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CN-122016971-A - PNA-based electrochemical-photothermal dual-mode biosensor and method for detecting SRB DsrC genes

CN122016971ACN 122016971 ACN122016971 ACN 122016971ACN-122016971-A

Abstract

The invention belongs to the field of a biosensor preparation method, and particularly relates to an electrochemical-photothermal dual-mode biosensor based on PNA and a method for detecting SRB DsrC genes. The sensor electrode structure is CHA/PNA/ITO, and PNA probes capture the CHA product triggered by the target gene and incubate MB and ICG as electrochemical and photothermal signaling probes, respectively, for detecting SRB DsrC genes. Compared with a single-mode biosensor, the dual-mode biosensor has the advantages of rapidness, sensitivity, low background signal and strong anti-interference capability of electrochemical sensing and photo-thermal sensing, and improves the detection accuracy through mutual verification of the two modes.

Inventors

  • ZHANG SHIQI
  • ZHAI XIAOFAN
  • JU PENG
  • DUAN JIZHOU
  • HOU BAORONG

Assignees

  • 中国科学院海洋研究所

Dates

Publication Date
20260512
Application Date
20260120

Claims (10)

  1. 1. A PNA-based electrochemical/photothermal dual-mode biosensor is characterized in that the electrode structure of the sensor is CHA/PNA/ITO, a PNA probe captures CHA products initiated by target genes, and MB and ICG are respectively incubated as electrochemical and photothermal signal probes.
  2. 2. The PNA-based electrochemical/photothermal dual-mode biosensor according to claim 1, wherein the PNA probe is immobilized on the surface of the Au/ITO electrode.
  3. 3. A PNA-based electrochemical/photothermal dual-mode biosensor according to claim 1 for detecting SRB DsrC genes.
  4. 4. The method for detecting SRB DsrC genes, which is characterized by the application of the method according to claim 3, wherein the PNA-based electrochemical/photo-thermal dual-mode biosensor is constructed, and the current value and the system temperature change value measured by square wave voltammetry are respectively output as sensor signals to perform electrochemical-photo-thermal dual-mode quantitative analysis of SRB DsrC genes.
  5. 5. The method of claim 4, wherein the PNA-based electrochemical/photothermal dual-mode biosensor electrochemical mode detection method comprises the steps of: PNA-based electrochemical/photothermal dual-mode biosensor electrodes were incubated with MB solution at 20-25 ℃, then the electrodes were washed with PBS buffer to remove excess MB, followed by electrochemical signal detection by square wave voltammetry at a potential ranging from-0.4V to 0.1V.
  6. 6. The method of claim 4, wherein the PNA-based electrochemical/photothermal dual-mode biosensor photo-thermal mode detection method comprises the steps of: The PNA-based electrochemical/photothermal dual-mode biosensor electrode was incubated with ICG solution at 20-25 ℃, then the electrode was washed with PBS buffer to remove excess ICG, then photothermal detection was performed using 808nm near infrared light source (1W), on-time was 300s, and temperature change values were recorded using a temperature sensor.
  7. 7. The method of claim 5 or 6, wherein the concentration of DsrC genes is measured, the square wave voltammetric current value (I) of the sensor is positively correlated with DsrC gene concentration, and the temperature change value (DeltaT) is positively correlated with DsrC gene concentration.
  8. 8. A method of preparing a PNA-based dual electrochemical/photothermal mode biosensor according to claim 1, comprising the steps of: Step 1, preparing an Au/ITO electrode, namely cleaning the ITO electrode, drying the ITO electrode by nitrogen, immersing the ITO electrode into HAuCl 4 and Na 2 SO 4 solution, performing electrodeposition by using a CV method and a current-time curve method, and then drying the ITO electrode; Incubating a target fragment and CHA hairpin probes H1 and H2, and performing target circulation CHA amplification; And 3, incubating the sensor, namely dripping a PNA probe on the surface of the Au/ITO electrode obtained in the step 1, washing the sensor by using PBS buffer solution, closing the electrode by using MCH, alternately washing the sensor by using ethanol and ultrapure water, and incubating the sensor electrode and the CHA product obtained in the step 2 to obtain the CHA/PNA/ITO sensor.
  9. 9. The preparation method according to claim 8, wherein the CV method is carried out for 10 cycles in the potential range of 0 to +1.5V at a sweeping speed of 100 mV.s -1 and the current-time curve method is carried out for 300s at a constant potential of-0.2V.
  10. 10. The method of claim 8, wherein the probe (SH-PNA) of step 3 PNA is thiol-modified and treated with TCEP at 20-25℃for 1 hour, and the concentration of PNA probe is 0.5-2. Mu.M.

