MoS2 / CeZnCuSx / AuNR-based thrombin photoelectrochemical aptamer sensor as well as preparation method and application thereof

Abstract

The invention discloses a thrombin photoelectrochemical aptamer sensor based on MoS / CeZnCuSx / AuNR and a preparation method and application thereof.The thrombin photoelectrochemical aptamer sensor is constructed by modifying the surface of an FTO electrode with a MoS / CeZnCuSx composite material and then attaching AuNR, and the preparation method comprises the following steps that S1, MoS is prepared; s2, preparation of CeZnCuSx is carried out; s3, preparation of a MoS / CeZnCuSx solution is carried out; s4, preparation of FTO (Fluorine-doped Tin Oxide) / MoS2 / CeZnCuS < x > / AuNR; and S5, preparing the thrombin sensing platform. The sensor utilizes the narrow band gap of MoS / CeZnCuSx and the plasma effect of AuNR to synergistically enhance the photoelectric property, realizes label-free one-step detection through a signal weakening strategy, has the advantages of high sensitivity, rapid response and strong stability, and is suitable for efficient detection of thrombin in a biological sample.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of photoelectrochemical sensors, and particularly relates to a thrombin photoelectrochemical aptamer sensor based on MoS2 / CeZnCuS x / AuNR and a preparation method and application thereof. BACKGROUND

[0002] Thrombin (TMB) can convert fibrinogen into fibrin, and after local application, the blood acting on the surface of the lesion rapidly forms a stable clot, which is used for controlling capillary, venous bleeding, or as an adhesive and fixing agent for skin and tissue grafts, and is involved in various diseases, such as thromboembolic diseases, leukemia and inflammatory reactions. In order to adapt to global health problems, especially in developing countries, it is very desirable to develop a simple, low-cost and effective method for quantitative detection of thrombin. Some conventional methods for protein analysis, such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), but these methods cannot break through the limitations of in-situ detection or clinical application, and still have defects such as false negative, false positive, poor reproducibility, and thus highlight the urgent need for technological innovation and upgrading.

[0003] Traditional optical detection methods have complex optical elements, high equipment maintenance costs, low sensitivity of background signal detection and strong interference; electrochemical detection methods also have expensive equipment and complicated detection techniques, and therefore there is an urgent need to develop a high-sensitivity, high-specificity, stable and reliable and low-cost protein biomarker detection method, which makes the research of a new type of biosensing method imminent, and therefore the innovative PEC photoelectric biosensing method developed by combining electrochemical sensing technology and optical detection method becomes the current research direction.

[0004] The basic principle of photoelectrochemical sensing technology is based on the photoelectrochemical active material under light absorption to generate photoelectron-hole pairs, and the chemical reaction of electron-hole pairs with electron donors or electron acceptors in the solution makes the electron-hole pairs separate, and then generates carriers and appears charge separation and transfer, forming a photocurrent. PEC technology is a forward-looking technology derived from photocatalytic technology, which combines photoelectron process and electrochemical technology, and applies external voltage to the photoelectrode to improve its efficiency by promoting charge separation and inhibiting secondary pollution on the electrode surface. PEC sensor is a new type of sensing technology developed on the basis of electrochemical sensor, which realizes through the photoelectric conversion capacity of light active material under light. According to the change of target concentration, the changes of interface electron transfer, electrolyte environment, electron donor / acceptor mass transfer and light absorption can be induced to achieve the purpose of detection and analysis. Among them, PEC technology is outstanding among many detection technologies due to its fast response speed, weak background signal (derived from the separation of excitation light source and output signal photocurrent), high sensitivity, and easy miniaturization. However, there are still problems such as insufficient sensitivity, stability, light absorption characteristics, detection recovery rate in the existing research of PEC biosensor, so it is necessary to find suitable materials to improve the functional characteristics of PEC biosensor, and to establish a simple and convenient method for detecting thrombin with high sensitivity, which has a profound influence on medicine and clinic. SUMMARY

[0005] The technical problem to be solved: In view of the above problems, the purpose of the present application is to provide a thrombin photoelectrochemical aptamer sensor based on MoS2 / CeZnCuS x / AuNR and a preparation method thereof, which attaches MoS2 / CeZnCuS x material on FTO glass electrode to prepare FTO / MoS2 / CeZnCuS x / AuNR composite material on the electrode surface, that is, to prepare FTO / MoS2 / CeZnCuSx / AuNR thrombin photoelectrochemical aptamer sensor, which realizes the ultra-sensitive detection of thrombin.

[0006] Technical scheme: A thrombin photoelectrochemical aptamer sensor based on MoS2 / CeZnCuS x / AuNR, the thrombin photoelectrochemical aptamer sensor is composed of MoS2 / CeZnCuS x / AuNR composite material and FTO electrode; the MoS2 / CeZnCuS x / AuNR composite material is a nano-hollow micro-flower structure with a diameter of 2-5 μm, an inner hole diameter of 0.2-0.8 μm, and a specific surface area of 20-30 m 2 / g.

[0007] The above one is based on MoS2 / CeZnCuS x Preparation method of a thrombin photoelectrochemical aptamer sensor based on FTO / MoS2 / CeZnCuS S1. Preparation of MoS2: CTAB, CH4N2S and Na2MoO4 are added to deionized water, stirred uniformly, hydrothermally reacted, centrifuged, washed and dried to obtain MoS2; S2. Preparation of CeZnCuS x : Ce(CH3CO2)3, Zn(CH3CO2)2 and Cu(CH3CO2)2 are dispersed in ethanol, ultrasonic treatment is performed for 20-50 min to prepare a dispersion solution, a mixed solution of C2H5NS and ethanol is added, stirring is performed at room temperature for 2-5 h, and then solvent thermal reaction is performed, and MoS2 / CeZnCuS x is obtained by taking out, washing and drying; S3. Preparation of MoS2 / CeZnCuS x solution: MoS2, Ce(CH3CO2)3, Zn(CH3CO2)2 and Cu(CH3CO2)2 prepared above are dispersed in ethanol to prepare a dispersion solution, a mixed solution of ethanol and C2H5NS is added, stirring is performed for 2-4 h, and then solvent thermal reaction is performed, and MoS2 / CeZnCuS x is obtained by taking out, washing and drying, and then dissolved in deionized water to obtain a MoS2 / CeZnCuS x solution; S4. Preparation of FTO / MoS2 / CeZnCuS x / AuNR: MoS2 / CeZnCuS x / AuNR solution is taken, stirred, centrifuged, and vacuum dried to obtain MoS2 / CeZnCuS x / AuNR, which is dropped onto a FTO glass electrode, calcined for 20-50 min to obtain FTO / MoS2 / CeZnCuS x / AuNR; S5. Preparation of a thrombin sensing platform: 10 μL of 1 μM thiol-modified TMB aptamer apt is dropped on the surface of FTO / MoS2 / CeZnCuS x / AuNR, stored at 4°C overnight to perform apt self-assembly, and MoS2 / CeZnCuS x / AuNR / apt is obtained; MoS2 / CeZnCuS xAfter the / AuNR / apt was washed with PBS, 10 μL 5% BSA was added to block for 40 min, the electrode was immersed in 200 μM MB for 30 min, and after washing, the photocurrent response was recorded in blank 0.1 M pH 7.4 PBS; 10 uL TMB of different concentrations was added to the electrode surface, and after incubation at 37℃ for 1 h, the photocurrent response value was recorded.

