CN113552199A

CN113552199A - Based on FeS2Molecular imprinting electrochemical sensor of/C/MQDs/GCE modified electrode and preparation method thereof

Info

Publication number: CN113552199A
Application number: CN202110866986.0A
Authority: CN
Inventors: 鲁志伟; 孙萌萌; 李婷; 王妍媖; 饶含兵; 杜鑫
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2021-07-29
Filing date: 2021-07-29
Publication date: 2021-10-26
Anticipated expiration: 2041-07-29
Also published as: CN113552199B

Abstract

The invention provides a FeS-based method₂Molecular imprinting electrochemical sensor of/C/MQDs/GCE modified electrode and preparation method thereof, FeS₂The preparation method of the/C/MQDs/GCE modified electrode sequentially comprises the following steps: mixing MQDs with FeS₂Respectively dispersing in chitosan-acetic acid solution; dripping the MQDs solution on GCE, and drying to obtain MQDs/GCE; FeS is prepared₂Dripping the/C solution on MQDs/GCE, and drying to obtain FeS₂the/C/MQDs/GCE modifies the electrode. The invention also discloses the modified electrode prepared by the method, a molecularly imprinted electrochemical sensor based on the modified electrode, and a preparation method and application of the molecularly imprinted electrochemical sensor. The method has good reproducibility, acceptable stability and high selectivity, simultaneously effectively realizes the simultaneous determination of the dipyridamole and the quinine sulfate, and solves the problems of complex and expensive detection mode of the dipyridamole and the quinine sulfate and the like.

Description

Based on FeS2Molecular imprinting electrochemical sensor of/C/MQDs/GCE modified electrode and preparation method thereof

Technical Field

The invention belongs to the technical field of double-template molecularly imprinted electrochemical sensors, and particularly relates to a FeS-based molecularly imprinted electrochemical sensor₂A molecular imprinting electrochemical sensor of a/C/MQDs/GCE modified electrode and a preparation method and application thereof.

Background

Dipyridamole (DIP) is a coronary-dilating and antithrombotic agent widely used in the treatment of cardiovascular diseases and also in the control of cancer cell proliferation. However, uncontrolled use of DIP can lead to psychiatric illness and serious secondary effects and serious health risks. Meanwhile, DIP can also be used as an excitant in sports, and causes deceptive results in sports occasions. Thus, the use of DIP is of great significance in medical and sporting events. Quinine Sulfate (QS) is a quinoline derivative that binds to DNA of the malarial parasite to form a complex, inhibiting DNA replication and RNA transcription, and thus, protein synthesis in protozoa. Can be used for treating various kinds of malignant malaria, leg cramp, pain, and fever. Quinine, however, has potential toxicity and can cause cinchona reaction, acute hemolysis (black urine heat), death, rash, itching and asthma. Therefore, it is necessary to develop a simple and inexpensive detection method to achieve highly selective and highly sensitive detection of two substances. In recent years, many analytical methods have been reported to achieve high sensitivity detection of DIP and QS, including High Performance Liquid Chromatography (HPLC), spectrophotometry, chemiluminescence, fluorescence, and the like. However, the application of the above-mentioned techniques to rapid low-cost detection is limited due to the complicated apparatus, the long pretreatment process or the expensive apparatus cost. On the contrary, the electrochemical method has the characteristics of fast response, low cost, high analysis sensitivity and the like. Recently, different electrochemical methods, such as differential pulse voltammetry, cyclic voltammetry, etc., have been used to determine DIP and QS. In addition, the selectivity and sensitivity of these voltammetry techniques can be further improved by modifying the electrode surface with various materials. Therefore, chemically modified electrodes are widely used in voltammetry to determine the sensitivity and selectivity of various analytes. Abnormal DIP and QS concentrations may lead to a range of diseases associated with human health management and treatment. Therefore, it is meaningful to construct a simple and sensitive electrochemical sensing platform to detect these substances simultaneously.

Molecular Imprinting (MIT) is a polymerization method that mimics the antigen-antibody effect. Due to its specific recognition ability for template molecules, it has received much attention in recent years. Therefore, MIT-based Molecularly Imprinted Polymers (MIPs) have higher selectivity for predetermined target molecules than other structurally similar compounds, and thus become hot spots for new sensor preparation in recent years. The manufacturing mechanism of MIPs is based on the principle of lock and key matching, including designing a polymer matrix containing cavities (locks) that are complementary to the target molecules (keys) in different ways, such as size, shape or other chemical interactions. During synthesis of MIPs, first the template molecule (key) and the functional monomer form an interacting polymer; the template is then removed by polar or acid-base solvents or electrolytic techniques to form a three-dimensional microporous structure with specific binding sites of matching size and shape. Thus, MIPs have excellent selectivity and can specifically recognize and bind substances having similar structures and properties to imprinted pores.

MIPs have high efficiency in analyte selection and retention and therefore have great potential for use in the field of biosensors. However, the combination of MIP and electrochemical methods can enhance the sensitivity and specific recognition of the template molecule by enhancing surface adsorption and binding capacity as well as electrocatalytic effects. In addition, the double-template molecular imprinting technology can realize simultaneous determination of two target analytes, greatly improves the utilization rate of the sensor, saves the determination time and improves the determination efficiency. Recently, Mirzajani et al reported an electrochemical sensor based on graphene oxide functionalized aminopropyltriethoxysilane surface MIP for the determination of DIP. However, there is no report on the detection of QS by electrochemical molecular imprinting sensors. Therefore, it is a challenge to simultaneously measure a plurality of target molecules by a molecularly imprinted electrochemical biosensor.

