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CN-118169198-B - Preparation method and application of photoelectrochemistry-surface-enhanced Raman dual-mode integrated sensor

CN118169198BCN 118169198 BCN118169198 BCN 118169198BCN-118169198-B

Abstract

The invention belongs to the technical field of biosensing detection, and particularly relates to a preparation method and application of a photoelectrochemistry-surface enhanced Raman dual-mode integrated sensor. The invention takes Au@Ag NPs/H-WO 3 as a substrate material, utilizes a difunctional probe molecule MB with photoelectrochemistry and Raman sensitivity, can enhance photoelectric and Raman signals of MB at the same time under low-power 532nm Raman excitation light, constructs a dual-mode integrated platform, is further applied to the proposed photoelectrochemistry-surface enhanced Raman biosensing measurement, and is forced to separate from a double-chain after incubation with MC-LR by the specific combination of an aptamer and the MC-LR, the MB is separated from an electrode interface, and the photocurrent and SERS intensity are changed, so that MC-LR detection is realized.

Inventors

  • LIU DONG
  • LI YUQING
  • LIU SHUDA
  • MENG SHUYUN

Assignees

  • 江苏大学

Dates

Publication Date
20260512
Application Date
20240318

Claims (10)

  1. 1. The preparation method of the photoelectrochemistry-surface enhanced Raman dual-mode integrated sensor is characterized by comprising the following steps of: (1) Preparation of a base material: s1, H-WO 3 preparation: dissolving cetyl trimethyl ammonium bromide in ultrapure water, adding calcium chloride, continuously stirring, adding sodium tungstate solution under the condition of continuous stirring, performing ultrasonic treatment at a certain temperature, centrifuging, washing with ethanol, drying, and marking as CaWO 4 ; Dissolving CaWO 4 in a nitric acid solution and stirring, fully reacting the two to obtain a yellow precipitate, and collecting the precipitate, washing the precipitate with ethanol, drying and calcining the precipitate to obtain the product H-WO 3 ; S2, preparation of Au@Ag NPs: Firstly, adding chloroauric acid solution into H 2 O, placing the solution in an oil bath, adding trisodium citrate solution after the temperature is raised to a certain temperature, and obtaining gold nanoparticle solution after reaction; (2) Adding TCEP solution into the cDNA solution to obtain activating solution, adding aptamer solution into the activating solution, vibrating, raising to a certain temperature, maintaining for a period of time, lowering to a certain temperature, maintaining for a certain time to obtain a final product, and marking as the Apt-cDNA double-stranded structure; (3) Boiling the indium tin oxide electrode in sodium hydroxide solution, taking out the indium tin oxide electrode, sequentially ultrasonically cleaning the indium tin oxide electrode in absolute ethyl alcohol and ultrapure water, and drying to obtain a pretreated indium tin oxide electrode; (4) Adding H-WO 3 prepared in the step (1) into absolute ethyl alcohol to obtain H-WO 3 solution, modifying the H-WO 3 solution on the surface of the indium tin oxide electrode pretreated in the step (3), and drying at room temperature, wherein the product is marked as H-WO 3 /ITO; (5) Modifying the Au@Ag NPs prepared in the S2 in the step (1) to the electrode interface of H-WO 3 /ITO in the step (4), and drying at room temperature, wherein the dried product is marked as Au@Ag NPs/H-WO 3 /ITO; (6) Modifying the double-stranded structure of the Apt-cDNA prepared in the step (2) to an electrode interface of Au@Ag NPs/H-WO 3 /ITO in the step (5), incubating at room temperature, leaching with Tris-HCl after incubation, and marking the leached product as Apt-cDNA/Au@Ag NPs/H-WO 3 /ITO; (7) Modifying Methylcyclohexane (MCH) solution to the electrode interface of Apt-cDNA/Au@Ag NPs/H-WO 3 /ITO in the step (6), incubating at room temperature, leaching by using H 2 O after incubation, and marking the leached product as MCH/Apt-cDNA/Au@Ag NPs/H-WO 3 /ITO; (8) And (3) modifying a Methylene Blue (MB) solution to an electrode interface of MCH/Apt-cDNA/Au@Ag NPs/H-WO 3 /ITO in the step (7), incubating at room temperature, and eluting by using H 2 O after incubation, wherein the eluted product is the photoelectrochemistry-surface enhanced Raman dual-mode integrated sensor, and is marked as MB/MCH/Apt-cDNA/Au@Ag NPs/H-WO 3 /ITO.
  2. 2. The method for preparing the photoelectrochemistry-surface enhanced Raman dual-mode integrated sensor according to claim 1, wherein in the S1 of the step (1), the dosage relationship of the cetyltrimethylammonium bromide, the ultrapure water, the calcium chloride and the sodium tungstate solution is 1g to 50mL to 0.1665g to 5mL, the stirring speed is 460rmp, the Na 2 WO 4 concentration is 0.3M, the certain temperature is 34-42 ℃, the ultrasonic time is 1h, the centrifugation speed is 10000rmp, the centrifugation time is 10min, the drying temperature is 60 ℃, the concentration of the CaWO 4 and the HNO 3 is 4M, the stirring time is 18h, the drying temperature is 60 ℃, and the calcining operation is that a vacuum tube furnace is utilized to heat up to 400 ℃ at a heating rate of 2- -1 ℃ and the heating time is 1h at 400 ℃; In S2 of the step (1), the dosage relationship of H 2 O, chloroauric acid solution and trisodium citrate solution is 25mL:0.2mL:0.25mL, wherein the concentration of chloroauric acid solution is 0.1M, the concentration of Na 3 C 6 H 5 O 7 is 100mg/mL, the temperature is raised to 150 ℃, the reaction time is 15min, the dosage relationship of gold nanoparticle solution, agNO 3 solution, na 3 C 6 H 5 O 7 solution and AA solution is 10mL:1.6mL:0.4mL:0.1mL, the concentration of AgNO 3 solution is 10mM, the concentration of Na 3 C 6 H 5 O 7 solution is 38.8mM, the concentration of AA solution is 0.1M, the stirring speed is 500rmp, and the continuous stirring time is 20min.