[0008] Further, the mass-volume ratio of CTAB, CH4N2S, Na2MoO4 and deionized water in step S1 is (0.08-0.12) g:(0.66-0.70) g:(0.70-0.74) g:(40-60) mL; the temperature of the hydrothermal reaction is 200-240℃, and the time is 20-30 h.

[0009] Further, the mass-volume ratio of Ce(CH3CO2)3, Zn(CH3CO2)2, Cu(CH3CO2)2 and ethanol in step S2 is (1.0-1.2) g:(0.73-0.77) g:(0.55-0.58) g:(60-70) mL; the mass-volume ratio of C2H5NS and ethanol in the mixed solution is (0.91-0.95) g:(12-25) mL; the volume ratio of the mixed solution to the dispersion is 1:(2-5); the temperature of the solvothermal reaction is 160-200℃, and the time is 22-26 h.

[0010] Further, the mass-volume ratio of MoS2, Ce(CH3CO2)3, Zn(CH3CO2)2, Cu(CH3CO2)2 and ethanol in step S3 is (0.11-0.15) g:(0.93-1.25) g:(0.74-0.77) g:(0.56-0.59) g:(60-70) mL; the mass-volume ratio of C2H5NS and ethanol in the mixed solution is (0.71-1.11) g:(12-25) mL; the volume ratio of the mixed solution to the dispersion is 1:(2-5); the temperature of the solvothermal reaction is 160-200℃, and the time is 22-26 h.

[0011] Further, the preparation steps of the AuNR solution in step S4 are as follows: (1) Preparation of gold seed solution: uniformly mix CTAB solution and HAuCl4 solution, then add NaBH4 solution, continuously stir for 1-4 min, and stand for aging for 20-50 min to obtain gold seed solution; (2) Preparation of AuNR solution: after mixing the CTAB solution and the NaOL solution uniformly, the HAuCl4 solution is added, and the mixed solution is obtained by adjusting the pH to 6-8 under water bath at 20-50 DEG C for 70-100 min; then the ascorbic acid solution, the AgNO3 solution and the gold seed solution are sequentially added to the mixed solution, and the product is collected after stirring for 20-50 s and standing for 10-15 h under 20-50 DEG C, and then purified by centrifugation at 6000-12000 rpm for 10-40 min and at 3000-8000 rpm for 8-15 min, and dispersed in deionized water to obtain the AuNR solution.

[0012] Further, the molar concentration ratio of the CTAB solution, the HAuCl4 solution and the NaBH4 solution in the step (1) is 40:1:(1-2).

[0013] Further, the molar concentration ratio of the CTAB solution, the NaOL solution and the HAuCl4 solution in the step (2) is 200:16:(4-6); and the concentration volume ratio of the mixed solution, the ascorbic acid solution, the AgNO3 solution and the gold seed solution is (18-24) mL:(0.05-0.15) M:(10-12) mL.

[0014] Further, the volume ratio of the MoS2 / CeZnCuS x solution to the AuNR solution in the step S4 is 4:(1-2).

[0015] The application of the prepared thrombin photoelectrochemical aptamer sensor in detecting thrombin. Beneficial effects

[0016] The novel quaternary metal sulfide CeZnCuS x developed by the application has the advantages of structural stability (high temperature resistance) and suitable band gap (2.61 eV), which is different from the traditional technology, and the metal semiconductor material (quaternary cerium zinc copper sulfide) has the advantages of adjustable energy band, high chemical stability, easy synthesis, visible light response and excellent photocatalytic performance; the molybdenum disulfide (MoS2) as a layered metal sulfide semiconductor has the advantages of van der Waals force of S-Mo-S atomic layer, stacking structure, high specific surface area, narrow band gap (excellent visible light absorption) and excellent photoelectric performance, and the single-layer / few-layer MoS2 nanosheet exhibits stronger photocatalytic performance due to more unsaturated sulfur active sites exposed; the MoS2 / CeZnCuS x by accurately adjusting the band gap and reasonably improving the material concentration, the sensitivity of the sensor is improved.

[0017] The MoS2 / CeZnCuS xThe AuNR is attached to the basis of the material, because the AuNR has better surface plasmonic effect, and the plasmonic material has larger optical interaction cross section at resonance frequency, they can absorb energy in a wide wavelength range, and combined with photoelectric active material, signal amplification effect can be provided, so that the prepared MoS2 / CeZnCuS x / AuNR composite material has excellent photoelectric performance.

[0018] The PEC sensor constructed in the application is different from a sandwich type sensor, the sandwich type sensor is composed of three main parts, i.e., two support layers at the upper and lower parts and a sensor element layer in the middle part, however, such a structure has problems of complicated operation steps and incapability of one-step identification; and the sensor prepared by adopting the signal weakening type strategy in the application reflects the relationship between the photocurrent and the concentration of the target object, that is, under the influence of the target object, the change of the interface resistance or other properties causes the process of photoexcited electron generation and transfer to be inhibited, and then the photocurrent signal is reduced, so that the correlation between the photocurrent value and the concentration of the target object is realized, and the advantages of label-free, one-step identification and rapid response are achieved.