Although specific binding of MIP can effectively improve selectivity of the sensor, as a polymer membrane material, its poor conductivity often limits its application in electrochemical sensing. To overcome these disadvantages and to improve the conductivity of the sensor, the choice of a conductive matrix with a high specific surface area structure as a component of the functional device has attracted a great deal of attention in sensor manufacturing. In addition, the method can be used for producing a composite materialThe sensitivity of the MIP sensor depends on the number of recognition sites on the electrode surface. Thus, preparing MIPs on the surface of materials with high specific surface areas can produce a large number of recognition sites, thereby increasing the accumulation of analytes on the electrode surface. The metal-based nano material has a mixed composite function and a high specific surface area, so that the conductivity and the electron transfer rate can be effectively improved. In addition, modifying the electrode surface with nanomaterials can amplify the electrochemical response signal. Ti₃C₂MXene Quantum Dots (MQDs) have high chemical inertness, excellent biocompatibility, and excellent photoluminescence properties. The amino and hydroxyl on the surface are more beneficial to the combination of materials and the full exertion of conductivity. Metal Organic Frameworks (MOFs) are porous coordination polymers formed by self-assembly of metal ions and organic ligands. Iron-based metal organic framework materials (Fe-MOFs) are an important branch of MOF, and have high specific surface area, high porosity, multiple active sites and excellent electrocatalytic activity. In order to further improve the conductivity of the Fe-MOFs, the Fe-MOFs can be vulcanized at high temperature to form FeS with larger specific surface area, high electrochemical activity and good biocompatibility₂a/C nanocomposite material. Finally, modifying FeS on the surface of the electrode₂the/C can amplify electrochemical signals and increase MIP recognition sites.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a FeS-based method₂Ti/MQDs/GCE modified electrode-based molecularly imprinted electrochemical sensor and preparation method thereof₃C₂Mxene Quantum Dots (MQD) and FeS₂/C nano material modified glassy carbon electrode (FeS)₂the/C/MQDs/GCE) and the application thereof in preparing a molecularly imprinted dual-template electrochemical sensor for simultaneously detecting Dipyridamole (DIP) and Quinine Sulfate (QS) have good reproducibility, acceptable stability and high selectivity, simultaneously effectively realize the simultaneous determination of dipyridamole and quinine sulfate, and solve the problems of complex and expensive detection modes of the two substances and the like.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows: provides a FeS₂Preparation method of/C/MQDs/GCE modified electrodeThe method sequentially comprises the following steps:

(1) mixing MQDs with FeS₂Dispersing the/C in 0.1-0.3 wt% chitosan-acetic acid solution to obtain MQDs solution and FeS solution₂Solution C;

(2) dripping 1-2 μ L MQDs solution onto GCE, and drying at 50-70 deg.C to obtain MQDs/GCE;

(3) 7-8 mu L of FeS₂Dripping the/C solution on MQDs/GCE, and drying at 50-70 deg.C to obtain FeS₂the/C/MQDs/GCE modifies the electrode.

Further, after the MQDs material is coated on the surface of the electrode, FeS is dripped in₂C; MQDs solution and FeS₂The concentration of the solution/C was 2 mg/mL.

The above FeS₂FeS prepared by preparation method of/C/MQDs/GCE modified electrode₂the/C/MQDs/GCE modifies the electrode.

The above FeS₂The application of the/C/MQDs/GCE modified electrode in the preparation of the molecularly imprinted electrochemical sensor.

Based on FeS₂The preparation method of the molecular imprinting electrochemical sensor of the/C/MQDs/GCE modified electrode comprises the following steps:

the FeS is prepared₂Soaking the/C/MQDs/GCE modified electrode in 0.01-0.02M phosphate buffer solution containing dipyridamole, quinine sulfate and beta-cyclodextrin, performing CV electropolymerization through a three-electrode system, soaking in mixed solution of methanol and acetic acid in a volume ratio of 7-9:2, stirring for 6-10min to remove template molecules, and obtaining the FeS-based modified electrode₂Molecular imprinting electrochemical sensor of/C/MQDs/GCE modified electrode, marked as FeS₂/C/MQDs/MIP/GCE。

Further, the molar ratio of dipyridamole, quinine sulfate and beta-cyclodextrin is 1:1: 3.

Further, methanol and acetic acid were mixed in a volume ratio of 8: 2.

Further, the range of the electropolymerization potential is-0.1-0.9V, the number of polymerization cycles is 20 cycles, and the scanning speed is 90 mV/s.

Based on FeS as described above₂FeS-based molecularly imprinted electrochemical sensor prepared by preparation method of/C/MQDs/GCE modified electrode₂the/C/MQDs/GCE modifies the molecular imprinting electrochemical sensor of the electrode.

Based on FeS as described above₂The application of the molecular imprinting electrochemical sensor of the/C/MQDs/GCE modified electrode in dipyridamole and quinine sulfate detection.

In summary, the invention has the following advantages:

1. the invention is based on titanium carbide Mxene Quantum Dots (MQD) and FeS₂/C nano material modified glassy carbon electrode (FeS)₂the/C/MQDs/GCE) and the application thereof in preparing a molecularly imprinted dual-template electrochemical sensor for simultaneously detecting Dipyridamole (DIP) and Quinine Sulfate (QS) have good reproducibility, acceptable stability and high selectivity, simultaneously effectively realize the simultaneous determination of dipyridamole and quinine sulfate, and solve the problems of complex and expensive detection modes of the two substances and the like.

2. The FeS-based catalyst obtained by the invention₂The molecular imprinting electrochemical sensor of the/C/MQDs/GCE modified electrode shows excellent analysis performance, the detection limits of DIP and QS are respectively (LOD) 8.68nM (S/N ═ 3) and 0.072 μ M (S/N ═ 3), and the linear ranges are respectively 0.05-1000 μ M and 0.4-1000 μ M. In addition, the dual-template MIP electrochemical sensor has good reproducibility, acceptable stability and high selectivity. Moreover, the sensor is also used for measuring DIP and QS in biological samples (human serum and urine) and DIP in tablets, has satisfactory recovery rate (90.12% -107.89%) and relative standard deviation (RSD, 0.38% -6.36%), and shows that the sensor can successfully measure analytes in complex samples and has wide practical application prospect in electroanalysis.

