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Electrochemical biosensing composition, working solution, electrochemical biosensor and application thereof

A biosensor and biosensing technology applied in the field of electrochemical biosensing composition to achieve high sensitivity and high specificity

Pending Publication Date: 2022-04-29
ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Guided by crRNA, Cas12 can recognize target dsDNA and initiate trans-cleavage activity, but it requires a protospacer-adjacent motif (PAM) sequence adjacent to the target dsDNA, which may limit its broader HCR application

Method used

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  • Electrochemical biosensing composition, working solution, electrochemical biosensor and application thereof
  • Electrochemical biosensing composition, working solution, electrochemical biosensor and application thereof
  • Electrochemical biosensing composition, working solution, electrochemical biosensor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] The preparation of embodiment 1 biosensor

[0051] Preparation of MB probe-modified biosensing electrodes

[0052] Dip the bare gold electrode (GE) into the piranha solution (H 2 SO 4 / H 2 o 2 = 3:1) for 20 minutes to remove impurities, then scrub with absorbent cotton balls soaked in deionized water. The electrode was polished with 0.05 μM alumina powder until the surface was smooth, and then scrubbed with absorbent cotton balls. Thiolated MB probes were dissolved in 1 mM phosphate buffered saline (PBS) solution (containing 14 mM NaCl, 0.3 mM KCl and 10 mM TCEP, pH 7.4). Then, 10 μL of 0.3 μM MB probe was added to the pretreated electrode surface and incubated overnight at room temperature. In addition, 10 μL of MCH (1 μM) was dropped onto the GE surface and incubated at 37° C. for 1 hour. Finally, ultrapure water was used to remove unbound material on the electrode surface for electrochemical measurements and subsequent experiments.

[0053] Amplification of s...

Embodiment 2

[0066] Embodiment 2 Actual sample determination

[0067] Different Salmonella typhimurium densities (10 4 、10 5 and 10 6 CFU / mL) was added to sterilized milk samples for recovery analysis. HEPES buffer was used as a negative control.

[0068] Characterization of Effect Example 1 HCR Formation

[0069] The reaction efficiency of HCR is closely related to the sensitivity of the assay. Therefore, a PAGE assay was performed to verify the formation of the HCR product ( figure 2 ). The concentration of the initiator is related to the average molecular weight of the polymer produced by HCR. Too much trigger (Trigger) may cause hybridization between H1 and H2, which cannot form a long enough DNA polymer chain. Therefore, different concentrations of triggers are used to optimize the best performance of the HCR system. like figure 2 As shown, when the Trigger concentration is low, there is almost no high-band polymer in row 1. This finding suggests that HCR does not occur d...

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Abstract

The invention relates to the field of electrochemical biosensors, in particular to a CRISPR (clustered regularly interspaced short palindromic repeats) system of Cpf1 (Cpf1) from bacteria of the hispiraceae, and the electrochemical biosensor for detecting pathogenic bacteria salmonella typhimurium is developed by combining the CRISPR system with a hybrid chain reaction. The autonomous cross opening of the functional DNA hairpin structure of the HCR produces a double-stranded DNA line of a polymer consisting of a plurality of single-stranded DNA, and the plurality of functional single-stranded DNA initiate the trans-cleavage activity of CRISPR-Cas12a, so that the DNA hairpin ring of the MB modified on the surface of the gold electrode is selectively cleaved. The process will cause a change in electron transfer of the electrochemical tag. And the polymer double-stranded DNA of the HCR is fixed on the DBs through a salmonella typhimurium aptamer. The established method can selectively and sensitively quantify salmonella typhimurium in a sample, and the detection limit is 20 CFU / mL.

Description

technical field [0001] The invention relates to the field of electrochemical biosensors, in particular to electrochemical biosensing compositions, working fluids, electrochemical biosensors and applications thereof. Background technique [0002] Food safety problems caused by foodborne pathogens have attracted more and more attention worldwide. Among them, Salmonella typhimurium (Salmonella typhimurium) is one of the main food-borne pathogens, mainly threatening people's health by eating contaminated food. The existing gold standard methods for detection of Salmonella typhimurium mainly include culture method, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) and so on. However, these methods suffer from a number of disadvantages. For example, culturing is time-consuming, typically requiring 2-3 days. PCR often requires complex DNA extraction procedures. ELISA lacks sufficient sensitivity. And these methods require highly trained technicians and...

Claims

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Application Information

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IPC IPC(8): G01N27/26G01N27/30G01N27/327
CPCG01N27/26G01N27/30G01N27/327
Inventor 万家余卜胜君刘文森李忠义郝镯
Owner ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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