[0019] The application reflects the relationship between the photocurrent and the concentration of the target object by the material properties, i.e., S 2- The negatively charged Ce 3+ The characteristics are further improved by hydrothermal synthesis, and the stability of the material is further improved, and the heterojunction, i.e., CeZnCuS x Nanoparticles are in-situ grown on the surface of MoS2 to form 0D / 3D heterojunction, and such a structure can significantly improve the photocatalytic activity and stability; wherein, in the preparation of the CeZnCuS x Material, an ultrasonic method is used to increase the dispersity of the material to further reduce the phenomenon of cluster stacking, and then the surface roughness is reduced; finally, the FTO / MoS2 / CeZnCuS x / AuNR composite material is synthesized by the method, and the experimental conditions can be more accurately controlled to further synthesize stable composite materials.

[0020] The application is based on the MoS2 / CeZnCuS x / AuNR composite material has excellent photoelectric performance, and a super-sensitivity label-free PEC thrombin sensor is successfully constructed, the linear range of TMB detection is 0.02 pM-5.0 nM, and the lower limit of detection is 8.5 fM (S / N=3), so the composite material has important application potential in the field of advanced PEC sensing, and when applied to the detection of thrombin in human serum samples, the recovery rate is 98.5%-104%, so the PEC enzyme sensor prepared in the application has the potential to detect thrombin in actual samples. BRIEF DESCRIPTION OF DRAWINGS Figure 1Flow chart for the construction of the thrombin PEC sensing platform.

[0021] Figure 2 For FTO / ZnS, FTO / CuS, FTO / ZnCuS x and FTO / CeZnCuS x Electrode photocurrent response graph.

[0022] Figure 3 For the optimization experiment of different Ce doping amounts on the photoelectric performance of the material, the photocurrent response graph is obtained respectively with different amounts of Ce doping of 1%, 2%, 5%, 10%, and 15%.

[0023] Figure 4 For the photocurrent response graph of different modified electrodes, where a is bare FTO; b is FTO / MoS2; c is FTO / CeZnCuS x ; d is FTO / MoS2 / CeZnCuS x photocurrent spectrum.

[0024] Figure 5 For the X-ray powder diffraction pattern of CeZnCuS x , MoS2 and MoS2 / CeZnCuS x .

[0025] Figure 6 For the EIS spectrum of different modified electrodes in a 2mM K4[Fe(CN)6] solution containing 0.1M KCl, where a is FTO / CeZnCuS x ; b is FTO / MoS2 / CeZnCuS x ; c is FTO / MoS2 / CeZnCuS x / AuNR; d is FTO / MoS2; e is FTO / MoS2 / CeZnCuS x / AuNR / apt; f is FTO / MoS2 / CeZnCuS x / AuNR / apt / BSA; g is FTO / MoS2 / CeZnCuS x / AuNR / apt / BSA / MB.

[0026] Figure 7 For the photocurrent response graph of the biological recognition process of the preparation of thrombin photoelectrochemical aptamer sensor, where a is FTO / MoS2 / CeZnCuS x / AuNR; b is FTO / MoS2 / CeZnCuS x / AuNR / apt; c is FTO / MoS2 / CeZnCuS x / AuNR / apt / BSA; d is FTO / MoS2 / CeZnCuS x / AuNR / apt / BSA / MB; e is FTO / MoS2 / CeZnCuS x / AuNR / apt / BSA / MB / TMB; The electrode is measured at a bias of 0.1 V (vs SCE) in 0.1 M ascorbic acid (AA), pH 7.4 phosphate buffer.

[0027] Figure 8 are scanning electron microscopy and energy dispersive X-ray microscopy images, wherein A is a transmission electron microscopy image of AuNR, B is an ultraviolet-visible spectrum of AuNRs with longitudinal plasmonic bands at 30 nm; C is MoS2, D is CeZnCuS x , E is MoS2 / CeZnCuS x , and F is MoS2 / CeZnCuS x are scanning electron microscopy images of AuNR, G is MoS2 / CeZnCuS x are energy spectrum analysis images of AuNR.

[0028] Figure 9 are UV-Vis DRS and Mott-Schottky graphs, wherein in graph A, a is MoS2, b is CeZnCuS x , c is MoS2 / CeZnCuS x ultraviolet-visible diffuse reflection; B and C are MoS2 and CeZnCuS x tauc graphs; D, E are Mott-Schottky graphs of MoS2 and CeZnCuS x ; F is a band structure diagram.

[0029] Figure 10 are thrombin detection graphs, wherein A is the photocurrent response of different concentrations of TMB, B is the detection curve, a-h are in turn: 0.02, 0.08, 0.8, 8, 80, 800, 2500, 5000 pM.

[0030] Figure 11 are stability measurement graphs of the electrode under continuous on / off illumination, wherein a is FTO / MoS2 / CeZnCuS x ; b is FTO / MoS2 / CeZnCuS x / AuNR; c is FTO / MoS2; d is FTO / CeZnCuS x .

[0031] Figure 12For selectivity and stability profile, the sensor's specificity to thrombin compared to interfering proteins: bovine serum albumin, fibrinogen, rabbit gamma globulin G and human immunoglobulin G at a concentration of 500 pM. DETAILED DESCRIPTION

[0032] The application will be further described in conjunction with the accompanying drawings and examples. The following examples are intended to explain the application and are not intended to limit the application to the examples described. Example 1

