Preparation method and application of photoelectrochemical-surface enhanced raman dual-mode integrated sensor

Abstract

The application belongs to the technical field of biosensing detection, and particularly relates to a preparation method of a photoelectrochemical-surface enhanced Raman dual-mode integrated sensor and application thereof. The Au@Ag NPs / H-WO3 is used as a base material, and a dual-function probe molecule MB sensitive to photoelectrochemistry and Raman is used. Under low-power 532nm Raman excitation light, the base material can simultaneously enhance the photoelectric and Raman signals of the MB, a dual-mode integrated platform is constructed, and the proposed photoelectrochemical-surface enhanced Raman biosensing detection is further applied. After incubation with the MC-LR, the specific binding of the aptamer and the MC-LR is forced to separate from the double-stranded, the MB is separated from the electrode interface, the photocurrent and the SERS intensity change, and thus the detection of the MC-LR is realized. In addition, the sensor has high sensitivity, good selectivity and good stability, and the detection linear range is 0.3-100ng / mL.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biosensing detection, and particularly relates to a preparation method of a photoelectrochemical-surface enhanced Raman dual-mode integrated sensor and application thereof. BACKGROUND

[0002] Microcystin-LR (MC-LR) is one of the algal toxins released by cyanobacterial cells during lysis, which usually exists in eutrophic lakes. During the outbreak of water bloom, the large accumulation of MC-LR in water will cause water pollution and damage to the adjacent soil, seriously affecting the balance of the ecological system; further, it will cause losses to aquaculture and agricultural production, bring great pressure to environmental protection, and even threaten human health and safety. Therefore, in order to achieve the goal of early warning of cyanobacterial bloom source, it is of great significance to detect MC-LR in water.

[0003] At present, the detection methods of MC-LR developed are mostly single-method detection. Single-method detection shows limited anti-interference ability and high risk of false positive. Especially when detecting complex water samples, it is necessary to explore more accurate biosensing strategies. Coupling multiple detection methods is one of the effective ways to improve accurate sensing. The mutual verification between multiple signals can realize accurate detection of MC-LR in complex samples and avoid the generation of false positive signals. The dual-mode sensors developed at present need to be measured respectively to obtain two signals, which is complicated and time-consuming, and a dual-signal detection method that can be obtained simultaneously is urgently needed to improve the accuracy and efficiency. SUMMARY

[0004] In view of the deficiencies of the prior art, the application aims to couple photoelectrochemistry and surface enhanced Raman sensing technology to construct a photoelectrochemical-surface enhanced Raman (PEC-SERS) sensor for detecting MC-LR based on silver-coated gold nanoparticles / hollow tungsten trioxide (Au@Ag NPs / H-WO3). The Au@Ag NPs / H-WO3 is used as a substrate material, and a bifunctional probe molecule methylene blue (MB) which is sensitive to photoelectrochemistry and Raman is used. Under low-power 532nm Raman excitation light, the substrate material can simultaneously enhance the photoelectric and Raman signals of MB, construct a dual-mode integrated platform, and further apply it to the proposed photoelectrochemical-surface enhanced Raman biosensing determination. After incubation with MC-LR, the specific binding of aptamer (Apt) and MC-LR is forced to separate from the double-stranded, MB is separated from the electrode interface, and the photocurrent and SERS intensity change, so as to realize the detection of MC-LR.

[0005] In order to achieve the above technical purposes, the application adopts the following steps:

[0006] A method for fabricating an integrated photoelectrochemical-surface-enhanced Raman dual-mode sensor includes the following steps:

[0007] (1) Preparation of substrate material:

[0008] Preparation of S1 and H-WO3:

[0009] Hexadecyltrimethylammonium bromide (CTAB) was dissolved in ultrapure water and stirred. Calcium chloride (CaCl2) was added and stirring continued. While stirring continuously, sodium tungstate (Na2WO4) solution was added. The mixture was then sonicated at a certain temperature, centrifuged, washed with ethanol, and dried. This solution was labeled CaWO4.

[0010] CaWO4 was dissolved in nitric acid (HNO3) solution and stirred. After the two reacted completely, a yellow precipitate was obtained. The precipitate was collected, washed with ethanol, dried, and calcined to obtain the product H-WO3.

[0011] Preparation of S2 and Au@Ag NPs:

[0012] First, chloroauric acid solution was added to H2O and placed in an oil bath. After reaching a certain temperature, trisodium citrate (Na3C6H5O7) solution was added. After the reaction, gold nanoparticles (Au NPs) solution was obtained. Then, under stirring, silver nitrate solution (AgNO3), Na3C6H5O7 solution and ascorbic acid (AA) solution were added dropwise to the Au NPs solution in sequence, and the mixture was stirred continuously to obtain the product Au@Ag NPs.

[0013] (2) Preparation of Apt-cDNA double-stranded structure: TCEP solution is added to cDNA solution for activation reaction to obtain activation solution; then Apt solution is added to activation solution, shaken and raised to a certain temperature and held for a period of time, then lowered to a certain temperature and held for a period of time to obtain the final product, which is denoted as Apt-cDNA double-stranded structure.