3. MQDs with rich amino functional groups are synthesized by a hydrothermal method; FeS₂the/C composite material is prepared by adopting Fe-MOFs in-situ high-temperature vulcanization technology. Then, MQDs and FeS₂the/C is assembled on the surface of the GCE layer by layer to form FeS₂the/C/MQDs/GCE, so as to enhance the conductivity and the active specific surface area of the sensor. And then, selecting functional monomers through Density Functional Theory (DFT) calculation, predicting a proper monomer to be beta-cyclodextrin (beta-CD), and providing a basis for reasonably designing the double-template MIPs. Double-template molecular imprinting electrochemical transducerSensors (FeS)₂/C/MQDs/MIP/GCE) are prepared by electropolymerization in the presence of the functional monomer beta-cyclodextrin and the template molecules DIP and QS. Differential Pulse Voltammetry (DPV), Cyclic Voltammetry (CV), and Electrochemical Impedance Spectroscopy (EIS) were used to characterize the electrochemical properties of the fabricated sensors; FeS with high affinity for DIP and QS₂the/C/MQDs/MIP/GCE sensor exhibits excellent pre-concentration capability. In addition, the prepared sensor also has good stability, reproducibility, repeatability and specificity. Moreover, the biosensor has also been successfully applied to the measurement of DIP and QS in biological samples, and also to the measurement of DIP in tablets.

Drawings

FIG. 1 is a TEM image (inset: HRTEM image) (A) of MQDs and FTIR analysis (B) of MQDs powder, FeS₂[ insertion: TEM image ] (C), FeS₂/C/MQDs/GCE(D)，FeS₂/C/MQDs/MIP/GCE(E)，FeS₂SEM and FeS of/C/MQDs/NIP/GCE (F)₂EDS-element map (G) of O, N, C, Ti, Fe, S and F in/C/MQDs;

FIG. 2 shows UV-Vis absorption spectra (Abs) and photoluminescence spectra of MQDs (ultrapure water (a) and MQDs aqueous solution (b) under 365nm UV lamp and MQDs aqueous solution under sunlight (c));

FIG. 3 is FeS₂XRD pattern of/C;

FIG. 4 shows XPS survey (A) of MQDs, C1s XPS survey (B) of MQDs, N1sXPS survey (C) of MQDs, Ti 2p XPS survey (D) of MQDs, FeS₂XPS full Spectrum for/C (E), FeS₂C1s XPS spectrum (F), FeS,/C₂Fe 2p XPS spectrum (G), FeS,/C₂S2 p XPS spectrum (H) of/C;

FIG. 5 shows (A) naked GCE (a), MQDs/GCE (b), FeS₂/C/MQDs/GCE(c)、FeS₂Before elution (d), FeS,/C/MQDs/MIP/GCE₂After elution of/C/MQDs/MIP/GCE (e), FeS₂Modified electrodes such as/C/MQDs/NIP/GCE (f) containing 5.0mM [ Fe (CN)₆]^3-/4-CV response in 0.5M KCl solution; (B) naked GCE (a), MQDs/GCE (b), FeS₂/C/MQDs/GCE(c)、FeS₂/C/MQDs/MIP/GCE(d)、FeS₂DPV reaction in 0.01M PBS buffer (pH 3.5) (40. mu.M DIP and QS); (C) naked GCE (a), MQDs/GCE (b))、FeS₂/C/MQDs/GCE(c)、FeS₂Modified electrodes before elution of/C/MQDs/MIP/GCE (d), after elution of FeS2/C/MQDs/MIP/GCE (e) containing 5.0mM [ Fe (CN)₆]^3-/4-EIS response in 0.5M KCl solution; (D) FeS₂EIS response of/C/MQDs/NIP/GCE (f) (a-e is consistent with C);

FIG. 6 shows a FeS sensor under different experimental conditions₂DPV Current response of/C/MQDs/MIP/GCE in 0.01M PBS solution containing 40. mu.M DIP and QS (in case of background subtraction): template 1: template 2: the molar ratio of functional monomers (A), the number of electropolymerization cycles (B), the pH value (C), the elution time (D), the incubation time (E) and the scanning rate (F);

fig. 7 shows the effect of eluent type: methanol: acetic acid 8:2(v/v) (a), methanol: water 1:1(v/v) (b), methanol: modified electrode FeS eluted by 5 eluents of dimethyl sulfoxide ═ 1:1(v/v) (c), hydrochloric acid (d), sodium hydroxide (e) and ethanol (f)₂DPV current response of/C/MQDs/MIP/GCE to 40 μ M DIP and QS;

FIG. 8 shows FeS₂DPV response of/C/MQDs/MIP/GCE in 0.01M PBS (pH 3.5) with different concentrations of DIP and QS: 0-500 μ M DIP in the presence of 250 μ M QS (A); a calibration curve (B) between DIP concentration and peak current; QS is 0-500. mu.M in the presence of 100. mu.M DIP (C); a calibration curve between QS concentration and peak current (D); sensor FeS₂DPV response curves of/C/MQDs/MIP/GCE in the presence of DIP (E) only and QS (G) only and corresponding linear regression equations (F) and (H);

FIG. 9 shows the passage of FeS in PBS buffers (pH 3.5) containing varying concentrations of DIP and QS₂DPV response curves (A) in concentration ranges of 0.05-1000 μ M and 0.4-1000 μ M and peak current calibration curves (B) for DIP and QS in corresponding concentration variation ranges obtained by a/C/MQDs/MIP/GCE test;

FIG. 10 shows that 50. mu.M DIP (red) and QS (black) were contained in the PBS solution (A), human urine sample (B) and human serum sample (C). In the absence of other interferents (a) and in the presence of the following interferents: glucose (b), KCl (c), MgSO₄(d)，NaCl(e)，CaCl₂(f) Sucrose (g), citric acid (h), urea (i), uric acid (j), oxalic acid (k), VC (l), DA (m), aspirin(n) DPV response in DIP and QS;

FIG. 11 shows the results of the analysis of glucose concentration at 500. mu.M, KCl, MgSO₄，NaNO₃，CaCl₂DPV response of 50 μ M DIP and QS in the presence of sucrose, citric acid, urea, uric acid, oxalic acid, VC, DA, aspirin (with background subtracted);

FIG. 12 shows FeS₂C/MQDs/MIP/GCE and FeS₂DPV response of/C/MQDs/NIP/GCE to 50 μ M DIP (A) and QS (B) and the corresponding 50 μ M structural analogs (hypoxanthine (Hx), DPV current response of DPV Ascorbic Acid (AA), Azithromycin (AZI), ceftazidime active ester (BPTA), phenol (Ph), tachyuria (INN));

FIG. 13 shows FeS in the presence of 500. mu.M DIP and QS₂(A) reproducibility and (B) reproducibility of/C/MQDs/MIP/GCE.