[0033] A preparation method of a thrombin photoelectrochemical aptamer sensor based on MoS2 / CeZnCuS x / AuNR, and the specific preparation steps are as follows: S1. Preparation of MoS2: 0.088 g of CTAB, 0.664 g of CH4N2S and 0.706 g of Na2MoO4 were added to 50 mL of deionized water, stirred uniformly, and hydrothermally reacted at 200°C for 30 h. After centrifugation, ethanol washing was performed three times, and vacuum drying was performed overnight to obtain MoS2. S2. Preparation of CeZnCuS x : 1.063 g of Ce(CH3CO2)3, 0.743 g of Zn(CH3CO2)2 and 0.561 g of Cu(CH3CO2)2 were dispersed in 60 mL of ethanol, ultrasonically treated for 30 min to prepare a dispersion liquid, and then 0.919 g of C2H5NS and 5 mL of ethanol were added. After stirring at room temperature for 3 h, solvent thermal reaction was performed at 160°C for 28 h. After removal, ethanol washing and vacuum drying overnight, a black sample was collected to obtain CeZnCuS x . S3. Preparation of MoS2 / CeZnCuS x solution: 0.114 g of MoS2 and 1.063 g of Ce(CH3CO2)3, 0.743 g of Zn(CH3CO2)2 and 0.561 g of Cu(CH3CO2)2 prepared above were dispersed in 60 mL of ethanol to prepare a dispersion liquid. While stirring, 20 mL of ethanol and 0.939 g of C2H5NS were added dropwise to the above dispersion liquid. After stirring for 3 h, solvent thermal reaction was performed at 180°C for 24 h. After removal, washing and drying, a black solid MoS2 / CeZnCuS x sample was obtained, which was collected for standby use. The above medicines were weighed and dissolved in deionized water, and then standby use.

[0034] S4. Preparation of AuNR solution: (1) Preparation of gold seed solution: 10 mL of 0.1 M CTAB solution was mixed with 0.1 mL of 25 mM HAuCl4 solution, then 0.6 mL of 0.1 M NaBH4 solution was added, stirring for 2 min, and aging for 20 min to obtain the gold seed solution; (2) Preparation of AuNR solution: 0.8 mL of 25 mM HAuCl4 solution was added to 20 mL of mixed solution containing 0.1 M CTAB and 16 mM NaOL, and the mixed solution was obtained by stirring at 20°C for 100 min, adjusting the pH to 7 with concentrated HCl, and then adding 64 μL of 0.1 M ascorbic acid solution, 0.75 mL of 10 mM AgNO3 solution and 16 μL of gold seed solution to the mixed solution, stirring for 20 s, and then aging at 30°C for 12 h. The product was collected by centrifugation at 6000 rpm for 40 min and then at 3000 rpm for 15 min, and dispersed in deionized water to obtain the AuNR solution; S5. Preparation of FTO / MoS2 / CeZnCuS / AuNR x S5. Preparation of FTO / MoS2 / CeZnCuS / AuNR x S5. Preparation of FTO / MoS2 / CeZnCuS / AuNR x S5. Preparation of FTO / MoS2 / CeZnCuS / AuNR x S5. Preparation of FTO / MoS2 / CeZnCuS / AuNR x S5. Preparation of FTO / MoS2 / CeZnCuS / AuNR S6. Preparation of thrombin sensing platform: 10 μL of 1 μM aptamer TMB aptamer was dropped on the surface of FTO / MoS2 / CeZnCuS / AuNR, and stored at 4°C overnight for aptamer self-assembly to obtain MoS2 / CeZnCuS / AuNR / apt; x S6. Preparation of thrombin sensing platform: 10 μL of 1 μM aptamer TMB aptamer was dropped on the surface of FTO / MoS2 / CeZnCuS / AuNR, and stored at 4°C overnight for aptamer self-assembly to obtain MoS2 / CeZnCuS / AuNR / apt; x S6. Preparation of thrombin sensing platform: 10 μL of 1 μM aptamer TMB aptamer was dropped on the surface of FTO / MoS2 / CeZnCuS / AuNR, and stored at 4°C overnight for aptamer self-assembly to obtain MoS2 / CeZnCuS / AuNR / apt; x S6. Preparation of thrombin sensing platform: 10 μL of 1 μM aptamer TMB aptamer was dropped on the surface of FTO / MoS2 / CeZnCuS / AuNR, and stored at 4°C overnight for aptamer self-assembly to obtain MoS2 / CeZnCuS / AuNR / apt;

[0035] Example 2 A MoS 2 / CeZnCuS x The preparation method of the thrombin photoelectrochemical aptamer sensor based on AuNR is as follows: S1. Preparation of MoS2: 0.128 g of CTAB, 0.704 g of CH4N2S, and 0.746 g of Na2MoO4 were added to 50 mL of deionized water, stirred uniformly, and subjected to hydrothermal reaction at 240°C for 20 h. After centrifugation, ethanol washing was performed three times, and vacuum drying was performed overnight to obtain MoS2; S2. Preparation of CeZnCuS x : 1.103 g of Ce(CH3CO2)3, 0.763 g of Zn(CH3CO2)2, and 0.571 g of Cu(CH3CO2)2 were dispersed in 60 mL of ethanol, ultrasonically treated for 30 min to prepare a dispersion liquid, and then a mixed solution of 0.959 g of C2H5NS and 15 mL of ethanol was added. After stirring at room temperature for 3 h, solvent thermal reaction was performed at 200°C for 22 h. After removal, ethanol washing, and vacuum drying overnight, a black sample was collected to obtain CeZnCuS x ; S3. Preparation of MoS2 / CeZnCuS x solution: 0.154 g of MoS2 and 1.103 g of Ce(CH3CO2)3, 0.753 g of Zn(CH3CO2)2, and 0.561 g of Cu(CH3CO2)2 prepared above were dispersed in 60 mL of ethanol to prepare a dispersion liquid. While stirring, 20 mL of ethanol and 0.939 g of C2H5NS were added dropwise to the above dispersion liquid. After stirring for 3 h, solvent thermal reaction was performed at 180°C for 24 h. After removal, washing, and drying, a black solid MoS2 / CeZnCuS x sample was obtained and collected for standby. The above medicine was weighed, dissolved in deionized water, and then standby.