[0014] (3) After boiling the indium tin oxide (ITO) electrode in sodium hydroxide (NaOH) solution, the ITO electrode was taken out and ultrasonically cleaned in anhydrous ethanol and ultrapure water in sequence, and then dried to obtain the pretreated ITO electrode.

[0015] (4) Dissolve the H-WO3 prepared in S1 of step (1) in anhydrous ethanol to obtain H-WO3 solution; modify the surface of the ITO electrode after the pretreatment in step (3) with H-WO3 solution and dry it at room temperature. At this time, the product is labeled as H-WO3 / ITO.

[0016] (5) Modify the Au@Ag NPs prepared in S2 of step (1) onto the electrode interface of H-WO3 / ITO in step (4) and dry it at room temperature. At this time, the product is marked as Au@Ag NPs / H-WO3 / ITO.

[0017] (6) Modify the Apt-cDNA double-stranded structure prepared in step (2) onto the electrode interface of Au@Ag NPs / H-WO3 / ITO in step (5), and incubate at a certain temperature. After incubation, rinse with Tris-HCl. The rinsed product is labeled as Apt-cDNA / Au@Ag NPs / H-WO3 / ITO.

[0018] (7) Modify the electrode interface of Apt-cDNA / Au@Ag NPs / H-WO3 / ITO in step (6) with methylcyclohexane (MCH) solution and incubate at room temperature. After incubation, rinse with H2O. The rinsed product is labeled as MCH / Apt-cDNA / Au@Ag NPs / H-WO3 / ITO.

[0019] (8) Modify the electrode interface of MCH / Apt-cDNA / Au@Ag NPs / H-WO3 / ITO in step (7) with methylene blue (MB) solution and incubate at room temperature. After incubation, rinse with H2O. The product after rinsing is the photoelectrochemical-surface-enhanced Raman dual-mode integrated sensor, denoted as MB / MCH / Apt-cDNA / Au@Ag NPs / H-WO3 / ITO.

[0020] Preferably, in step (1) S1, the ratio of the amounts of CTAB, H2O, CaCl2 and Na2WO4 solution is 1g:50mL:0.1665g:5mL, the stirring speed is 460rpm, the Na2WO4 concentration is 0.3M, the temperature is 34-42℃, the sonication time is 1h, the centrifugation speed is 10000rpm, the centrifugation time is 10min, and the drying temperature is 60℃.

[0021] The ratio of CaWO4 to HNO3 was 0.3 g: 20 mL, the concentration of HNO3 was 4 M, the stirring time was 18 h, and the drying temperature was 60 °C. The calcination operation was carried out using a vacuum tube furnace with a heating rate of 2 °C / min. -1 Raise the temperature to 400°C and heat at 400°C for 1 hour.

[0022] Preferably, in step (1) S2, the ratio of H2O, chloroauric acid solution, and Na3C6H5O7 solution is 25mL:0.2mL:0.25mL, wherein the concentration of chloroauric acid solution is 0.1M, the concentration of Na3C6H5O7 is 100mg / mL, the temperature is raised to a certain level of 150℃, and the reaction time is 15min; the ratio of Au NPs solution, AgNO3 solution, Na3C6H5O7 solution, and AA solution is 10mL:1.6mL:0.4mL:0.1mL, wherein the concentration of AgNO3 solution is 10mM, the concentration of Na3C6H5O7 solution is 38.8mM, the concentration of AA solution is 0.1M, the stirring speed is 500rpm, and the stirring time is 20min.

[0023] Preferably, in 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 μM and the concentration of the TCEP solution is 3 mM; the activation reaction is carried out at room temperature for 1 h; the concentration of the Apt solution is 3 μM and the volume ratio of the activation solution to the Apt solution is 1:1; the temperature is raised to 95°C and the reaction time is 3 min; the temperature is lowered to 25°C and the holding time is 3 min.

[0024] The aptamer is the aptamer of MC-LR, with the following sequence: 5'-HS-SH-GGC CGG AAA CAG GAC CACCAT GAC AAT TAC CCA TAC CAC CTC ATT ATG CCC CAT CTC CGC-3';

[0025] The cDNA sequence is: 5'-GCG GAG ATG GGG CAT AAT GAG GTG GTA-3'.

[0026] Preferably, in step (3), the diameter of the indium tin oxide electrode is 6 mm, the concentration of the NaOH solution is 0.3 M, the boiling time is 30 min, the ultrasonic time is 15 min, and the drying temperature is 37 °C.

[0027] Preferably, in step (4), the concentration of H-WO3 solution is 2 mg / mL; H-WO3 solution is used to modify the surface of the pretreated indium tin oxide electrode twice, and the amount of H-WO3 solution used each time is 6 μL.

[0028] Preferably, the amount of Au@Ag NPs used in step (5) is 20 μL.

