Dual-mode separation type immunosensor based on enzyme-induced biological etching and preparation method thereof

An immune sensor and separate technology, applied in the field of immune sensors, can solve the problems of inaccurate detection results, long operation time, low sensitivity, etc., achieve good commercial application prospects, improve detection sensitivity, and accurately and reliably read out the results.

Active Publication Date: 2019-07-12
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These detection methods are often limited in practical applications due to problems such as inaccurate de

Method used

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  • Dual-mode separation type immunosensor based on enzyme-induced biological etching and preparation method thereof
  • Dual-mode separation type immunosensor based on enzyme-induced biological etching and preparation method thereof
  • Dual-mode separation type immunosensor based on enzyme-induced biological etching and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] A preparation method based on enzyme-induced bioetching dual-mode separation immunosensor, comprising the following steps:

[0036] S1. Preparation of CdS / ZnO NRs / r-GO: first synthesize graphene oxide hydrogel (GO), and prepare cadmium sulfide / zinc oxide nanorod array CdS / ZnO NRs on the reduced graphene oxide r-GO to obtain CdS / ZnO NRs / r-GO composites;

[0037] Preparation of HRP-liposome-Ab 2 Complex: preparation of liposome-encapsulated horseradish peroxidase HRP by thin-film dispersion method,

[0038] The HRP-liposome complex was obtained, and the HRP-liposome complex was combined with the second antibody Ab by glutaraldehyde cross-linking method 2 connect,

[0039] HRP-Liposome-Ab 2 Complex;

[0040] Preparation of gold nanobicones: Au NBPs prepared by seed growth method;

[0041] The specific operation for preparing CdS / ZnO NRs / r-GO is as follows: add 160mL concentrated sulfuric acid into a dry three-necked flask, slowly add 4g graphite powder and 14g potas...

Embodiment 2

[0050] Example 2 Characterization of CdS / ZnO NRs / r-GO composites

[0051] Synthesis of graphene oxide hydrogel (GO): Add 160mL concentrated sulfuric acid into a dry three-necked flask, slowly add 4g graphite powder and 14g potassium permanganate under stirring in an ice-water bath, and keep stirring the mixture at 35°C for 24h . After the reaction was completed, dilute with 400 mL of ice water, and then add hydrogen peroxide until the color of the mixture did not change and no bubbles were generated. Stirring was continued for 2 h to remove unreacted potassium permanganate, and then centrifuged at 5000 rpm for 3 minutes. Centrifuge and wash with 300mL HCl (1mol / L) three times and then wash with water until the supernatant is neutral. The precipitate after centrifugation was dialyzed for one week, and the product was divided into two equal parts, which were centrifuged and washed with water and ethanol respectively, and finally dispersed in water or ethanol and stored for late...

Embodiment 3

[0054] Example 3 HRP-liposome-Ab 2 Characterization of the complex

[0055] The liposome-encapsulated horseradish peroxidase (HRP) complex is prepared by a film dispersion method, and the process is briefly described as follows: Weigh dipalmitoylphosphatidylcholine (DPPC), cholesterol and dipalmitoylphosphatidylethanolamine (DPPE ) in a total of 30 mg (molar ratio 10:10:1), mixed and dissolved in 4 mL of chloroform / methanol mixture (volume ratio 6:1), and then transferred to a round-bottomed flask by rotary evaporation at 45 ° C to form a uniform layer Lipid film, then add 2 mL of phosphate buffer solution (PBS, 0.01 M, pH 7.4) containing 2.5 mg / mL HRP, and incubate at 35 °C for 2 h. The mixture was then sonicated for 5 min in an ice-water bath, repeating three cycles. Finally, the prepared HRP-liposome complex was centrifuged to remove unencapsulated HRP.

[0056] The complexes prepared above were combined with the second antibody (Ab) by glutaraldehyde cross-linking metho...

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Abstract

The invention discloses a dual-mode separation type immunosensor based on enzyme-induced biological etching. A cadmium sulfide/zinc oxide nanorod array CdS/ZnO NRs covers/is prepared at the surface ofa three-dimensional reduced graphene oxide r-GO as a photoelectrode; gold nanoparticle dual-cone Au NBPs are adopted as a multicolor chromogenic substrate; horse radish peroxidase (HRP) is used to connect with photo-electrochemical immunoassay and colorimetric detection, wherein Cd/ZnO NRs/r-GO generates biological etching through the enzymic catalytic reaction induced by HRP so as to form lightcurrent change, and hydroxyl radical generated by catalytic oxidation of the hydrogen peroxide by the HRP is used for biological etching of Au NBPs to form gold nanoparticles with different sizes andshapes so as to display the color change and the blue shift of an LSPR peak. The method adopts a lipidosome to package a lot of HRPs and load more Ab2 to effectively amplify response signals so as tofurther improve the accuracy of the detection.

Description

technical field [0001] The invention belongs to the technical field of immunosensors, and more specifically relates to an enzyme-induced bioetching-based dual-mode separation immunosensor and a preparation method thereof. Background technique [0002] Photoelectrochemical (PEC) detection, as an emerging and rapidly developing detection technology, has attracted widespread attention. Compared with the traditional ELISA, PEC detection uses light as the excitation light source and photocurrent as the detection signal. Thanks to two different forms of energy conversion, the PEC immunoassay has a lower detection limit and higher sensitivity. In addition, relatively simple and low-cost devices are more conducive to the development of portable and miniaturized instruments. However, even though PEC detection has many of the advantages mentioned above, the traditional single-signal PEC immunoassay platform has been gradually difficult to meet the growing detection needs of people. ...

Claims

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Application Information

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IPC IPC(8): G01N27/327G01N21/78G01N27/30G01N33/543
CPCG01N21/78G01N27/305G01N27/3278G01N33/5432
Inventor 刘英菊刘莹魏婕申浩然陈华明
Owner SOUTH CHINA AGRI UNIV
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