Detailed Description

The experimental reagents used in the invention are as follows: 2-methylimidazole (C)₄H₆N₂Not less than 98.0%), ferrous chloride, tetrahydrate (FeCl)₂·4H₂O, > 99.0%), acetone (C)₃H₆O, more than or equal to 99.5%); sublimed sulfur (S, more than or equal to 99.9%); titanium aluminum carbide (Ti)₃AlC₂Not less than 98.0%); hydrofluoric acid (HF, more than or equal to 99.5%); chitosan (C)₆H₁₁NO₄) N, the deacetylation degree is more than or equal to 95.0%); uric acid (C)₅H₄N₄O3, 99.0% or more) was purchased from Mecang reagent, Inc., Shanghai, China. Potassium ferricyanide (K)₃[Fe(CN)₆]Not less than 99.5%), potassium chloride (KCl, not less than 99.5%), potassium ferrocyanide (K)₄[Fe(CN)]·3H₂O, > 99.5%), glucose (C)₆H₁₂O₆Not less than 99.0%), ethanol (CH)₃CH₂OH > 99.5%), methanol (CH)₃OH > 99.5%), acetic acid (CH)₃COOH, not less than 99.5%), ascorbic acid (C)₆H₈O₆Not less than 99.7%), ethanol (CH)₃CH₂OH > 99.5%), ammonia (NH)₃·H₂25.0% -28.0% of O, nitric acid (HNO)₃65.0% -68.0%), ceftazidime active ester (C)₂₀H₂₂N₄O₄S₃Not less than 97 percent) dipyridamole (C)₂₄H₄₀N₈O₄Not less than 98.0%); quinine sulfate (C)₄₀H₄₈N₄O₄·H₂SO₄·2H₂O); beta-cyclodextrin (C)₄₂H₇0O₃₅Not less than 99.0%); ultrapure water (18.25 M.OMEGA.. cm) was used throughout the experiment^-1)。

The experimental equipment used in the invention is as follows: electrochemical testing was performed at room temperature using a three-electrode system. The modified glassy carbon electrode is a working electrode (diameter is 5mm), the platinum column electrode is an auxiliary electrode, and the Ag/AgCl electrode is a reference electrode. All electrodes were purchased from the limited public agency of the technology of the Tianjin British union (Tianjin, China). The apparatus for electrochemical testing was purchased from Shanghai Chenghua workstation CHI 660E. The constant temperature water bath and the ultrasonic cleaner were purchased from Shanghai Ningbo Xinzhi Biotech Co., Ltd, and the high speed refrigerated centrifuge was purchased from Zhongjia scientific instruments Co., Ltd, Zhonghui, Anhui. A vacuum freeze dryer was purchased from huaxing. The air-blast drying box is purchased from Shanghai-Hengchun scientific instruments, Inc.; the high-temperature tube furnace is purchased from Henan Chengyi experiment equipment, Inc., and the constant-temperature heating stirrer is purchased from Prohua instruments, Inc. Scanning (SEM) and Transmission Electron Microscopy (TEM) were performed using Zeiss Supra 55(Carl Zeiss AG) and JEOL2100f (JEOL), respectively. X-ray powder diffraction (XRD) patterns were recorded using a DX-2700XRD instrument (Dandong, China). X-ray photoelectron spectroscopy (XPS) was recorded using Escalab250Xi (AMICUS, Shimadzu, Japan). FTIR spectra were collected by a Fourier transform infrared spectrometer (FTIR-8400S, Shimadzu, Japan). Energy dispersive X-ray spectroscopy (EDS) and elemental mapping were characterized using JEOL2100f (JEOL).

Example 1

FeS₂Preparation of/C/MQDs/GCE modified electrode

FeS₂the/C and MQDs are synthesized according to reported literature. Thereafter, 2mg of MQDs and 2mg of FeS were added₂the/C were dispersed in 1mL of 0.2% chitosan-acetic acid solution (CS), respectively. mu.L of MQDs solution was dropped onto GCE and dried at 60 ℃. Then 7.5. mu.L of FeS₂the/C solution was dropped onto the obtained MQDs/GCE and dried at 60 deg.CAfter drying, FeS is obtained₂the/C/MQDs/GCE modifies the electrode.

Based on FeS₂Preparation of molecularly imprinted electrochemical sensor of/C/MQDs/GCE modified electrode

Will modify the electrode (FeS)₂/C/MQDs/GCE) was immersed in 0.01M phosphate buffer solution (PBS, pH 3.5) containing 1mM DIP, 1mM QS and 3mM β -CD as functional monomers, and then a modified electrode was immersed therein, and FeS was obtained by electropolymerization of CV in a three-electrode system₂C/MQDs/MIP/GCE. The electropolymerization potential range is-0.1-0.9V, the polymerization cycle number is 20 circles, and the scanning speed is 90 mV/s. After electropolymerization to form a MIP film, it is immersed in methanol: in acetic acid (v/v-8/2) solution, stirring slowly for 8min to remove the template molecule and form the imprinted cavity for DIP and QS specific recognition. Non-molecularly imprinted polymers (NIPs) are prepared in the same way as MIPs, but the polyelectrolyte solution is free of DIP and QS.