[0029] Preferably, in step (6), the amount of Apt-cDNA double-stranded structure used is 20 μL, the incubation time is 12 h, and the temperature is 4 °C; the composition of Tris-HCl buffer is: 10 mM Tris, 120 mM NaCl, 1 mM KCl, adjusted to pH = 7.4 with HCl.

[0030] Preferably, in step (7), the concentration of the MCH solution is 1 mM, the amount of modification is 20 μL, and the incubation time is 1 h.

[0031] Preferably, the MB concentration in step (8) is 30 μM, the modification amount is 20 μL, and the incubation time is 30 min.

[0032] This invention also relates to the application of an integrated photoelectrochemical-surface-enhanced Raman dual-mode sensor for detecting MC-LR, the steps of which are as follows:

[0033] (1) Prepare MC-LR standard solutions of different concentrations, modify the photoelectrochemical-surface-enhanced Raman dual-mode integrated sensor interface (MB / MCH / Apt-cDNA / Au@Ag NPs / H-WO3 / ITO) respectively, and incubate at room temperature for a period of time; then rinse the product with H2O, and label the rinsed product as MC-LR / MB / MCH / Apt-cDNA / Au@Ag NPs / H-WO3 / ITO;

[0034] (2) Using the MC-LR / MB / MCH / Apt-cDNA / Au@Ag NPs / H-WO3 / ITO obtained in step (1) as the working electrode, the Ag / AgCl electrode as the reference electrode, and the platinum wire as the counter electrode, the working electrode was placed in the electrolyte for simultaneous photoelectrochemical and surface-enhanced Raman detection; the electrode interface was irradiated with a 532nm Raman laser source to obtain and record the photoelectrochemical-surface-enhanced Raman signal, and two standard curves were constructed using the photocurrent, Raman intensity and the logarithm of MC-LR concentration, respectively.

[0035] (3) Detection of MC-LR in actual samples: First, obtain the sample solution, and then operate according to the methods in steps (1) and (2). The difference is that the MC-LR standard solution in step (1) is replaced with the sample solution. Finally, the photocurrent and Raman intensity are obtained by photoelectrochemical and surface-enhanced Raman detection. The corresponding values ​​are substituted into the standard curve constructed in step (2) to obtain the concentration of MC-LR in the sample, thus realizing the purpose of detecting MC-LR in unknown samples.

[0036] Preferably, in step (1), the concentration of the MC-LR standard solution is 0.3-100 ng / mL, the volume of the MC-LR standard solution is 20 μL, and the incubation period is 40 min.

[0037] Preferably, in step (2), the electrolyte is composed of the following components: 0.1M NaH2PO4, 0.1M Na2HPO4 and 0.1M AA; the photoelectrochemical and surface-enhanced Raman detection is performed as follows: the photoelectrochemical signal is recorded using a Princeton VersaSTAT 3F electrochemical workstation, the Raman signal is recorded using an AOPTINC ATP5020 system, and an external bias voltage of +0.1V is applied.

[0038] Preferably, in step (3), the sample solution is prepared by filtering through a 500-mesh stainless steel sieve and then through a 0.22μm filter membrane to obtain the sample solution.

[0039] The beneficial effects of this invention are:

[0040] (1) In this invention, MB is selected as a dual-function probe and applied to the photoelectrochemical-surface-enhanced Raman dual-mode integrated platform. It can simultaneously cause changes in photoelectrochemical and Raman signals, thereby realizing the synergistic effect of photoelectrochemical-surface-enhanced Raman.

[0041] (2) This invention utilizes Au@Ag NPs / H-WO3 composite material as a bifunctional reinforcing substrate. Under the low-power excitation of Raman laser at 532nm, the photocurrent signal and Raman signal of the bifunctional probe molecule can be simultaneously and significantly amplified.

[0042] (3) The present invention introduces the MC-LR aptamer as a specific recognition element, which can improve the selectivity of the sensor, reduce the interference of other fungal toxins, construct an integrated photoelectrochemical-surface-enhanced Raman dual-mode aptamer sensor, and realize the specific analysis of MC-LR.

[0043] (4) This invention combines the Aopu Tiancheng ATP5020 with the Princeton VersaSTAT 3F electrochemical workstation, using a 532nm Raman laser as a dual-mode signal excitation device to construct an integrated photoelectrochemical-surface-enhanced Raman dual-mode platform. By combining two different signal response mechanisms and relatively independent signal transduction modes, two different types (electro-optic) signal feedbacks are obtained; the two different modes of signals are acquired on the same electrode, and the two output signals can be mutually referenced, effectively reducing the interference of solution matrix and environmental factors on the electrode, avoiding interference from the same type of signal output, significantly improving the sensor's detection accuracy, and achieving accurate MC-LR analysis while improving the dual-mode detection efficiency.