  3. 3. The method for preparing the photoelectrochemistry-surface enhanced Raman dual-mode integrated sensor according to claim 1, wherein in the step (2), the volume ratio of the cDNA solution to the TCEP solution is 10:1, wherein the concentration of the cDNA solution is 3 mu M, the concentration of the TCEP solution is 3mM, the temperature of the activation reaction is room temperature for 1h, the temperature is raised to a certain temperature of 95 ℃, the reaction time is 3min, the temperature is lowered to a certain temperature of 25 ℃, and the retention time is 3min; Wherein the aptamer is MC-LR aptamer with the following sequence of 5'-HS-SH-GGC CGG AAA CAG GAC CAC CAT GAC AAT TAC CCA TAC CAC CTC ATT ATG CCC CAT CTC CGC-3'; the cDNA sequence is 5'-GCG GAG ATG GGG CAT AAT GAG GTG GTA-3'.
  4. 4. The method for preparing the photoelectrochemistry-surface enhanced Raman dual-mode integrated sensor according to claim 1, wherein in the step (3), the diameter of the indium tin oxide electrode is 6mm, the concentration of NaOH solution is 0.3M, the boiling time is 30min, the ultrasonic time is 15min, and the drying temperature is 37 ℃.
  5. 5. The preparation method of the photoelectrochemistry-surface enhanced Raman dual-mode integrated sensor, which is disclosed in claim 1, is characterized in that in the step (4), the concentration of H-WO 3 solution is 2mg/mL, the surface of an indium tin oxide electrode after pretreatment is modified for 2 times by the H-WO 3 solution, the modification dosage is 6 mu L, and the dosage of Au@Ag NPs in the step (5) is 20 mu L.
  6. 6. The method for preparing the photoelectrochemistry-surface enhanced Raman dual-mode integrated sensor according to claim 1, wherein the dosage of the Apt-cDNA double-stranded structure in the step (6) is 20 mu L, the incubation time is 12h, the incubation temperature is 4 ℃, the composition of Tris-HCl buffer solution is 10mM Tris,120mM NaCl,1mM KCl, and the pH value is regulated to be 7.4 by HCl.
  7. 7. The preparation method of the photoelectrochemistry-surface enhanced Raman dual-mode integrated sensor, which is disclosed in claim 1, is characterized in that the concentration of MCH solution in the step (7) is 1mM, the modification amount is 20 mu L, the incubation time is 1h, the MB concentration in the step (8) is 30 mu M, the MB amount is 20 mu L, and the incubation time is 30min.
  8. 8. Use of a photoelectrochemical-surface enhanced raman dual mode integrated sensor prepared according to any of claims 1-7 for detecting MC-LR, characterized by the steps of: (1) Preparing MC-LR standard solutions with different concentrations, respectively modifying the interface of the photoelectrochemistry-surface enhanced Raman dual-mode integrated sensor, and incubating for a period of time at room temperature, and then leaching the product by using H 2 O, wherein the leached product is marked as MC-LR/MB/MCH/Apt-cDNA/Au@Ag NPs/H-WO 3 /ITO; (2) The MC-LR/MB/MCH/Apt-cDNA/Au@Ag NPs/H-WO 3 /ITO obtained in the step (1) is used as a working electrode, an Ag/AgCl electrode is used as a reference electrode, a platinum wire is used as a counter electrode, and the working electrode is placed in electrolyte to carry out photoelectrochemistry and simultaneous detection of surface enhanced Raman; (3) And (3) detecting MC-LR in the actual sample, namely firstly obtaining a sample liquid, and then operating according to the methods of the steps (1) and (2), wherein the difference is that the MC-LR standard solution in the step (1) is replaced by the sample liquid, and finally, photocurrent and Raman intensity are obtained through photoelectrochemistry and surface-enhanced Raman detection, and are correspondingly substituted into the standard curve constructed in the step (2), so that the concentration of MC-LR in the sample can be obtained, and the purpose of MC-LR detection in the unknown sample is realized.
  9. 9. The method according to claim 8, wherein in the step (1), the concentration of the MC-LR standard solution is 0.3-100ng/mL, the dosage of the MC-LR standard solution is 20 mu L, the incubation period is 40min, in the step (2), the electrolyte consists of 0.1M NaH 2 PO 4 、0.1M Na 2 HPO 4 and 0.1M AA, the detection of photoelectrochemistry and surface enhanced Raman is that an electrochemical workstation of Prlington VERSASTAT F is used for recording photoelectrochemical signals, an ATP5020 system is used for recording Raman signals, and the external bias voltage is +0.1V.
  10. 10. The method according to claim 8, wherein in the step (3), the sample solution is prepared by filtering with a 500-mesh stainless steel sieve and then filtering with a 0.22 μm filter membrane.

