Copper nanoparticle synthetic method using rolling circle amplification product as template and application thereof in electrochemical detection

A copper nanoparticle, rolling circle amplification reaction technology, applied in the field of biomedicine, can solve problems such as hindering long-term monitoring applications

Active Publication Date: 2016-05-11
SHANDONG UNIV
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However, the fluorescence emission of a single CuNP can only last for 20 minutes, and then t...

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  • Copper nanoparticle synthetic method using rolling circle amplification product as template and application thereof in electrochemical detection
  • Copper nanoparticle synthetic method using rolling circle amplification product as template and application thereof in electrochemical detection
  • Copper nanoparticle synthetic method using rolling circle amplification product as template and application thereof in electrochemical detection

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Embodiment 1

[0042] 1. Experimental part

[0043] 1.1. Reagents and materials

[0044] Human prostate-specific antigen (PSA), monoclonal antibody anti-PSA (anti-PSA) extracted from mice, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) were purchased from Shanghai Lingchao Bioscience Co., Ltd. (China ). 5'-thiol-modified PSA-specific DNA aptamer (5'-SH-TTATTATTAAATTAAAGCTCGCCATCAAATAGCTTT-3'), 5'-thiol-modified primer (5'-SH-TTATTATTATGTCCGTGCTAGAAGGAAACAGTTAC-3'), circular template (5' -p-TAGCACGGACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGTAACTGTTTCCTTC-3'), bovine serum albumin (BSA), thrombin and human immunoglobulin G (H-IgG) were obtained from Shanghai Sangon Biotechnology Co., Ltd. (China). Phi29 DNA polymerase, T4 DNA ligase and dNTPs were purchased from Thermol Fermentas (Lithuania). Human serum samples were collected from Qilu Hospital of Shandong University. Prepare PBS with 0.01M disodium hydrogen phosphate, 0.01M disodium hydrogen phosphate and 0.9% sodium chlor...

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Abstract

The invention provides a copper nanoparticle synthetic method using a rolling circle amplification product as a template and application thereof in electrochemical detection. A prostate specific antigen (PSA) is used as a model target molecule. The establishment of a primer-nanogold-aptamer/PSA/PSA antibody sandwich structure initiates a rolling circle amplification reaction, thus generating tens of thousands of poly-thymine repetitive sequences that serve as a template for synthesis of copper nanoparticles; the formed copper nanoparticles are dissolved further using nitric acid, copper ions obtained by dissolution are detected by using an electrochemical method, and finally target PSA content is determined. Benefited from a designed signal-level cascade amplifying strategy, a linear range of the PSA detected by the invention is between 0.05 fg/mL and 500 fg/mL, and a detection limit is 0.020+/-0.001 fg/mL PSA. Finally, the practicality of the method is verified by detecting the PSA content in a clinical serum sample. With the supersensitivity, specificity and ability to detect an actual sample, the method has great potential in disease diagnosis application.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for synthesizing copper nanoparticles using a rolling circle amplification product as a template and its application in electrochemical detection. Background technique [0002] The detection of disease biomarkers based on the specific interaction between target molecules and biorecognition molecules is of great significance for clinical diagnosis. The conversion of this biological binding into a measurable signal is typically achieved optically and electrochemically. Among them, the optical method has high sensitivity. However, this signal conversion method not only requires high-precision and expensive instruments, but also involves complex numerical algorithms to analyze the data, thus limiting the wide application of this method. Electrochemical devices can also convert biological reactions into detectable electrical signals, and provide a simple, accurate and cheap platfo...

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q2531/125C12Q2525/205C12Q2563/137
Inventor 朱烨王慧娟朱静
Owner SHANDONG UNIV
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