[0036] S4. Preparation of AuNR solution: (1) Preparation of gold seed solution: 10 mL of 0.1M CTAB solution was uniformly mixed with 0.1 mL of 25 mM HAuCl4 solution, and then 0.6 mL of 0.1 M NaBH4 solution was added. After continuous stirring for 2 min, aging was performed for 50 min to obtain gold seed solution; (2) Preparation of AuNR solution: 0.8 mL of 25 mM HAuCl4 solution was added to 20 mL of a mixed solution containing 0.1 M CTAB and 16 mM NaOL, and the mixture was incubated at 50 °C in a water bath for 70 min. The pH was adjusted to 7 with concentrated HCl to obtain a mixed solution. 64 μL of 0.1 M ascorbic acid solution, 0.75 mL of 10 mM AgNO3 solution, and 16 μL of gold seed solution were added to the mixed solution in sequence. After stirring for 50 s, the mixture was allowed to react at 30 °C for 12 h. The mixture was first centrifuged at 12000 rpm for 10 min and then at 8000 rpm for 8 min for purification. The product was collected and dispersed in deionized water to obtain the AuNR solution. S5.FTO / MoS2 / CeZnCuS x Preparation of MoS2 / AuNR: FTO glass electrode was ultrasonically cleaned with acetone, ethanol, and ultrapure water for 10 min, and dried in an oven for 30 min. Then 20 μL of 4 mg / mL MoS2 / CeZnCuS x The solution was added dropwise onto the FTO glass electrode and dried at 60 °C for 2 h to prepare FTO / MoS2 / CeZnCuS x electrode, and then 10 μL of 0.8 mM AuNR solution was added dropwise to the FTO / MoS2 / CeZnCuS x The electrode surface was calcined for 30 min to obtain FTO / MoS2 / CeZnCuS x / AuNR; S6. Preparation of thrombin sensing platform: 10 μL of 1 μM thiol-modified TMB aptamer apt was dropped on FTO / MoS2 / CeZnCuS x / AuNR surface, stored at 4 ° C overnight, and then apt self-assembled to obtain MoS2 / CeZnCuS x / AuNR / apt; MoS2 / CeZnCuS x After washing the / AuNR / apt with PBS, 10 μL of 5% BSA was taken to block for 40 min, and the electrode was immersed in 200 μM MB for 30 min. After washing, the photocurrent response was recorded in blank 0.1 M pH 7.4 PBS; 10 uL of different concentrations of TMB were applied to the electrode surface, incubated at 37°C for 1 h, and the photocurrent response value was recorded.

[0037] Example 3 A MoS-based 2 / CeZnCuS x The preparation method of the thrombin photoelectrochemical aptamer sensor based on AuNR is as follows: S1. Preparation of MoS2: 0.108 g CTAB, 0.684 g CH4N2S and 0.726 g Na2MoO4 were added into 50 mL deionized water, stirred uniformly, hydrothermally reacted at 220 °C for 24 h, centrifuged, washed with ethanol for 3 times, vacuum dried overnight, and MoS2 was obtained; S2. Preparation of CeZnCuS x : 1.083 g Ce(CH3CO2)3, 0.763 g Zn(CH3CO2)2 and 0.581 g Cu(CH3CO2)2 were dispersed in 60 mL ethanol, ultrasonically treated for 30 min to prepare a dispersion, and then 0.939 g C2H5NS and 10 mL ethanol were added. After stirring at room temperature for 3 h, solvent-thermal reaction was carried out at 180 °C for 24 h. After taking out, washing with ethanol and vacuum drying overnight, a black sample of CeZnCuS was collected. x ; S3. Preparation of MoS2 / CeZnCuS x : 0.134 g MoS2 prepared above, 1.083 g Ce(CH3CO2)3, 0.743 g Zn(CH3CO2)2 and 0.561 g Cu(CH3CO2)2 were dispersed in 60 mL ethanol to prepare a dispersion, and 20 mL ethanol and 0.939 g C2H5NS were added dropwise to the above dispersion while stirring. After stirring for 3 h, solvent-thermal reaction was carried out at 180 °C for 24 h. After taking out, washing and drying, a black solid sample of MoS2 / CeZnCuS was obtained. x The above medicines were weighed, dissolved in deionized water, and then used.

[0038] S4. Preparation of AuNR solution (1) Preparation of gold seed solution: 10 mL 0.1 M CTAB solution was uniformly mixed with 0.1 mL 25 mM HAuCl4 solution, and then 0.6 mL 0.1 M NaBH4 solution was added. After continuous stirring for 2 min, the solution was allowed to stand for aging for 30 min to obtain gold seed solution; (2) Preparation of AuNR solution: 0.8 mL 25 mM HAuCl4 solution was added to 20 mL mixed solution containing 0.1 M CTAB and 16 mM NaOL, and the mixed solution was subjected to water bath at 30 °C for 90 min. The pH value of the mixed solution was adjusted to 7 by using concentrated HCl. Then, 64 μL 0.1 M ascorbic acid solution, 0.75 mL 10 mM AgNO3 solution and 16 μL gold seed solution were sequentially added to the mixed solution. After stirring for 30 s, the solution was allowed to stand for reaction at 30 °C for 12 h. The product was purified by centrifugation at 8000 rpm for 20 min and then at 5000 rpm for 10 min, and then dispersed in deionized water to obtain AuNR solution. S5. Preparation of FTO / MoS2 / CeZnCuS x Preparation of FTO / MoS2 / CeZnCuS / AuNR: Take FTO glass electrode, clean with acetone, ethanol, ultrapure water respectively for 10 min, and dry in the oven for 30 min. Then take 20 μL of 4 mg / mL MoS2 / CeZnCuS solution and drop it on the FTO glass electrode. x Preparation of FTO / MoS2 / CeZnCuS / AuNR: Take FTO glass electrode, clean with acetone, ethanol, ultrapure water respectively for 10 min, and dry in the oven for 30 min. Then take 20 μL of 4 mg / mL MoS2 / CeZnCuS solution and drop it on the FTO glass electrode. x Preparation of FTO / MoS2 / CeZnCuS / AuNR: Take FTO glass electrode, clean with acetone, ethanol, ultrapure water respectively for 10 min, and dry in the oven for 30 min. Then take 20 μL of 4 mg / mL MoS2 / CeZnCuS solution and drop it on the FTO glass electrode. x Preparation of FTO / MoS2 / CeZnCuS / AuNR: Take FTO glass electrode, clean with acetone, ethanol, ultrapure water respectively for 10 min, and dry in the oven for 30 min. Then take 20 μL of 4 mg / mL MoS2 / CeZnCuS solution and drop it on the FTO glass electrode. x Preparation of FTO / MoS2 / CeZnCuS / AuNR: Take FTO glass electrode, clean with acetone, ethanol, ultrapure water respectively for 10 min, and dry in the oven for 30 min. Then take 20 μL of 4 mg / mL MoS2 / CeZnCuS solution and drop it on the FTO glass electrode. S6. Preparation of thrombin sensing platform: Take 10 μL of 1 μM aptamer apt and drop it on the FTO / MoS2 / CeZnCuS / AuNR surface. x Preparation of thrombin sensing platform: Take 10 μL of 1 μM aptamer apt and drop it on the FTO / MoS2 / CeZnCuS / AuNR surface. x Preparation of thrombin sensing platform: Take 10 μL of 1 μM aptamer apt and drop it on the FTO / MoS2 / CeZnCuS / AuNR surface. x Preparation of thrombin sensing platform: Take 10 μL of 1 μM aptamer apt and drop it on the FTO / MoS2 / CeZnCuS / AuNR surface.