Example 2

2.4 characterization of nanomaterials

SEM and TEM were used to characterize the morphology of the composite nanomaterial during the preparation of the modified electrode. From the TEM in fig. 1A, it can be seen that the MQDs are uniformly dispersed with an average size of about 3 nm. HRTEM images (inset in fig. 1A) show the lattice characteristics of MQD particles. The lattice spacing was 0.23nm, corresponding to the 008 lattice plane of MQD. Meanwhile, Fourier transform infrared spectroscopy (FTIR) is used for characterizing the functional group types on the surface of MQDs. As shown in fig. 1B, functional groups such as-OH, -NH, -C ═ O, -C-F, and Ti-O were observed, respectively. MQD at 3105cm^-1The vibration at (a) was due to-NH groups, indicating that the surface of the MQDs was passivated with-NH groups. It is due to the presence of these functional groups that the subsequent substance binding and active site increase is facilitated.

In addition, the optical properties of the MQDs in FIG. 2 indicate that the ultraviolet absorption peak of the MQDs is about 280 nm. Meanwhile, MQDs has a fluorescence emission peak at 425nm at an excitation wavelength of 320 nm. The inset in FIG. 5 shows blue fluorescence under 365nm UV light, which becomes transparent in sunlight. Conversion of early-formed Fe-MOF to carbon-doped FeS after high-temperature sulfidation₂. It can be seen that after high temperature thiosulfation, part of the MOF morphology is preservedLeaving, part of the structure collapsed (fig. 1C). FeS₂Peaks at equal positions at 2 θ of 28.51 °, 33.08 °, 37.11 °, 40.78 °, 47.71 °, and 50.49 ° correspond to FeS, respectively₂Diffraction on (1-1-1), (2-0-0), (2-1-1), (2-2-0), (2-2-1)) crystal planes (FIG. 3) (PDF # 42-1340).

In addition, X-ray photoelectron spectroscopy (XPS) tests were also performed to further analyze the elemental composition and chemical bond status of the different samples. Fig. 4A shows three peaks of MQDs at 284.82eV, 402.60eV, and 459.64eV, which are attributed to C1s, N1s, and Ti 2p, respectively. It can also be seen from the total spectrum (fig. 4A) that the Ti content is relatively low. This is due to the high temperature and hydrothermal etching of some of the Ti during the material synthesis. The C1s spectrum (fig. 4B) of MQDs consists of C-C (284.80eV), C-N (286.28eV), C ═ O (288.54eV), and C-F (288.54eV) peaks, where C-C is from the MQDs and carbon impurities in the test bands. In the N1s spectrum (fig. 4C), 401.46eV, 401.90eV, and 407.14eV correspond to N-H, ═ NH, respectively₂ ⁺and-NO₃. Wherein the N-H band is obtained from the reaction between ammonia and a carboxyl group. However, the Ti 2p spectrum (FIG. 4D) shows two main 2p spectra at 452.05eV and 459.33eV_3/2Peaks, which are mainly due to Ti-C and Ti-O, respectively. 2p shown at 463.98eV_1/2The peak is due to Ti-O, whereas O is from dissolved oxygen. FIG. 3E shows FeS₂The three peaks of/C at 284.50eV, 163.04eV, and 709.20eV are attributed to C1S, S2 p, and Fe 2p, respectively. The C1s spectrum (fig. 4F) consisted of peaks for C-C (284.80eV), C-O (286.39eV), and O-C ═ O (288.94 eV). Notably, O-C ═ O is associated with Fe-MOF precursors. The binding energies of 707.60eV, 711.12eV, 720.35eV, and 724.35eV in the Fe 2p spectrum (FIG. 4G) correspond to (Fe-S)2p_3/2、Fe 2p_3/2、(Fe-S)2p_1/2And Fe 2p_1/2. In the S2 p spectrum (FIG. 7H), 162.87eV and 164.05eV are attributed to S ^2-2p of_3/2And 2p_1/2. The peaks at 168.95eV and 170.12eV are due to SO₄ ^2-Is caused by the formation of (a). The above results indicate that MQDs and FeS₂Successful synthesis of/C. However, in MQDs and FeS₂the/C is uniformly dispersed, and after being coated on the surface of GCE, MQDs/FeS₂the/C distribution was homogeneous (FIG. 1D), indicating a complexThe stability of the composite material film is superior to FeS₂/C, which is attributed to the rational use of chitosan. In addition, FeS shown in FIG. 1G₂EDS spectra of/C/MQDs/GCE indicated that GCE was successfully modified. In addition, FeS in FIG. 1G₂The elemental mapping of/C/MQDs/GCE shows a uniform distribution of O, N, C, Ti, Fe, S and F elements. The results show that the prepared nano material is successfully modified on the surface of the electrode. Subsequently, FeS₂C/MQDs/MIP/GCE and FeS₂SEM of/C/MQDs/NIP/GCE (after elution) are shown in FIG. 1E and FIG. 21F, respectively. It can be seen that after the MIP is formed, the electrode surface is more uniform and smooth than in fig. 1D. The MIP formed has some luminal structure and is coarser than the NIP due to the elution of the template molecules in the MIP to form the imprint cavities. Morphological and elemental analysis showed that FeS₂the/C/MQDs/MIP/GCE biosensor has been successfully prepared.

2.5 electrochemical characterization of modified electrodes

In the presence of 5.0mM [ Fe (CN)₆]^3-/4-In 0.5M KCl solution, the GCE and other modified electrodes were subjected to CV scanning (scan rate of 100mV/s) at a range of-0.2 to 0.6V to analyze the conductivity of the various modified electrodes. As shown in FIG. 5, when MQDs are modified on the electrode (curve b), the peak current intensity of MQD/GCE is improved compared to GCE (curve a). Furthermore, FeS₂After dropping the/C material into the MQDs/GCE modified electrode, the peak current increased again (curve C). The results further prove that the two prepared nano materials have good conductivity and stronger electron transfer capability. Curves d and e show the current response intensity before and after MIP elution. After electropolymerization, the peak current is significantly reduced due to poor conductivity of the MIP film formed. However, the elution process of the template molecules results in the formation of imprint cavities to expose the built-in material, thereby improving the electron transfer capability of the modified electrode surface. Thus, a significant increase in the peak current of curve d relative to the peak current of curve e can be observed. The peak current of the modified electrode after electropolymerization to form the NIP film was reduced compared to before and after the molecular imprinting elution (curve f). This is because the polymer film does not conduct a film, which prevents electrons from entering the electrode surface.