Description

Preparation method and application of photoelectrochemistry-surface-enhanced Raman dual-mode integrated sensor Technical Field The invention belongs to the technical field of biosensing detection, and particularly relates to a preparation method and application of a photoelectrochemistry-surface enhanced Raman dual-mode integrated sensor. Background Microcystin LR (MC-LR) is one of the toxins released by cyanobacteria cells during the lysis process, commonly found in eutrophic lakes. When water bloom breaks out, a large amount of MC-LR accumulates in water, which can cause water pollution and adjacent soil damage, seriously affect the balance of an ecological system, further cause the loss of aquaculture and agricultural production, bring greater pressure to environmental protection and even threaten the health and safety of human beings. Therefore, in order to finish the aim of blue algae bloom source early warning, the detection of MC-LR in water is very important. The detection method of MC-LR developed at present is mostly single-side detection. Unilateral detection shows limited anti-jamming capability and high risk of false positives. Especially when detecting water samples with complex matrices, more accurate biosensing strategies need to be explored. Coupling multiple detection methods is one of effective ways for improving accurate sensing, mutual verification among multiple signals can be realized, accurate detection of MC-LR in complex samples can be realized, and false positive signals are avoided. The existing developed dual-mode sensor needs to be measured respectively to obtain two signals, is complex and tedious in operation and long in time consumption, and a dual-signal detection method capable of being obtained simultaneously is needed, so that the accuracy is improved, and meanwhile, the detection efficiency is improved. Disclosure of Invention Aiming at the defects of the prior art, the invention aims to couple photoelectrochemistry and a surface-enhanced Raman sensing technology and construct a photoelectrochemistry-surface-enhanced Raman (PEC-SERS) sensor for detecting MC-LR based on silver-coated gold nano particles/hollow tungsten trioxide (Au@Ag NPs/H-WO 3). The Au@Ag NPs/H-WO 3 is used as a substrate material, and the substrate material can simultaneously enhance photoelectric and Raman signals of MB under low-power 532nm Raman excitation light by utilizing a difunctional probe molecule Methylene Blue (MB) with photoelectrochemistry and Raman sensitivity, so that a dual-mode integrated platform is constructed, and the dual-mode integrated platform is further applied to the proposed photoelectrochemistry-surface enhanced Raman biosensing measurement. After incubation with MC-LR, the aptamer (Apt) specifically binds to MC-LR, is forced to separate from the double strand, MB breaks away from the electrode interface, and the photocurrent and SERS intensity change, thus realizing detection of MC-LR. In order to achieve the technical purpose, the invention adopts the following steps: a preparation method of a photoelectrochemistry-surface enhanced Raman dual-mode integrated sensor comprises the following steps: (1) Preparation of a base material: s1, H-WO 3 preparation: Dissolving Cetyl Trimethyl Ammonium Bromide (CTAB) in ultrapure water, stirring, adding calcium chloride (CaCl 2), continuously stirring, adding sodium tungstate (Na 2WO4) solution under the condition of continuous stirring, performing ultrasonic treatment at a certain temperature, centrifuging, washing with ethanol, drying, and marking as CaWO 4; dissolving CaWO 4 in nitric acid (HNO 3) solution, stirring, fully reacting to obtain yellow precipitate, collecting the precipitate, washing with ethanol, drying, and calcining to obtain H-WO 3; S2, preparation of Au@Ag NPs: Firstly, adding chloroauric acid solution into H 2 O, placing in an oil bath, heating to a certain temperature, adding trisodium citrate (Na 3C6H5O7) solution, reacting to obtain gold nanoparticle (Au NPs) solution, then dropwise adding silver nitrate solution (AgNO 3)、Na3C6H5O7 solution and Ascorbic Acid (AA) solution) into the Au NPs solution under stirring condition, and continuously stirring to obtain the Au@Ag NPs product; (2) Adding TCEP solution into the cDNA solution to perform activation reaction to obtain activation solution, adding the Apt solution into the activation solution, vibrating and raising the temperature to a certain temperature to keep the temperature for a certain period of time, then lowering the temperature to a certain temperature to keep the temperature for a certain period of time, and obtaining a final product, namely the Apt-cDNA double-stranded structure; (3) Boiling an Indium Tin Oxide (ITO) electrode in a sodium hydroxide (NaOH) solution, taking out the ITO electrode, sequentially ultrasonically cleaning the ITO electrode in absolute ethyl alcohol and ultrapure water, and drying to obtain a pretreated ITO electrode; (4) H-