Molecularly imprinted electrochemical sensor for detecting tacrolimus and preparation method thereof

Abstract

The application discloses a molecular imprinting electrochemical sensor for detecting tacrolimus. An electrochemical polymerization method is adopted, tacrolimus is used as a template molecule, o-phenylenediamine and chloroauric acid are used as functional monomers, an electrochemical copolymerization reaction occurs on the surface of a gold electrode, and poly-o-phenylenediamine and gold nanoparticles are generated. Then, the template molecule is eluted, and an organic / inorganic hybrid containing a target molecular imprint is prepared on the surface of the gold electrode. The gold electrode with the hybrid modified on the surface is immersed into a bottom solution containing potassium ferricyanide, a three-electrode system of an electrochemical workstation is adopted to detect redox signals by using electrochemical cyclic voltammetry and differential pulse voltammetry. Samples containing different concentrations of tacrolimus are added dropwise on the surface of the modified electrode, and then the redox signal intensity is measured by immersing into the bottom solution. The relationship between the concentration of tacrolimus and the corresponding electrochemical signal intensity is established, and thus a molecular imprinting electrochemical sensor is developed for efficient detection of tacrolimus.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of tacrolimus detection methods and the preparation of molecularly imprinted electrochemical sensors. Specifically, it relates to the preparation of an organic / inorganic hybrid containing target molecular imprints using an electrochemical polymerization method, and the use of this hybrid to prepare a molecularly imprinted electrochemical sensor for the efficient detection of tacrolimus in serum samples. Background Technology

[0002] Tacrolimus (or FK506) is a potent macrolide immunosuppressant with high lipid solubility and low water solubility. Its mechanism of action involves specifically interacting with intracellular FK506-binding proteins to form a complex, thereby inhibiting calcineurin activity, blocking the nuclear translocation of T cell nuclear factors, and ultimately inhibiting the transcription and release of key cytokines such as interleukin-2. Tacrolimus has a potent inhibitory effect on T cell-mediated immune responses and has been developed as a first-line immunosuppressant after solid organ transplantation (liver, kidney, heart, etc.), significantly reducing the incidence of acute rejection. Therefore, accurate detection of tacrolimus in physiological samples is of great significance for monitoring human health. Currently, existing detection technologies face many challenges; the effective concentration of tacrolimus is as low as a few nanograms per milliliter, requiring highly sensitive methods for detection. Major metabolites of tacrolimus, such as 13-O-demethyltacrolimus, can interfere with the specificity of the detection. Furthermore, the high lipid solubility and protein binding rate of tacrolimus also increase the difficulty of achieving accurate detection in whole blood. Therefore, developing novel and efficient tacrolimus detection methods that are convenient, sensitive, and resistant to interference has become a key technological requirement for realizing personalized medication and monitoring the prognosis of transplant patients.

[0003] In recent years, research on the detection of tacrolimus blood concentration has made progress, mainly involving detection methods such as high-performance liquid chromatography (HPLC), mass spectrometry (MS), immunoassay, spectroscopic analysis, and sensor analysis techniques. For example, Yang Zhe et al. disclosed a tacrolimus blood concentration detection device based on hyperspectral imaging (patent application publication number CN119125011A); Xie Jie et al. developed a method for rapid detection of tacrolimus content in plasma using paper spray mass spectrometry (patent application publication number CN120314497A); Chang Qingyao et al. designed a rapid tacrolimus drug concentration detection device based on microfluidic chips and spectrometers (patent application publication number CN120801217A); and Hu Yongyue et al. developed a reagent kit for tacrolimus detection based on chemiluminescent immunoassay (patent application publication number CN120761629A). These methods have certain advantages in the detection of tacrolimus blood concentration, but they also have problems such as complex sample pretreatment procedures, long processing time, expensive instruments and high maintenance costs, low specificity, and complex operation. In contrast, molecularly imprinted electrochemical sensors offer significant advantages in the high sensitivity and specificity of target molecule detection. Molecularly imprinted technology (MIP) is a technique for constructing artificial molecular recognition materials through biomimetic design. Its core principle lies in mimicking the "lock-and-key mechanism" of biological systems. MIP uses the target molecule as a template, forming a complex through covalent or non-covalent bonding between functional monomers (such as acrylic acid and vinylpyridine) and the template molecule. This complex is then polymerized and solidified under the action of a crosslinking agent (such as ethylene glycol dimethacrylate), forming a three-dimensional rigid network structure. After eluting the template molecule, the imprinted cavities remaining in the polymer backbone exhibit a high degree of matching with the target molecule in terms of spatial configuration and functional group distribution, endowing MIP materials with specific recognition capabilities.