According to previous reports, DIP andQS is all electrochemically active substances. FeS₂The sensing mechanism of the/C/MQDs/MIP/GCE electrode pair DIP and QS follows two steps: first, the imprinted cavities formed on the MIP film bind specifically to the template molecules. Secondly, in FeS₂The electrochemical reduction process of DIP and QS is realized on the surface of the/C/MQDs/MIP/GCE electrode. Since this process involves electron transfer between DIP and QS, the electrochemical behavior of DIP and QS was evaluated by DPV (fig. 5B). The template molecule has a reduction peak (very weak in QS, relatively stronger in DIP) on the exposed GCE surface, with a lower current response than nanomaterial-modified GCE (curve a). This property can be attributed to the good catalytic ability of the nanomaterials on DIP and QS electroreduction processes. After MIP elution, a blot cavity matching the template size, shape and functional groups is formed. Thus, DIP and QS can be specifically recognized and bound to functional groups within the blot cavity through hydrogen bonding. Thus, the peak currents of DIP and QS were significantly enhanced after incubation (curve d). In contrast, the prepared NIP film did not have a template cavity and thus did not promote reduction of DIP and QS at the electrode surface (curve e). Thus, it can be observed that the current response of the NIP to the test subject is the lowest.

EIS was used to evaluate different modified electrodes at 5.0mM [ Fe (CN) ] in 0.5M KCl₆]^3-/4-Resistance in solution. As shown in fig. 5C, the semi-circle diameter of the Nyquist spectrum of the GCE (curve a) is significantly larger than the MQDs-modified electrode (curve b). The results indicate that GCE has a large charge transfer resistance (Rct). Modification of FeS on MQDs/GCE electrode₂after/C, the Rct value decreased significantly (curve C). This is due to FeS₂the/C has good conductivity, and the high adsorption capacity can enhance the electron transfer capacity of the composite material. Rct after MIP elution (curve e) is less than before elution (curve e) because the blot cavity formed after elution is K₄[Fe(CN)₆]/K₃[Fe(CN)₆]Provides a channel for the redox reaction. FIG. 5D shows FeS₂the/C/MQDs/NIP/GCE has the largest Rct value (curve f) since NIP consists of poly-beta-CD and no imprinted cavity. The above results are consistent with CV characterization conclusions.

Example 3

In order to improve the measurability of imprinted nanocompositesA series of experimental factors such as the molar ratio of the template to the monomer, the number of polymerization cycles, the pH value, the elution and incubation time, the scanning rate and the like are respectively researched. By making FeS under single factor parameters₂[ the electrochemical response of DIP and QS was measured by DPV in PBS buffer (pH 3.5) containing 40. mu.M of a mixture of DIP and QS/C/MQDs/MIP/GCE. The results of the optimization section are shown below.

The number and strength of molecular imprinting cavities formed by CV electropolymerization are related to the ratio of template molecules to functional monomers. As shown in FIG. 6A, the peak current response is strongest when the ratio of template to functional monomer is 1:1:3, between 1:1:0.5 and 1:1: 7. Thus, the optimal molar ratio of the template molecules DIP and QS to the functional monomer is 1:1: 3. In addition to this, the number of electropolymerization cycles is an important factor that directly affects the MIP film thickness and stability. As shown in fig. 6B, the number of polymerization turns was between 5 and 30, and it was found that the peak current was strongest when the number of polymerization turns was 20. Therefore, the optimum number of polymerization cycles is 20 cycles.

The pH of the test environment affects the extent of dissociation of the MIP from the DIP and QS functionalities, and thus the interaction of the MIP with the template molecule functionality. As shown in fig. 6C, in 0.01M PBS, the DPV peak currents for both DIP and QS increased and then decreased as the pH changed from 2 to 7. The peak current of DIP was at pH 3.0. QS, however, has the strongest peak current response at pH 4.0, which may be related to the strength of hydrogen bonds formed by specific recognition binding of template molecules to the molecularly imprinted cavity under different pH environments. Therefore, a PBS solution of 0.01M, pH 3.5.5 was determined to be the best condition for electrochemical molecular imprinting for detecting DIP and QS.

Elution time has a large influence on the number and structure of the blot cavities formed. As shown in fig. 6D, the DPV peak current gradually decreased with increasing elution time. When the elution time reached 8min, there was almost no DPV response, indicating that the template molecules were almost removed. Therefore, the optimal elution time of template molecules in the molecularly imprinted membrane was 8 min.

The length of incubation determines the number of template molecules bound to the blot cavity. As shown in fig. 6E, the DPV response of DIP and QS gradually increased between 15min and 40min, with the DPV response of DIP and QS having reached a steady state at 35min, since the cavity at the electrode surface is already in saturation. Therefore, the optimum incubation time was set to 35 min.

The selection of the scanning rate is an important factor in the preparation of molecularly imprinted membranes. As shown in FIG. 6F, DPV responses were strongest in DIP and QS at 90mV/s, between 30-130 mV/s. Therefore, the optimal scanning speed for forming the molecularly imprinted membrane is 90 mV/s.

3.7 selection of eluent

The eluent can affect the elution degree of the template molecules and cause certain damage to the molecularly imprinted membrane. As shown in FIG. 7, the eluents commonly used in 6 molecular imprinting directions were selected for comparative studies. The peak response was highest for DIP and QS when the eluent was methanol to acetic acid to 8:2 (v/v). The reason is that other eluents cause certain damage to the modified electrode due to the characteristics of the eluents in the elution process, so that the specific recognition cavity positions of the molecular imprinting membrane of the detection substance are reduced. Thus, the eluent selected in the present invention is methanol: acetic acid ═ 8:2 (v/v).