[0039] Comparative Example 1 The difference between this comparative example and Example 3 is that FTO / ZnS is prepared. Comparative Example 2

[0040] The difference between this comparative example and Example 3 is that FTO / CuS is prepared. Comparative Example 3

[0041] The difference between this comparative example and Example 3 is that FTO / ZnCuS is prepared. x . Comparative Example 4

[0042] The difference between this comparative example and Example 3 is that FTO / CeZnCuS is prepared. x .

[0043] Figure 2 The photoelectrochemical properties of the prepared materials are shown in Figure 5. The FTO / MoS2 / CeZnCuS / AuNR showed higher photocurrent density than FTO / ZnS, FTO / CuS and FTO / ZnCuS alone.x Compared with the electrode, FTO / CeZnCuS x The electrode generates a larger photocurrent, indicating that the doping of Ce elements helps to separate the electron-hole pairs and has good photoelectric response performance. x The photocurrent is 20.06 μA / cm 2 , which is about 23.61 times that of single FTO / ZnS (0.84 μA / cm 2 ), 6.59 times that of FTO / CuS (3.04 μA / cm 2 ) and 2.41 times that of FTO / ZnCuS (8.39 μA / cm 2 ).

[0044] like Figure 3 At the same time, the optimization experiment of different Ce doping amounts on the photoelectric performance of the material was carried out. By optimizing the five Ce doping amounts, it was found that excessive or small amounts of doping would lead to a decrease in the current value. Therefore, 5% CeZnCuS x The photocurrent response value is the best.

[0045] Comparative Example 5 The difference between this comparative example and Example 3 is that the modified electrode is removed, as follows: Figure 4 The photocurrent response diagram of different modified electrodes is shown in Figure 2. The bare FTO has almost no photocurrent signal (curve a), the photocurrent of the MoS2 modified FTO electrode is 2.38 mA (curve b), and the CeZnCuS x The photocurrent of the modified FTO electrode is 1.98 mA (curve c), and the photocurrent of FTO / MoS2 / CeZnCuS x The photocurrent response of the electrode is significantly increased to 7.46 mA, which is 3.1 times that of the MoS2 electrode (curve d). The above experimental results show that FTO / MoS2 / CeZnCuS x Electrode with FTO / MoS2 and FTO / CeZnCuS x Compared with the electrode, it has better charge separation ability, can effectively prevent the recombination of carriers, improve the utilization rate of carriers, and promote the photoelectric conversion efficiency.

[0046] Select FTO / MoS2 / CeZnCuS prepared in Example 3 x / AuNR thrombin photoelectrochemical aptamer sensor for subsequent related measurements.

[0047] Performance testing: (1) X-ray powder diffraction (XRD) analysis Analysis of CeZnCuS by X-ray powder diffraction (XRD) x, MoS2 and MoS2 / CeZnCuS x Crystalline characteristics of the catalyst. Figure 5 As shown in Figure 1, it can be observed that MoS2 has four different characteristic diffraction peaks at 13.8°, 33.2°, 39.7° and 59.2°, which correspond to the (002), (100), (103) and (110) crystal planes of MoS2 (PDF#17-0744). At the same time, we can observe that CeZnCuS x The diffraction peaks of ZnS (PDF#06-0464), ZnS (PDF#05-0566) and CeS (PDF#04-0688) are CuS (PDF#06-0464), ZnS (PDF#05-0566) and CeS (PDF#04-0688). 2+ (0.74Å) and Cu 2+ The radii of Ce (0.73Å) and Ce (1.01Å) are different, and atomic doping will change the lattice spacing, resulting in CeZnCuS x There is a slight shift in the diffraction peak, indicating that CeZnCuS was successfully prepared. x In addition, peaks matching the (002) and (100) planes of MoS2 appeared at 13.8 nm and 33.2 nm, indicating that MoS2 / CeZnCuS x Heterostructure formation.

[0048] (2) Electrochemical impedance spectroscopy (EIS) measurement Electrochemical impedance spectroscopy was used to investigate the electrode surface modification process and the aptasensor construction process. Figure 6 The EIS spectra of different modified electrodes in 2mM K4[Fe(CN)6] solution containing 0.1M KCl are shown. The semicircle diameter of the EIS spectrum is the electron transfer resistance (Ret), which is the electron transfer kinetic parameter of the redox probe on the electrode surface. The Ret value of the FTO / MoS2 electrode is about 249Ω, and the FTO / CeZnCuS x The Ret value of the electrode is about 75Ω, FTO / MoS2 / CeZnCuS x The Ret value of the electrode is about 70Ω, while FTO / MoS2 / CeZnCuS x The Ret value of the FTO / MoS2 / CeZnCuS / AuNR electrode is about 50Ω. x / AuNR has high conductivity and can accelerate the electron transfer process on the electrode surface. xAfter TMB aptamer (apt) and BSA were sequentially assembled on the AuNR surface, the Ret value increased (curves e and f). This is because substances such as aptamers and proteins hindered the transfer of the redox probe to the electrode surface, causing the EIS spectrum semicircle to expand. After positively charged methylene blue (MB) was adsorbed on the apt-modified electrode, the Ret value decreased to 758, indicating the successful adsorption of the MB probe (curve g). After thrombin (TMB) was incubated on the electrode, the Ret value increased (specifically, curve ef). This is because the spatial resistance of the G-tetrad formed by TMB and the aptamer reduced the contact area between the anti-acid (AA) and the base photosensitive material to a certain extent, reducing the AA reduction h. + The increase in Ret value also matches the decrease in photocurrent, and the EIS data further confirm the successful construction of the label-free sensor.

