide through a specific molecular recognition effect, recovering a fluorescence signal of the dye, and establishing a linear working curve based on changes of BNP concentration and fluorescence intensity; and finally, through testing a fluorescence intensity change value after the sample is added and substituting into the working curve, calculating and acquiring accurate concentration of the BNP in a clinical sample. Various immunoassays have cross reaction due to usage of antibody recognition. By using the method of the invention, the above disadvantage is overcome; interferences of related peptides and the like are avoided; the method has advantages of simpleness, speediness, low cost, high sensitivity, high selectivity and the like; and the method can be used for quantitative detection of the BNP in blood of patients in clinical application, and can be used for timely diagnosis, treatment guidance and prognosis prompting.

Description

technical field [0001] The invention belongs to the technical field of medical examination, and in particular relates to a new method for highly sensitive and highly specific fluorescent detection of brain natriuretic peptide. Background technique [0002] Cardiovascular disease is one of the diseases with the highest morbidity and disability rate in modern society, which seriously threatens human health. Heart failure (HF) is the terminal stage of cardiovascular disease, and its mortality rate and readmission rate are high, but many deaths caused by heart failure can be avoided through early detection and timely treatment, so heart failure Early diagnosis is of great significance. Monitoring of physiological indicators (such as electrocardiogram, chest X-ray examination, echocardiography) and biochemical indicators (such as specific biomarkers) is the main method for clinical diagnosis of heart failure. Compared with the monitoring of physiological indicators, the monitor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N33/53G01N33/533G01N33/577G01N33/68
CPCG01N21/6486G01N33/53G01N33/533G01N33/577G01N33/6821
Inventor 张普涂爱萍尚京川尹一兵刘来成
Owner CHONGQING MEDICAL UNIVERSITY
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