3.7MIP response characteristics and calibration curves

Under the best detection conditions, different concentrations of DIP and QS in 0.01M PBS buffer (pH 3.5) were evaluated and measured simultaneously by the DPV method. The background value obtained by the blank concentration test is subtracted to construct a standard curve. Simultaneous determination of DIP and QS is a major goal of this work. First, the effect of DIP and QS on the electrochemical response is performed with a fixed concentration of one species and an increased concentration of the other species. FIGS. 8A and 8B show DPV curves and fitted linear curves (after background subtraction) for DIP concentrations in PBS buffer (pH 3.5) in the presence of 250. mu.M QS over the interval 1-500. mu.M, respectively. The linear regression equation for DIP is: ip (μ a) ═ 0.0664C (μ M) -1.27967 (R)²0.99008). FIGS. 8C and 8D are DPV plots (after background subtraction) of QS at various concentrations (1-500. mu.M) in 100. mu.M DIP PBS buffer (pH 3.5) with a linear regression equation for QS of Ip (. mu.A) ═ 0.07176C (. mu.M) -0.45664 (R.mu.M)²0.99876). The result shows that one detection object shows linear corresponding relation along with the change of concentration in the gradient range, and the other detection object simultaneouslyThere was no significant change in the signal. This indicates that DIP and QS are utilizing FeS₂The electrochemical measurement process of the/C/MQDs/MIP/GCE has no mutual interference. Subsequently, simultaneous sensing capability was achieved by varying the concentrations of DIP and QS. Fig. 8E to 8H depict DPV curves and corresponding linear regression equations for the presence of two analytes on the biosensor alone. The linear regression equations respectively corresponding to DIP and QS are: ip (μ a) ═ 0.06966C (μ M) -1.18037 (R)²＝0.99032)，Ip(μA)＝-0.07206C(μM)-0.7109(R²0.99489). The feasibility of simultaneous detection of two detection objects is further verified by the slope comparison of the linear regression equation.

The results showed that the reduction signal peaks of DIP and QS appeared at-0.79V and-1.02V, and the DPV response concentrations of DIP and QS were 0.05-1000. mu.M and 0.4-1000. mu.M, respectively. As expected, the current response signals for DIP and QS were positively correlated with their concentrations (fig. 9B). For the range of concentration gradients, DIP and QS concentrations were regressive with current changes and are expressed as: ip (μ a) ═ 0.07138C (μ M) -0.62353 (R)²0.9919) and Ip (μ a) -0.06248C (μ M) -1.27574 (R)²0.9910). The limit of detection (LOD) for DIP and QS was calculated based on 3. sigma./S using a linear regression curve as 8.68nM and 0.072. mu.M, respectively. Wherein σ is FeS₂The standard deviation of the peak current at the lower limit of detection of the corresponding substance (n ═ 3) is/C/MQDs/MIP/GCE, and S is the slope of the calibration curve for the detected substance. The above results show that FeS₂Simultaneous detection of DIP and QS is feasible and reliable for/C/MQDs/MIP/GCE. Table 1 shows the performance characteristics of the sensors in this study compared to other reported methods or other methods in the literature. It can be seen that the sensitivity and wide linearity range of DIP and QS is almost higher in this work than other methods. It is noteworthy that there is little method for electrochemical molecular imprinting to measure QS, and the sensitivity of electrochemical assays is lower than this work. In conclusion, the method has wider linear operation range and lower LOD, and is more suitable for simultaneously measuring DIP and QS, which can be attributed to the molecular imprinting method used in the method.

TABLE 1 comparison of the preparation process according to the invention with other processes

Example 4

Selectivity, reproducibility and stability of biosensors

To evaluate FeS₂Selectivity of the/C/MQDs/MIP/GCE sensor 13 potential interferents were added to 0.01M PBS (pH 3.5) at fixed concentrations of DIP and QS, respectively, and the sensors were made in solutions containing only 50. mu.M of the analyte (a) and either 50. mu.M of the analyte and 500. mu.M glucose (b) or KCl (C), MgSO (MgSO) respectively, as shown in FIG. 10₄(d)，NaNO₃(e)，CaCl₂(f) Sucrose (g), citric acid (h), urea (i), uric acid (j), oxalic acid (k), VC (l) DA (m), aspirin (n) in different solutions. There was a slight change in the current response of DIP and QS compared to blank solution (a) ((<5%). DPV responses in DIP and QS were reduced by 9.7% and 2.2%, respectively, in the presence of 13 potential interference factors at the same time (fig. 11). The above results show that the prepared imprinted polymer nanocomposites have significant selectivity for DIP and QS, and the effect of interferents is almost negligible. Furthermore, the Imprinting Factor (IF) was used to further evaluate the selectivity of the proposed sensor. According to previous reports, IF is calculated by the following equation:

IF＝I_MIP/I_NIP (2)

wherein, I_MIPAnd I_NIPRepresents the current response of each analog detected electrochemically by imprinted and non-imprinted nanocomposite modified electrodes, respectively. As shown in FIG. 12, IF values for DIP and QS, which are 50. mu.M analytes, were calculated to be 2.917 and 2.646, respectively, demonstrating FeS₂the/C/MQDs/MIP/GCE can specifically recognize DIP and QS. Calculated imprinting factors (Hx: 1.186, AA: 1.33) for the corresponding analogs of the two test substances at a concentration of 50. mu.M0, AZI: 1.270; BPTA: 1.276, Ph: 1.280, INN: 0.937) was estimated to be 1, indicating that the sensor has nonspecific recognition ability for them. In summary, the above results indicate that the sensor system has good selectivity, which may be due to FeS₂The imprinted cavity formed on the surface of the/C/MQDs/MIP/GCE can better match the spatial structure of DIP and QS molecules.