[0049] (3) UV-Vis DRS and Mott-Schottky spectra The light absorption properties of the synthesized materials were determined using UV-Vis diffuse reflectance spectroscopy (UV-Vis DRS). Figure 9 As shown, compared with the individual MoS2, CeZnCuS x , composite MoS2 / CeZnCuS x The optical absorption at 250-550 nm is obviously red-shifted, indicating that the composite material has better visible light absorption. The band gap energy can be determined based on the Kubelka-Munk theory (αhν) 2 The correlation between the band gap Eg and the absorption coefficient α was obtained by calculating the correlation between MoS2 and CeZnCuS x The band gap energies of MoS2 and CZnCuS are 1.67eV and 2.61eV respectively. x The point where the tangent line intersects the X-axis represents the flat band potential. Figure 9 As shown, MoS2 and CeZnCuS x The CB values ​​relative to the standard calomel electrode can be measured to be -0.24 V and -0.39 V, respectively. The band gap width measured by combining UV diffuse reflectance E g and equation E g = E VB - E CB According to calculations, MoS2 and CeZnCuS x The relative valence band (VB) potentials of the two species are 1.43 V and 2.22 V (vs. standard calomel electrode), respectively.

[0050] (4) Photoelectric current response test Figure 7 For the photoelectric current response of the biological recognition process of the thrombin photoelectrochemical aptamer sensor, the electrode was measured at a bias voltage of 0.1 V (vs SCE) in a phosphate buffer of 0.1 M AA pH 7.4. In FTOMoS2 / CeZnCuS x The surface plasmon resonance effect generated by the further modification of AuNRs on the surface further improved the current to 13.6 mA (curve a). In FTOMoS2 / CeZnCuS x After apt (aptamer), BSA (bovine serum albumin) and TMB were sequentially assembled on the surface of the FTO / MoS2 / CeZnCuS / AuNR, the photoelectric current intensity of the corresponding modified electrode decreased (curves b, c, e) due to the hindering of the transfer of the redox probe to the electrode surface by substances such as aptamer, protein, etc., wherein curve e is higher than a (e is apt / BSA / MB / TMB modified), which indicates the successful immobilization of apt, BSA and TMB; after the positively charged MB was adsorbed on the apt modified electrode, the signal increased (curve d), indicating the successful adsorption of the MB probe; after the TMB analyte was specifically bound to its aptamer, the photoelectric current intensity decreased (e.g. curve e). The results show that the label-free TMB aptamer sensor is successfully prepared. Figure 7

[0051] As shown in Figure 8 -C, the original MoS2 presents a hollow microflow-like morphology with a diameter of about 3 μm, which is assembled by many two-dimensional nanosheets with a thickness of about 20 nm. According to Figure 8 -D, CeZnCuS x nanoparticles, Figure 8 in D have a particle size of about 30-60 nm. According to Figure 8 -E, CeZnCuS x nanoparticles are attached to the surface of MoS2, forming a 0D / 3D heterojunction, which improves the photoelectric performance. In addition, the MoS2 / CeZnCuS x composite catalyst was analyzed by energy dispersive X-ray (EDX) spectroscopy and mapping, and the results are shown in Figure 8 -G. The results show that Mo, Ce, Zn, Cu, S and Au elements coexist in the MoS2 / CeZnCuS x / AuNR composite material.

[0052] (5) TMB determination Figure 10 The change in the photoelectric current signal before and after the binding of TMB was detected by using transient photoelectric current test, and the photoelectric current response was measured in blank PBS. The linear relationship between the current drop and the logarithm of the TMB concentration, the linear regression equation is Δ​I =2.329 log c (TMB) +5.435, the linear detection range is 0.02-5000pM, and the detection limit is 8.5 fM (S / N=3).

[0053] (6) Analytical method selectivity test To demonstrate the selectivity of the analytical method, MoS2 / CeZnCuS x / AuNR electrodes can detect possible external interferences of common proteins, such as Figure 12 , including human immunoglobulin G, rat immunoglobulin, bovine serum albumin, fibrinogen, blank samples, and mixed samples. The results showed that only TMB showed a significant photocurrent response, while the other samples showed almost no significant signal change compared to TMB. The good selectivity of TMB can be attributed to the specific recognition of TMB by the aptamer.

[0054] In addition, the stability and reproducibility of the photoelectrochemical immunosensor were also investigated. Figure 11 , 5 parallel experiments were performed in blank PBS, and the relative standard deviation (RSD) was 2.31%-3.85%. Secondly, the prepared electrode was stored in a 4℃ refrigerator for 20 days, and its response was 89% of the original. In summary, FTO / MoS2 / CeZnCuS x / AuNR has good reproducibility and stability.

[0055] (7) Detection of thrombin in human serum samples To investigate the practical application capabilities of this thrombin biosensor, clinical serum samples were tested, as shown in Table 1. Prior to testing, the human serum samples were diluted 50-fold with pH 7.4 PBS buffer to the appropriate concentration. The recoveries ranged from 98.5% to 104%, demonstrating the potential of this PEC enzyme biosensor for detecting thrombin in real-world samples.

[0056] Table 1 Detection of TMB in clinical serum samples by standard addition method Samples Add (pM) Found (pM) RSD (%, n=3) Recovery (%) 1 0.5 0.52 3.85 104.0 2 2.0 1.97 2.64 98.5 3 10.0 9.98 2.31 99.8 The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any person skilled in the art can, without departing from the spirit and technical solution of the present invention, make many possible changes and modifications to the technical solution of the present invention using the methods and technical contents disclosed above, or modify it into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A MoS-based 2 / CeZnCuS x / AuNR thrombin photoelectrochemical aptamer sensor, characterized by: The thrombin photoelectrochemical aptamer sensor is composed of MoS2 / CeZnCuS x / AuNR composite material and FTO electrode; the MoS2 / CeZnCuS x The AuNR composite material is a nano hollow micro flower structure with a diameter of 2-5 μm, an inner pore diameter of 0.2-0.8 μm, and a specific surface area of ​​20-30 m 2 / g.