[0044] (5) The photoelectrochemical-surface-enhanced Raman dual-mode integrated aptamer sensor constructed in this invention is used for the detection of MC-LR. The sensor has high sensitivity, good selectivity, good stability, and a detection linear range of 0.3-100 ng / mL. Attached Figure Description

[0045] Figure 1 A schematic diagram of the detection mechanism of an integrated aptamer sensor based on silver-coated gold nanoparticles / hollow tungsten trioxide photoelectrochemical-surface-enhanced Raman dual-mode sensor.

[0046] Figure 2 (A) shows the Raman signal enhancement effect of MB using different substrates, where (a) blank, (b) H-WO3, (c) Au@Ag NPs, and (d) Au@Ag NPs / H-WO3; (B) shows the photoelectric signal enhancement effect of MB using different substrates, where (a) blank, (b) H-WO3, (c) Au@Ag NPs, and (d) Au@Ag NPs / H-WO3.

[0047] Figure 3 In the middle (A), the photoelectrochemical-surface-enhanced Raman signal response of the sensor to different concentrations of MC-LR is shown, where (a) 0 ng / mL, (b) 5 ng / mL, and (c) 30 ng / mL; (B) the Nyquist response of the sensor construction process is shown, where (a) ITO, (b) H-WO3 / ITO, (c) Au@AgNPs / H-WO3 / ITO, (d) Apt-cDNA / Au@AgNPs / H-WO3 / ITO, (e) MCH / Apt-cDNA / Au@AgNPs / H-WO3 / ITO, (f) MB / MCH / Apt-cDNA / Au@Ag NPs / H-WO3 / ITO, and (g) MC-LR / MB / MCH / Apt-cDNA / Au@Ag NPs / H-WO3 / ITO.

[0048] Figure 4 (A) is the response curve of the sensor to different concentrations of MC-LR (0.3 ng / mL, 0.5 ng / mL, 1 ng / mL, 3 ng / mL, 5 ng / mL, 10 ng / mL, 30 ng / mL, 50 ng / mL, 100 ng / mL); (B) is the linear regression curve of photocurrent (PEC), Raman signal (SERS) and the logarithm of MC-LR concentration; (C) is the selectivity of the sensor to 10 ng / mL MC-LR and 100 ng / mL interfering substances MC-YR, MC-RR, and AFB1 in surface-enhanced Raman mode; (D) is the selectivity of the sensor to 10 ng / mL MC-LR and 100 ng / mL interfering substances MC-YR, MC-RR, and AFB1 in photoelectric mode.

[0049] Figure 5 To test the surface-enhanced Raman mode reproducibility (A) and photoelectric mode reproducibility (B) of the MC-LR sensor in parallel 6 times, and to test the surface-enhanced Raman mode stability (C) and photoelectric mode stability (D) of the sensor within 7 days of storage. Detailed Implementation

[0050] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0051] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention.

[0052] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0053] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0054] The MC-LR aptamers and MC-LR aptamer complementary DNA (cDNA) used in this invention were purchased from Sangon Biotech (Shanghai) Co., Ltd.; this invention does not involve sequence listing inventions, and the primers used are conventional commercially available primers.

[0055] Example 1:

[0056] The construction method of the photoelectrochemical-surface-enhanced Raman dual-mode integrated aptamer sensor based on the Au@Ag NPs / H-WO3 substrate of this invention is as follows: Figure 1 As shown, the specific steps are as follows:

[0057] (1) Preparation of substrate material:

[0058] S1 and H-WO3 were prepared by ultrasonic-wet chemical method to prepare H-WO3;

[0059] 1 g of CTAB was added to 50 mL of ultrapure water and stirred for 5 min. Then, 0.1665 g of CaCl2 was dissolved in the above solution and stirred for another 15 min to obtain a mixture. Then, 5 mL of 0.3 M Na2WO4 solution was added to the mixture under slow stirring at 460 rpm. The mixture was continuously sonicated at 34-42 °C for 1 h, centrifuged at 10000 rpm for 10 min to collect the precipitate, washed with ethanol, and finally dried at 60 °C to obtain the CaWO4 precursor.

[0060] 0.3 g of the CaWO4 precursor prepared in the above steps was dissolved in 20 mL of 4 M HNO3 solution and stirred for 18 h to obtain a yellow precipitate. The precipitate was then washed with ethanol and dried. The dried product was placed in a covered alumina crucible and heated in a vacuum tube furnace under air atmosphere at 2 °C for 1 min. -1 The temperature was increased to 400℃ at a certain rate, held for 1 hour, and finally cooled to room temperature to obtain H-WO3.

[0061] Preparation of S2 and Au@Ag NPs:

[0062] First, add 0.2 mL of 0.1 M HAuCl4 solution to 25 mL H2O. Place the solution in an oil bath at 150 °C. After boiling, add 0.25 mL of 100 mg / mL Na3C6H5O7 solution and maintain for 15 min to obtain Au NPs solution.