[0004] Based on this, the present invention discloses a molecularly imprinted electrochemical sensor for detecting tacrolimus. Using an electrochemical polymerization method, tacrolimus is used as a template molecule, and o-phenylenediamine and chloroauric acid are used as functional monomers. An electrochemical copolymerization reaction occurs on the surface of a gold electrode (Au), generating poly(1,2-diaminobenzene) (pDAB) and gold nanoparticles (AuNPs). The template molecule is then eluted, preparing an organic / inorganic hybrid (Au / AuNPs / pDAB-MIP) with target molecular imprinting on the gold electrode surface. The gold electrode with this hybrid surface modified is immersed in a substrate containing potassium ferricyanide, and electrochemical cyclic voltammetry is performed using a three-electrode system of an electrochemical workstation to detect the redox signal. Samples containing different concentrations of tacrolimus are dropped onto the modified electrode surface and then immersed in the substrate to measure their redox signal intensity. The relationship between tacrolimus concentration and its corresponding electrochemical signal intensity was established, leading to the development of a molecularly imprinted electrochemical sensor for efficient tacrolimus detection. To date, there are no domestic or international patents or literature reports on the electrochemical copolymerization of tacrolimus molecularly imprinted polymers on electrode surfaces, or on the development of molecularly imprinted electrochemical sensors based on these polymers for efficient tacrolimus detection. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a molecularly imprinted electrochemical sensor for detecting tacrolimus and its preparation method. An organic / inorganic hybrid containing a target molecular imprint is prepared using an electrochemical polymerization method, which is then used to prepare a molecularly imprinted electrochemical sensor and applied to the efficient detection of tacrolimus in serum samples.

[0006] To achieve the above objectives, the present invention relates to a method for preparing a molecularly imprinted electrochemical sensor for detecting tacrolimus, the method comprising the following steps:

[0007] (1) Electrochemical copolymerization: The gold electrode was polished, washed with water and sonicated. Tacrolimus was used as the template molecule and o-phenylenediamine and chloroauric acid were used as functional monomers. They were added to the substrate containing acidic buffer solution and inserted into the gold electrode. Electrochemical polymerization was carried out on its surface. The applied cyclic potential was 0-1 V, the scan rate was 10-100 mV / s, and 10-50 cyclic voltammetric scans were performed. The copolymerization process was characterized by cyclic voltammetry curves.

[0008] (2) Elution of template molecules: After the scan, the surface of the gold electrode is immersed in the alcohol solution and treated with magnetic stirring for 10-30 minutes to remove the tacrolimus template molecules in the polymer system, and a polymer containing tacrolimus molecular imprint is obtained. The selective binding sites of tacrolimus are left in the molecular imprinted polymer matrix. The response before and after template molecule removal is characterized by the curve determined by cyclic voltammetry.

[0009] (3) Electrochemical signal response: An organic / inorganic hybrid of poly(o-phenylenediamine) (pDAB) coated with gold nanoparticles (AuNPs) and containing tacrolimus molecularly imprinted (MIP) was prepared on the surface of a gold electrode (Au). Its morphology was characterized by scanning electron microscopy. The modified electrode surface (Au / AuNPs / pDAB-MIP) was inserted into a tacrolimus methanol solution with a concentration of 0-100 ng / mL and incubated for 1-10 minutes. Then, the electrode surface was gently rinsed with methanol and then inserted into a substrate containing potassium ferricyanide. Electrochemical cyclic voltammetry was performed at a potential of 0-0.5 V and the scan rate was adjusted to 50-150 mV / s.

[0010] (4) Construction of molecularly imprinted electrochemical sensor: Au / AuNPs / pDAB-MIP was used as the working electrode interface. Differential pulse voltammetry (DPV) curves of the sensing interface were measured at different tacrolimus concentrations. The relationship between different tacrolimus concentrations and the corresponding peak current intensity of the DPV curve was established, and an excellent linear relationship was fitted. Thus, a molecularly imprinted electrochemical sensor for tacrolimus detection was constructed. The linear detection range of tacrolimus concentration was 0.0001-100 ng / mL, and the detection limit was 0.05-1 picogram / mL.