Description

PNA-based electrochemical-photothermal dual-mode biosensor and method for detecting SRB DsrC genes Technical Field The invention belongs to the field of a biosensor preparation method, and particularly relates to an electrochemical-photothermal dual-mode biosensor based on PNA and a method for detecting SRB DsrC genes. Background The marine industry and marine equipment have become the pillar industry indispensable for economic development. However, the marine environment is an extremely harsh corrosion environment for metals and other materials used in marine engineering, and marine organisms and their metabolites in the marine environment also synergistically affect the corrosion process of the materials. The problem of corrosion of metallic materials caused by the presence or growth metabolism of microorganisms, i.e. microbial corrosion, can account for 70% -80% of the damage of marine materials, is one of the key threats of the marine and offshore industry sectors. Marine corrosion microorganisms are of a wide variety, with sulfate-reducing bacteria (SRB) often found in metal corrosion of marine engineering equipment, being one of the most widely studied and most corrosive microorganisms in microbial corrosion. Development of detection methods and techniques for SRB is very necessary for monitoring population concentration of SRB in an environment in time, and performing corrosion mechanism research, microbial corrosion determination, sterilization effect detection and related research and exploration. The common SRB detection method mainly comprises a maximum likelihood (MPN) method, a polymerase chain reaction, a fluorescence method, an immunoassay method and the like, and has the problems of complex operation, long time consumption, low sensitivity, poor environmental suitability and the like. With the rapid development of fields such as nanotechnology and biosensing, the biosensor technology utilizes the metabolic substances, cell structures and characteristic genetic substances of SRB as identification objects, so that the detection efficiency and accuracy are effectively improved, but the current biosensing strategy is mostly limited to a single target or detection mode, and is difficult to meet the multiple verification and anti-interference requirements in a complex actual environment. Thus, there is an urgent need to develop more sensitive and specific SRB detection methods. Disclosure of Invention Aiming at the necessity of SRB detection and the limitation of the traditional detection method, the invention aims to provide an electrochemical-photo-thermal dual-mode biosensor based on PNA and a method for detecting SRB DsrC genes, which realize high-sensitivity and high-specificity detection of SRB DsrC genes. In order to achieve the above purpose, the invention adopts the following technical scheme: a PNA-based electrochemical/photothermal dual-mode biosensor has a sensor electrode structure of CHA/PNA/ITO, and PNA probes immobilized on the surface of an Au/ITO electrode capture CHA products initiated by target genes and incubate MB and ICG as electrochemical and photothermal signaling probes, respectively. PNA probes were immobilized on the Au/ITO electrode surface. One such PNA-based electrochemical/photothermal dual-mode biosensor is used to detect SRB DsrC genes. The SRB DsrC gene method is that the PNA-based electrochemical/photo-thermal dual-mode biosensor is constructed, and the current value and the system temperature change value which are measured by square wave voltammetry are respectively used as sensor signals to output, so as to carry out electrochemical-photo-thermal dual-mode quantitative analysis on SRB DsrC genes. The method mainly comprises the following detection steps: Electrochemical mode detection the sensor electrode was incubated with MB solution at 20-25℃for 1-1.5 hours, then the electrode was rinsed with PBS buffer to remove excess MB, followed by electrochemical signal detection by square wave voltammetry at a potential ranging from-0.4V to 0.1V. Photo-thermal mode detection, namely incubating a sensor electrode with ICG solution for 1-1.5 hours at 20-25 ℃, then washing the electrode with PBS buffer solution to remove redundant ICG, then carrying out photo-thermal detection by using a 808nm near infrared light source (1W), and recording the temperature change value of a system by using a temperature sensor, wherein the photo-thermal detection is carried out for 280-320 seconds. The DsrC genes with different concentrations are measured, the square wave volt-ampere current value (I) of the sensor is positively correlated with the DsrC gene concentration, and the temperature change value (delta T) is positively correlated with the DsrC gene concentration. The preparation method of the PNA-based electrochemical/photo-thermal dual-mode biosensor comprises the following steps: Step 1, preparing an Au/ITO electrode, namely cleaning the ITO electrode in an ultrasonic bath fo