[0063] Take 10 mL of Au NPs solution and slowly add 1.6 mL of 10 mM AgNO3 solution, 0.4 mL of 38.8 mM Na3C6H5O7 solution, and 0.1 mL of 0.1 M AA solution dropwise while stirring at 550 rpm. Continue stirring for 20 min to obtain Au@Ag NPs.

[0064] (2) Preparation of Apt-cDNA double-stranded structure formed by complementary base pairing between Apt and cDNA:

[0065] 3 mM TCEP was added to a 3 μM cDNA solution and activated at room temperature for 1 h to obtain an activation solution, in which the volume ratio of cDNA solution to TCEP was 10:1. Then, an Apt solution with a concentration of 3 μM and an equal volume to the activation solution was added. After thorough shaking, the solution was heated at 95 °C for 3 min to allow it to fully unwind. After cooling to 25 °C, the temperature was maintained for another 3 min to form an Apt-cDNA double-stranded structure through base pairing.

[0066] (3) Boil the 6 mm diameter indium tin oxide electrode in 0.3 M NaOH solution for 30 min, then sonicate it in anhydrous ethanol and ultrapure water for 15 min in sequence, and finally dry it at 37 °C to obtain the pretreated indium tin oxide electrode.

[0067] (4) Use anhydrous ethanol as a solvent to prepare a 2 mg / mL H-WO3 solution; then take 6 μL of H-WO3 solution to modify the surface of the indium tin oxide electrode after the pretreatment in step (3), modify it twice in total, and then dry it at room temperature. The product is labeled as H-WO3 / ITO.

[0068] (5) 20 μL of Au@Ag NPs prepared in S2 of step (1) was applied to the electrode interface of H-WO3 / ITO in step (4) and dried at room temperature. The product was labeled as Au@Ag NPs / H-WO3 / ITO.

[0069] (6) 20 μL of Apt-cDNA double-stranded structure was modified into the electrode interface of Au@Ag NPs / H-WO3 / ITO in step (5) and incubated at 4℃ for 12 h. Then the product was washed with Tris-HCl and labeled as Apt-cDNA / Au@AgNPs / H-WO3 / ITO.

[0070] (7) 20 μL of MCH solution was applied to the electrode interface of Apt-cDNA / Au@Ag NPs / H-WO3 / ITO in step (6) and incubated at room temperature for 1 h. The product was then rinsed with H2O and labeled as MCH / Apt-cDNA / Au@Ag NPs / H-WO3 / ITO.

[0071] (8) Modify 20 μL of MB solution onto the electrode interface of MCH / Apt-cDNA / Au@Ag NPs / H-WO3 / ITO in step (7) and incubate at room temperature for 30 min. MB will exist in the interlayer of the Apt-cDNA double-stranded structure and be adsorbed in small amounts by adenine on the DNA strand. Then rinse the product with H2O and label the product as MB / MCH / Apt-cDNA / Au@Ag NPs / H-WO3 / ITO.

[0072] (9) In step (8), 20 μL of MC-LR standard solutions of different concentrations (0.3 ng / mL, 0.5 ng / mL, 1 ng / mL, 3 ng / mL, 5 ng / mL, 10 ng / mL, 30 ng / mL, 50 ng / mL, 100 ng / mL) were added to the electrode interface of MB / MCH / Apt-cDNA / Au@Ag NPs / H-WO3 / ITO, respectively, and incubated at room temperature for 40 min. Then, the product was rinsed with H2O to obtain the photoelectrochemical-surface-enhanced Raman dual-mode integrated aptamer sensor, labeled as MC-LR / MB / MCH / Apt-cDNA / Au@Ag NPs / H-WO3 / ITO.

[0073] In the three-electrode system, the washed electrode was used as the working electrode, the Ag / AgCl electrode as the reference electrode, and the platinum wire as the counter electrode. A 532nm laser from the ATP5020 system of AOPTINIUS was used as the excitation source, and the photoelectrochemical signal was recorded using a Princeton VersaSTAT 3F electrochemical workstation. The tests were conducted in an electrolyte-containing solution with an applied bias voltage of +0.1V. The constructed sensor can simultaneously acquire photoelectric and Raman signals.

[0074] Figure 2 (A) shows the Raman signal enhancement effect of different substrates on MB, where (a) blank, (b) H-WO3, (c) Au@Ag NPs, and (d) Au@Ag NPs / H-WO3; (B) shows the photoelectric signal enhancement effect of different substrates on MB, where (a) blank, (b) H-WO3, (c) Au@Ag NPs, and (d) Au@Ag NPs / H-WO3. Figure 2 The photocurrent and Raman response of the sensing substrate to MB enhancement were shown; a weak photocurrent signal could be recorded under 532 nm laser excitation when only MB was present; simultaneously, MB at 1624 cm⁻¹... -1 A strong Raman peak was observed at this location, attributed to the vibration of —C=C— in the benzene ring, consistent with existing literature reports; therefore, this peak was chosen to quantify the concentration of MC-LR. Using Au@Ag NPs / H-WO3 as the substrate material to enhance the MB dual-mode signal, the photocurrent and Raman intensity were 5 times and 59 times that of MB itself, respectively, indicating that Au@Ag NPs / H-WO3 can effectively enhance the photocurrent and Raman response. This is because the enhanced electron transfer capability of Au@Ag NPs / H-WO3 under photoexcitation enables the amplification of photoelectric / Raman signals. These results demonstrate that Au@Ag NPs / H-WO3 can serve as an effective reinforcing substrate material for an integrated photoelectrochemical-surface-enhanced Raman dual-mode platform.