On the basis of optimized conditions, 5 batches of molecular imprinting biosensors were prepared to study FeS₂Reproducibility of/C/MQDs/MIP/GCE. These modified electrodes were tested by the DPV method after incubation in a solution containing 50 μ M DIP and QS. The Relative Standard Deviation (RSD) values of the two assays were found to be 3.9% and 2.54% respectively in the five test results (fig. 13A), indicating good reproducibility of the prepared sensors. By using the same FeS₂the/C/MQDs/MIP/GCE sensor 5 tests were performed on a solution containing 50. mu.M DIP and QS to determine the reproducibility of the sensing system, and each test procedure followed an elution-adsorption step. RSD values for DIP and QS were calculated as 3.3% and 3.0%, respectively (fig. 13B), showing good repeatability. Thereafter, the prepared FeS₂the/C/MQDs/MIP/GCE was stored at 4 ℃ for 20 days and then the stability of the sensor system was evaluated. From the change in DPV peak current, DIP and QS responses decreased by 5.4% and 5.3%, respectively, after 20 days of storage. The above results indicate that the sensing system is reliable for DIP and QS detection applications.

Example 5

Analysis and application of sensor in actual sample

Each sample was tested 3 times using standard addition methods to calculate recovery and RSD. As shown in table 2, the recovery of DIP and QS was 95.22% to 107.89% with RSD of 0.38% to 6.36%. Table 3 compares the methods for measuring DIP in tablets, both HPLC and DPV. The recovery rate is 96.50-106.97%, and the RSD is 0.94-4.38%. The accuracy of the method was compared in conjunction with HPLC (table 4) performed on the biological samples. From the end results, it can be found that a comparison of the two methods shows an acceptable difference. These results indicate that the biosensor can be reliably used for simultaneous measurements of DIP and QS.

Table 2 results of DIP and QS measurements in actual samples (n ═ 3)

Table 3 results of DPV and HPLC determination of DIP in dipyridamole tablets (n ═ 3)

Table 4 results of determination of DIP and QS by HPLC method (n ═ 3) in the actual sample

The invention constructs a novel electrochemical molecular imprinting double detection system, and realizes the simultaneous determination of DIP and QS in an actual sample based on a double-template MIP sensor. By modifying MQDs and FeS on the surface of GCE₂and/C, realizing the amplification effect of the electric signal. Thereafter, in FeS₂MIP film (FeS) is formed on the surface of the/C/MQDs/GCE₂/C/MQDs/MIP/GCE) to improve the selectivity and sensitivity of the biosensor and increase the number of recognition sites of the template molecules. Prepared FeS was studied by CV, DPV and EIS techniques₂Electrochemical behavior of/C/MQDs/MIP/GCE. In fact, MIP sensors have a higher capacity to recognize DIP and QS than non-imprinted sensors. Double blotting of MIP electrodes and FeS₂The electrocatalysis of the/C/MQDs enhances the pre-enrichment effect and improves the FeS₂Sensitivity of DIP and QS was measured by/C/MQDs/MIP/GCE. The electrochemical current signal responses of DIP and QS sensors over the linear measurement range were 0.05-1000. mu.M (R)²0.99008) and 0.4-1000 μ M (R)²0.99876) and has been successfully applied to the simultaneous determination of DIP and QS in serum and urine. The developed MIP sensing system has good reproducibility and repeatability, acceptable stability and high selectivity. In addition, the recovery of the samples and tablets ranged from 95.22% to 107.89%And the RSD value is between 0.38% and 6.36%, which shows that the method has good biomedical application prospect.

While the present invention has been described in detail with reference to the illustrated embodiments, it should not be construed as limited to the scope of the present patent. Various modifications and changes may be made by those skilled in the art without inventive step within the scope of the appended claims.

Claims

1. FeS₂The preparation method of the/C/MQDs/GCE modified electrode is characterized by sequentially comprising the following steps of:

2. The FeS of claim 1₂The preparation method of the/C/MQDs/GCE modified electrode is characterized in that after the MQDs material is coated on the surface of the electrode, FeS is dripped in₂C; MQDs solution and FeS₂The concentration of the solution/C was 2 mg/mL.

3. FeS according to any of claims 1-2₂FeS prepared by preparation method of/C/MQDs/GCE modified electrode₂the/C/MQDs/GCE modifies the electrode.

4. FeS according to claim 3₂The application of the/C/MQDs/GCE modified electrode in the preparation of the molecularly imprinted electrochemical sensor.

5. Based on FeS₂The preparation method of the molecular imprinting electrochemical sensor of the/C/MQDs/GCE modified electrode is characterized by comprising the following steps:

FeS according to claim 3₂Soaking the/C/MQDs/GCE modified electrode in 0.01-0.02M phosphate buffer solution containing dipyridamole, quinine sulfate and beta-cyclodextrin, performing CV electropolymerization through a three-electrode system, soaking in mixed solution of methanol and acetic acid in a volume ratio of 7-9:2, stirring for 6-10min to remove template molecules, and obtaining the FeS-based modified electrode₂Molecular imprinting electrochemical sensor of/C/MQDs/GCE modified electrode, marked as FeS₂/C/MQDs/MIP/GCE。

6. FeS-based according to claim 5₂The preparation method of the molecular imprinting electrochemical sensor of the/C/MQDs/GCE modified electrode is characterized in that the molar ratio of dipyridamole, quinine sulfate and beta-cyclodextrin is 1:1: 3.

7. FeS-based according to claim 5₂The preparation method of the molecular imprinting electrochemical sensor of the/C/MQDs/GCE modified electrode is characterized in that methanol and acetic acid are mixed according to the volume ratio of 8: 2.

8. FeS-based according to claim 5₂The preparation method of the molecular imprinting electrochemical sensor of the/C/MQDs/GCE modified electrode is characterized in that the range of the electropolymerization potential is-0.1-0.9V, the polymerization cycle number is 20 circles, and the scanning speed is 90 mV/s.

9. FeS-based according to any one of claims 5 to 8₂FeS-based molecularly imprinted electrochemical sensor prepared by preparation method of/C/MQDs/GCE modified electrode₂the/C/MQDs/GCE modifies the molecular imprinting electrochemical sensor of the electrode.

10. FeS-based according to claim 9₂The application of the molecular imprinting electrochemical sensor of the/C/MQDs/GCE modified electrode in dipyridamole and quinine sulfate detection.