Rolling circle amplification DNA enzyme and covalent organic framework-based electrochemical-ELISA immunosensor

A covalent organic framework and immunosensor technology, applied in the field of electrochemical detection, can solve the problems of high cost and easy inactivation of biological enzymes

Active Publication Date: 2020-01-31
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method has carried out nucleic acid signal amplification to ELISA, its signal still originates from biological enzyme (sucrase),

Method used

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  • Rolling circle amplification DNA enzyme and covalent organic framework-based electrochemical-ELISA immunosensor
  • Rolling circle amplification DNA enzyme and covalent organic framework-based electrochemical-ELISA immunosensor
  • Rolling circle amplification DNA enzyme and covalent organic framework-based electrochemical-ELISA immunosensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Electrochemical-ELISA Immunosensor Construction

[0055] (1) Primer-AuNPs-aptamer complex preparation:

[0056] Au-S bonds formed between AuNPs and sulfhydryl-modified nucleic acid chains to prepare primer-AuNPs-aptamer complexes: mix 1 mL of prepared AuNPs (7.5 μM) with 3 μL sulfhydryl-modified aflatoxin M1 aptamers (0.1 mM), 12 μL of sulfhydryl-modified primers (0.1 mM) and 2 μL of acetic acid (0.5 mM, pH 5.2) were mixed and incubated at room temperature for 12 h; then 20 μL of dNTP (0.1 mM) was added to block redundant active sites on the surface of AuNPs. Subsequently, 1 mL of NaCl (0.25M) was added and incubated at room temperature for 24 h to increase the stability of the complex. Excess reagents were removed by centrifugation at 10000rpm for 20min; the resulting precipitate was suspended in 1.5mL Tris-EDTA (TE, 0.1M, pH 7.4) buffer, and stored at 4°C for later use;

[0057] (2) Construction of primer-AuNPs-aptamer / aflatoxin M1 / antibody sandwich modifi...

Embodiment 2

[0079] Example 2 Constructing the standard curve of the target antigen aflatoxin M1

[0080] The standard solution is processed with reference to the sensor in Example 1:

[0081] Replace the aflatoxin M1 target solution in step (2) with different concentrations of aflatoxin M1 standard solutions (0.1, 0.25, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 120ng / mL), respectively, to obtain the ELISA plates modified by the primer-AuNPs-aptamer / aflatoxin M1 / antibody sandwich structure containing different concentrations of aflatoxin M1;

[0082] Then measure the corresponding reduction peak current value I according to (3), (4) steps respectively i , Measure the reduction peak current value I of blank sample (do not contain aflatoxin M1) simultaneously 0 , using I i / I 0 Construct a linear model with concentrations, such as Figure 7 shown in I i / I 0 is the ordinate, the concentration is the abscissa, the standard curve is y=2.076+0.039x, the correlation coefficient R 2...

Embodiment 3

[0084] Embodiment 3 sensor construction condition optimization experiment

[0085] (1) Effect of primer / aptamer ratio:

[0086] Primer / aptamer ratios were investigated with varying primer / aptamer ratios (2:1, 3:1, 4:1, 5:1, 6:1, and 7:1, with a fixed aptamer concentration of 1.5 μM) Impact on analysis performance.

[0087] Such as Figure 8 A, As the primer / aptamer ratio increases, the current signal increases, and when the primer / aptamer ratio is higher than 4:1, the current signal decreases. The reduction in current may be due to the extra primers occupying the binding sites of AuNPs that belong to aptamers, and may even lead to no aptamers in the complex. Therefore, primer-AuNPs-aptamer complexes were synthesized using a primer / aptamer ratio of 4:1.

[0088] (2) Optimization of rolling circle amplification reaction time:

[0089] The concentration of T4 DNA ligase and phi29 polymerase has a great influence on the rolling circle amplification reaction. Such as Figure...

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Abstract

The invention discloses a rolling circle amplification DNA enzyme and covalent organic framework-based electrochemical-ELISA immunosensor, and belongs to the technical field of electrochemical detection. An electrochemical method and enzyme-linked immunosorbent assay are combined, a nucleic acid signal is amplified on an ELISA plate, an electrode is modified with a functional nanometer material, linear dependence between an electrical signal of a reduction peak and aflatoxin M1 within a range being 0.5-80ng/mL is generated, an association coefficient is 0.993, and the detection limit is 0.15ng/mL (S/N=3). By the rolling circle amplification DNA enzyme and covalent organic framework-based electrochemical-ELISA immunosensor, the electrode passivation in traditional electrochemical analysis can be prevented, an ELISA signal also can be effectively amplified, amplification of a cascading signal is achieved, the detection limit is reduced, trace detection is achieved, and a high-sensitivity, high-selectivity and high-throughput electrochemical ELISA method is built.

Description

technical field [0001] The invention relates to an electrochemical-ELISA immunosensor based on a rolling circle amplification DNA enzyme and a covalent organic framework, and belongs to the technical field of electrochemical detection. Background technique [0002] Enzyme-linked immunosorbent assay (ELISA) relies on the specific recognition of antigens and antibodies, and has the advantages of strong specificity, good stability, and easy operation. gold standard. However, traditional ELISA usually uses biological enzymes (usually horseradish peroxidase or alkaline phosphatase) as a catalyst to catalyze the production of colored substances, and uses a spectrophotometer to read the signal, which limits the sensitivity of detection and cannot meet The need for detection of trace substances. Electrochemical biosensors can detect trace substances due to the high sensitivity of electrochemical methods, the ease of operation, and the combination of nanomaterials and nucleic acid ...

Claims

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Application Information

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IPC IPC(8): G01N27/327C12Q1/6804
CPCC12Q1/6804G01N27/3276G01N27/3277G01N27/3278C12Q2531/125C12Q2521/337
Inventor 庞月红郭露露沈晓芳
Owner JIANGNAN UNIV
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