[0075] Figure 3Figure (A) shows the photoelectrochemical-surface-enhanced Raman signal response of the sensor to different concentrations of MC-LR, where (a) 0 ng / mL, (b) 5 ng / mL, and (c) 30 ng / mL; Figure (B) shows the Nyquist response during sensor construction, where (a) ITO, (b) H-WO3 / ITO, (c) Au@Ag NPs / H-WO3 / ITO, (d) Apt-cDNA / Au@Ag NPs / H-WO3 / ITO, (e) MCH / Apt-cDNA / Au@Ag NPs / H-WO3 / ITO, (f) MB / MCH / Apt-cDNA / Au@Ag NPs / H-WO3 / ITO, and (g) MC-LR / MB / MCH / Apt-cDNA / Au@Ag NPs / H-WO3 / ITO. Figure 3 It can be seen that the constructed sensor has obvious signal changes for different concentrations of MC-LR, and the successful construction of the sensor was further verified by electrochemical impedance spectroscopy.

[0076] Figure 4 (A) shows the response curves of the sensor to different concentrations of MC-LR (0.3 ng / mL, 0.5 ng / mL, 1 ng / mL, 3 ng / mL, 5 ng / mL, 10 ng / mL, 30 ng / mL, 50 ng / mL, 100 ng / mL); Figure 4 As shown in (A), the photocurrent and surface-enhanced Raman response after the addition of the target material gradually decrease with increasing MC-LR concentration. The MC-LR concentration is quantified based on the photocurrent magnitude and surface-enhanced Raman intensity.

[0077] Figure 4 Figure (B) shows the linear regression curves of photocurrent (PEC), Raman signal (SERS), and MC-LR concentration. The curves in Figure (B) are respectively I PEC Linear regression curves of SERS values ​​and the logarithm of MC-LR concentration were plotted. The results showed that the linear range for MC-LR detection by the sensor was 0.3-100 ng / mL, with a detection limit of 0.16 ng / mL. The linear regression equations were Y1, Y2, and Y3, respectively. SERS =3649-1073lgC MC-LR (R 2 =0.992) and Y PEC =0.707-0.128lgC MC-LR (R 2 =0.998), indicating that the sensor performance is good.

[0078] To evaluate the feasibility of the constructed sensor, MC-YR, MC-RR, AFB1, and mixtures thereof were used as interfering agents. Figure 4Figure (C) shows the sensor's selectivity for 10 ng / mL MC-LR and 100 ng / mL interfering substances MC-YR, MC-RR, and AFB1 in surface-enhanced Raman mode; Figure (D) shows the sensor's selectivity for 10 ng / mL MC-LR and 100 ng / mL interfering substances MC-YR, MC-RR, and AFB1 in photoelectric mode. Figure 4 As can be seen from (C) and (D), the sensor does not have a significant response to other interfering objects, indicating that the sensor has good selectivity.

[0079] Reproducibility and stability are also important factors in evaluating the practicality of sensors. Figure 5 In (A) and (B), the sensor was used to detect MC-LR in six parallel runs. The RSDs of surface-enhanced Raman mode reproducibility and photoelectric mode reproducibility were 3.5% and 2.3%, respectively. The sensor was used to detect MC-LR continuously for seven days. Figure 5 As shown in (C) and (D), the RSDs of surface-enhanced Raman mode stability and photoelectric mode stability are 2.1% and 1.6%, respectively. These data indicate that the sensor exhibits good reproducibility and stability.

[0080] Meanwhile, the constructed sensor was used to analyze the actual sample, and the specific steps are as follows:

[0081] (1) The actual sample (Zhenjiang, Jiangsu) was pretreated (referring to the national standard method (GB / T 20466–2006)): the sample was filtered through a 500-mesh stainless steel sieve to remove plankton and suspended solids, and then filtered through a 0.22μm filter membrane.

[0082] (2) Take 20 μL of the treated sample and modify it in the sensor prepared in step (8) of Example 1. Incubate it at room temperature for 40 min, then rinse it with H2O and test the MC-LR concentration in the actual sample under the same conditions as in step (9).

[0083] (3) Substitute the acquired signal value into the standard curve constructed by the sensor to obtain the concentration of MC-LR in the sample (as shown in Table 1).

[0084] Table 1. Developed aptamer sensors and HPLC-MS / MS determination of MC-LR (n=3).

[0085]

[0086] "-" indicates that no detection was detected.