2. A MoS-based 2 / CeZnCuS x The preparation method of the thrombin photoelectrochemical aptamer sensor based on AuNR is as follows: S1. Preparation of MoS2: Add CTAB, CH4N2S, and Na2MoO4 to deionized water, stir well, perform hydrothermal reaction, centrifuge, wash, and dry to obtain MoS2. S2.CeZnCuS x Preparation: Ce(CH3CO2)3, Zn(CH3CO2)2, and Cu(CH3CO2)2 are dispersed in ethanol and ultrasonically treated for 20-50 minutes to obtain a dispersion. A mixed solution of C2H5NS and ethanol is then added. After stirring at room temperature for 2-5 hours, a solvent thermal reaction is carried out. The mixture is taken out, washed, and dried to obtain CeZnCuS x ; S3.MoS2 / CeZnCuS x Preparation of solution: Disperse the prepared MoS2, Ce(CH3CO2)3, Zn(CH3CO2)2 and Cu(CH3CO2)2 in ethanol to obtain a dispersion, then add a mixed solution of ethanol and C2H5NS, stir for 2-4 hours, and then perform solvent thermal reaction. Then, remove, wash and dry to obtain MoS2 / CeZnCuS x , dissolved in deionized water to obtain MoS2 / CeZnCuS x solution; S4.FTO / MoS2 / CeZnCuS x Preparation of / AuNR: Dissolve AuNR into MoS2 / CeZnCuS x The solution was stirred, centrifuged and vacuum dried to obtain MoS2 / CeZnCuS x / AuNR, add it dropwise onto the FTO glass electrode and calcine for 20-50 min to obtain FTO / MoS2 / CeZnCuS x / AuNR; S5. Preparation of thrombin sensing platform: Take the thiol-modified TMB aptamer apt and drop it on FTO / MoS2 / CeZnCuS x / AuNR surface, stored at 4 ° C overnight, to obtain MoS2 / CeZnCuS x / AuNR / apt, washed with PBS, blocked with BSA, immersed in MB, washed, and the photocurrent response was recorded in blank pH 7.4 PBS; TMB was applied to the electrode surface, incubated, and the photocurrent response value was recorded.

3. A method based on MoS2 / CeZnCuS according to claim 2 x The preparation method of the thrombin photoelectrochemical aptamer sensor of / AuNR is characterized in that: In step S1, the mass volume ratio of CTAB, CH4N2S, Na2MoO4 and deionized water is (0.08-0.12) g: (0.66-0.70) g: (0.70-0.74) g: (40-60) mL; the temperature of the hydrothermal reaction is 200-240° C. and the time is 20-30 h.

4. A MoS-based 2 / CeZnCuS x The preparation method of the thrombin photoelectrochemical aptamer sensor of / AuNR is characterized in that: In step S2, the mass volume ratio of Ce(CH3CO2)3, Zn(CH3CO2)2, Cu(CH3CO2)2 and ethanol is (1.0-1.2) g:(0.73-0.77) g:(0.55-0.58) g:(60-70) mL; the mass volume ratio of C2H5NS and ethanol in the mixed solution is (0.91-0.95) g:(12-25) mL; the volume ratio of the mixed solution to the dispersion is 1:(2-5); the temperature of the solvothermal reaction is 160-200°C and the time is 22-26 h.

5. A MoS-based 2 / CeZnCuS x The preparation method of the thrombin photoelectrochemical aptamer sensor of / AuNR is characterized in that: In step S3, the mass volume ratio of MoS2, Ce(CH3CO2)3, Zn(CH3CO2)2, Cu(CH3CO2)2, and ethanol is (0.11-0.15) g:(0.93-1.25) g:(0.74-0.77) g:(0.56-0.59) g:(60-70) mL; the mass volume ratio of C2H5NS and ethanol in the mixed solution is (0.71-1.11) g:(12-25) mL; the volume ratio of the mixed solution to the dispersion is 1:(2-5); the temperature of the solvothermal reaction is 160-200°C, and the time is 22-26 h.

6. A method based on MoS2 / CeZnCuS according to claim 2 x The preparation method of the thrombin photoelectrochemical aptamer sensor of / AuNR is characterized in that: The preparation steps of the AuNR solution in step S4 are as follows: (1) Preparation of gold seed solution: After evenly mixing CTAB solution and HAuCl4 solution, add NaBH4 solution, continue stirring for 1-4 minutes, and let it stand for 20-50 minutes to obtain a gold seed solution; (2) Preparation of AuNR solution: After the CTAB solution and NaOL solution are evenly mixed, HAuCl4 solution is added, and the mixture is placed in a water bath at 20-50°C for 70-100 min. The pH is adjusted to 6-8 to obtain a mixed solution. Then, ascorbic acid solution, AgNO3 solution and gold seed solution were added to the mixed solution in sequence. After stirring for 20-50 s, the mixture was allowed to react at 20-50°C for 10-15 h. The mixture was first centrifuged at 6000-12000 rpm for 10-40 min and then purified by centrifugation at 3000-8000 rpm for 8-15 min. The product was collected and dispersed in deionized water to obtain the AuNR solution.

7. A method based on MoS2 / CeZnCuS according to claim 6 x The preparation method of the thrombin photoelectrochemical aptamer sensor of / AuNR is characterized in that: The molar concentration ratio of the CTAB solution, HAuCl4 solution and NaBH4 solution in step (1) is 40:1:(1-2).

8. A method based on MoS2 / CeZnCuS according to claim 6 x The preparation method of the thrombin photoelectrochemical aptamer sensor of / AuNR is characterized in that: In step (2), the molar concentration ratio of the CTAB solution, the NaOL solution, and the HAuCl4 solution is 200:16:(4-6); and the volume ratio of the mixed solution, the ascorbic acid solution, the AgNO3 solution, and the gold seed solution is (18-24) mL:(0.05-0.15) M:(10-12) mL.

9. A method based on MoS2 / CeZnCuS according to claim 2 x The preparation method of the thrombin photoelectrochemical aptamer sensor of / AuNR is characterized in that: In step S4, MoS2 / CeZnCuS x The molar concentration ratio of the solution to the AuNR solution was 4:(1-2).

10. Use of the thrombin photoelectrochemical aptasensor prepared by the preparation method according to any one of claims 1 to 9 in detecting thrombin.

Images