[0087] Without spiking, MC-LR was not detected in the water sample. After spiking, the recovery rate of the dual-mode method of the aptamer sensor (PEC-SERS sensor) constructed in this invention was between 90% and 98%, which was basically consistent with the results of the national standard method. This shows that the method of this invention has reliability and accuracy and can be used to realize the detection of MC-LR in unknown samples.

[0088] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A method for fabricating an integrated photoelectrochemical-surface-enhanced Raman dual-mode sensor, characterized in that, Includes the following steps: (1) Preparation of substrate material: Preparation of S1 and H-WO3: Hexadecyltrimethylammonium bromide was dissolved in ultrapure water, calcium chloride was added and stirring was continued; while stirring was continued, sodium tungstate solution was added, and the mixture was ultrasonically treated at a certain temperature. After centrifugation, washing with ethanol and drying, it was labeled as CaWO4. CaWO4 was dissolved in nitric acid solution and stirred. After the two reacted completely, a yellow precipitate was obtained. The precipitate was collected, washed with ethanol, dried, and calcined to obtain the product H-WO3. Preparation of S2 and Au@Ag NPs: First, chloroauric acid solution was added to H2O and placed in an oil bath. After the temperature was raised to a certain level, trisodium citrate solution was added, and gold nanoparticle solution was obtained after the reaction. Then, silver nitrate solution, trisodium citrate solution and ascorbic acid solution were added dropwise to the gold nanoparticle solution under stirring, and the mixture was stirred continuously to obtain the product Au@Ag NPs. (2) Preparation of Apt-cDNA double-stranded structure: TCEP solution was added to cDNA solution for activation reaction to obtain activation solution; then aptamer solution was added to activation solution, shaken and raised to a certain temperature and held for a period of time, then lowered to a certain temperature and held for a certain time to obtain the final product, which is denoted as Apt-cDNA double-stranded structure. (3) Boil the indium tin oxide electrode in sodium hydroxide solution, then take out the indium tin oxide electrode and clean it by ultrasonication in anhydrous ethanol and ultrapure water in sequence, and then dry it to obtain the pretreated indium tin oxide electrode. (4) Add the H-WO3 prepared in S1 of step (1) to anhydrous ethanol to obtain H-WO3 solution; modify the surface of the indium tin oxide electrode after the pretreatment in step (3) with H-WO3 solution and dry it at room temperature. At this time, the product is marked as H-WO3 / ITO. (5) Modify the Au@Ag NPs prepared in S2 of step (1) onto the electrode interface of H-WO3 / ITO in step (4) and dry it at room temperature. The dried product is labeled as Au@Ag NPs / H-WO3 / ITO. (6) Modify the double-stranded Apt-cDNA structure prepared in step (2) onto the electrode interface of Au@Ag NPs / H-WO3 / ITO in step (5) and incubate at room temperature. After incubation, rinse with Tris-HCl. The rinsed product is labeled as Apt-cDNA / Au@Ag NPs / H-WO3 / ITO. (7) Modify the electrode interface of Apt-cDNA / Au@Ag NPs / H-WO3 / ITO in step (6) with methylcyclohexane (MCH) solution and incubate at room temperature. After incubation, rinse with H2O. The rinsed product is labeled as MCH / Apt-cDNA / Au@AgNPs / H-WO3 / ITO. (8) Modify the electrode interface of MCH / Apt-cDNA / Au@Ag NPs / H-WO3 / ITO in step (7) with methylene blue (MB) solution and incubate at room temperature. After incubation, rinse with H2O. The product after rinsing is the photoelectrochemical-surface-enhanced Raman dual-mode integrated sensor, denoted as MB / MCH / Apt-cDNA / Au@Ag NPs / H-WO3 / ITO.

2. The method for fabricating a photoelectrochemical-surface-enhanced Raman dual-mode integrated sensor according to claim 1, characterized in that, In step (1) S1, the ratio of hexadecyltrimethylammonium bromide, ultrapure water, calcium chloride, and sodium tungstate solution is 1g:50mL:0.1665g:5mL, the stirring speed is 460rpm, and the Na2WO4 concentration is 0.3M; the temperature is 34-42℃, the sonication time is 1h; the centrifugation speed is 10000rpm, the centrifugation time is 10min, and the drying temperature is 60℃; the ratio of CaWO4 to HNO3 is 0.3g:20mL, the HNO3 concentration is 4M, the stirring time is 18h, and the drying temperature is 60℃; the calcination operation is: using a vacuum tube furnace with a heating rate of 2℃ / min. -1 Raise the temperature to 400℃ and heat at 400℃ for 1 hour; In step (1) S2, the ratio of H2O, chloroauric acid solution, and trisodium citrate solution is 25 mL: 0.2 mL: 0.25 mL, where the concentration of chloroauric acid solution is 0.1 M, the concentration of Na3C6H5O7 is 100 mg / mL, the temperature is raised to 150 °C, and the reaction time is 15 min. The ratio of the gold nanoparticle solution, AgNO3 solution, Na3C6H5O7 solution, and AA solution is 10 mL: 1.6 mL: 0.4 mL: 0.1 mL, where the concentration of AgNO3 solution is 10 mM, the concentration of Na3C6H5O7 solution is 38.8 mM, the concentration of AA solution is 0.1 M, the stirring speed is 500 rpm, and the stirring time is 20 min.

3. The method for fabricating a photoelectrochemical-surface-enhanced Raman dual-mode integrated sensor according to claim 1, characterized in that, In 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 μM and the concentration of the TCEP solution is 3 mM; the activation reaction is carried out at room temperature for 1 h; the temperature is raised to 95 °C for 3 min; and the temperature is lowered to 25 °C for 3 min. The aptamer is the aptamer of MC-LR, with the following sequence: 5'-HS-SH-GGC CGG AAA CAG GAC CAC CATGAC 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. The method for fabricating a photoelectrochemical-surface-enhanced Raman dual-mode integrated sensor according to claim 1, characterized in that, In step (3), the diameter of the indium tin oxide electrode is 6 mm, the concentration of the NaOH solution is 0.3 M, the boiling time is 30 min, the ultrasonic time is 15 min, and the drying temperature is 37 °C.

5. The method for fabricating a photoelectrochemical-surface-enhanced Raman dual-mode integrated sensor according to claim 1, characterized in that, In step (4), the concentration of H-WO3 solution is 2 mg / mL; H-WO3 solution is used to modify the surface of the pretreated indium tin oxide electrode twice, with a modification amount of 6 μL each time; in step (5), the amount of Au@Ag NPs used is 20 μL.

6. The method for fabricating a photoelectrochemical-surface-enhanced Raman dual-mode integrated sensor according to claim 1, characterized in that, In step (6), the amount of Apt-cDNA double-stranded structure used is 20 μL, the incubation time is 12 h, and the incubation temperature is 4 °C. The composition of Tris-HCl buffer is: 10 mM Tris, 120 mM NaCl, 1 mM KCl, adjusted to pH 7.4 with HCl.

7. The method for fabricating a photoelectrochemical-surface-enhanced Raman dual-mode integrated sensor according to claim 1, characterized in that, In step (7), the concentration of MCH solution is 1 mM, the amount of modification is 20 μL, and the incubation time is 1 h; in step (8), the concentration of MB is 30 μM, the amount of MB is 20 μL, and the incubation time is 30 min.

8. The use of the photoelectrochemical-surface-enhanced Raman dual-mode integrated sensor prepared according to any one of claims 1-7 for detecting MC-LR, characterized in that, The steps are as follows: (1) Prepare MC-LR standard solutions of different concentrations, modify the interface of photoelectrochemical-surface-enhanced Raman dual-mode integrated sensor respectively, and incubate at room temperature for a period of time; then rinse the product with H2O, and label the rinsed product as MC-LR / MB / MCH / Apt-cDNA / Au@Ag NPs / H-WO3 / ITO; (2) Using the MC-LR / MB / MCH / Apt-cDNA / Au@Ag NPs / H-WO3 / ITO obtained in step (1) as the working electrode, the Ag / AgCl electrode as the reference electrode, and the platinum wire as the counter electrode, the working electrode was placed in the electrolyte for simultaneous photoelectrochemical and surface-enhanced Raman detection; the electrode interface was irradiated with a 532nm Raman laser source to obtain and record the photoelectrochemical-surface-enhanced Raman signal, and two standard curves were constructed using the photocurrent, Raman intensity and the logarithm of MC-LR concentration, respectively. (3) Detection of MC-LR in actual samples: First, obtain the sample solution, and then operate according to the methods in steps (1) and (2). The difference is that the MC-LR standard solution in step (1) is replaced with the sample solution. Finally, the photocurrent and Raman intensity are obtained by photoelectrochemical and surface-enhanced Raman detection. The corresponding values ​​are substituted into the standard curve constructed in step (2) to obtain the concentration of MC-LR in the sample, thus realizing the purpose of detecting MC-LR in unknown samples.

9. The use according to claim 8, characterized in that, In step (1), the concentration of the MC-LR standard solution is 0.3-100 ng / mL, the volume of the MC-LR standard solution is 20 μL, and the incubation time is 40 min; in step (2), the electrolyte is composed of the following components: 0.1 M NaH2PO4, 0.1 M Na2HPO4 and 0.1 M AA; the photoelectrochemical and surface-enhanced Raman detection is as follows: the photoelectrochemical signal is recorded using a Princeton VersaSTAT 3F electrochemical workstation, the Raman signal is recorded using an Aopu Tiancheng ATP5020 system, and the external bias voltage is +0.1 V.

10. The use according to claim 8, characterized in that, In step (3), the sample solution is prepared by filtering it through a 500-mesh stainless steel sieve and then filtering it through a 0.22μm filter membrane.

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