[0011] (5) Detection of tacrolimus in actual samples: Take a certain amount of real serum sample, add a certain amount of tacrolimus to it, and form a homogeneous mixture by shaking it thoroughly. The concentration of tacrolimus in the mixture is adjusted to 0-10 ng / mL. Then, using the molecularly imprinted electrochemical sensor method constructed above, the DPV curve and current peak intensity corresponding to different homogeneous mixtures are measured. Based on the linear relationship between tacrolimus concentration and current peak intensity fitted in step (4), the concentration of tacrolimus in the sample system corresponding to different current peak intensities is calculated. The consistency between the detected value and the spiked value of tacrolimus concentration is verified by repeated measurement experiments, as well as the high recovery rate and low relative standard deviation of the detected value, thereby confirming that the molecularly imprinted electrochemical sensor designed in this invention has the ability to efficiently detect tacrolimus in actual samples.

[0012] The present invention discloses a molecularly imprinted electrochemical sensor for detecting tacrolimus and its preparation method. Its significant features and advantages are mainly reflected in the following aspects:

[0013] 1. A highly sensitive and selective molecularly imprinted electrochemical sensor doped with gold nanoparticles was prepared for the detection of tacrolimus. The sensor was fabricated using a simple one-step electrochemical polymerization method followed by in-situ gold reduction. The incorporation of gold nanoparticles into the molecularly imprinted polymer layer increased the effective surface area of ​​the sensor, improved electron transfer on the polymer, and provided effective current modulation in response to the capture of the target analyte tacrolimus, thereby enhancing the detection sensitivity.

[0014] 2. This molecularly imprinted polymer uses tacrolimus as a template, forming hydrogen bonds with tacrolimus through the abundant amino groups in o-phenylenediamine, and then doping it with gold nanoparticles. Poly(o-phenylenediamine) possesses biocompatibility and mechanical stability, which is beneficial for fabricating a thin and dense polymer matrix required for rapid and stable sensing responses. The introduction of gold nanoparticle doping promotes rapid electron transfer across the molecularly imprinted polymer layer between the electrochemical redox probe and the electrode interface.

[0015] 3. The binding of target molecules within the imprinted cavity inhibits electron transfer, leading to a change in the current response. The presence of gold nanoparticles near the imprinted cavity allows the trapped target molecules to effectively block electron transfer through the gold nanoparticles, thereby improving detection sensitivity. This copolymerization process increases the effective surface area of ​​the molecularly imprinted polymer, endowing the molecularly imprinted polymer sensor with excellent detection sensitivity.

[0016] 4. This high-performance and stable tacrolimus molecularly imprinted sensor was demonstrated on discrete electrodes that are difficult to miniaturize. Molecular imprinting technology and electrochemical sensing methods can be applied to screen-printed electrodes in the future to facilitate device integration for point-of-care applications. Furthermore, molecular imprinting technology is easily modified and can selectively capture different targets to achieve multi-channel detection. The superior performance and fabrication method of the molecularly imprinted electrochemical sensor of this invention validate its great potential for low-cost and efficient detection of tacrolimus in biological samples. Attached Figure Description

[0017] Figure 1 A schematic diagram of the fabrication process of a molecularly imprinted electrochemical sensor for detecting tacrolimus.

[0018] Figure 2 Cyclic voltammetry response curves of the electropolymerization process of polymer AuNPs / pDAB on the gold electrode surface.

[0019] Figure 3 Cyclic voltammetry response curves of bare gold electrode, AuNPs / pDAB electrochemical copolymer-modified gold electrode, and copolymer-modified gold electrode after elution of tacrolimus template molecules.

[0020] Figure 4The gold electrode surfaces before and after modification with the AuNPs / pDAB-MIP electrochemical copolymer were characterized using scanning electron microscopy images.

[0021] Figure 5 UV-Vis absorption spectra of ethanol solution and eluent (ethanol solution after template removal).

[0022] Figure 6 The X-ray diffraction (XRD) pattern of the prepared AuNPs / pDAB-MIP was measured on conductive glass.

[0023] Figure 7 Cyclic voltammetry (CV) response curves of methanol solutions with different tacrolimus concentrations were determined using Au / AuNPs / pDAB-MIP as the working electrode interface.

[0024] Figure 8 Differential pulse voltammetry (DPV) curves of the sensing interface at different tacrolimus concentrations were measured (a), the relationship between different tacrolimus concentrations and the corresponding peak current intensity of the DPV curves was established, and an excellent linear relationship was fitted (b). Detailed Implementation

[0025] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0026] Example 1

[0027] This embodiment relates to a molecularly imprinted electrochemical sensor for detecting tacrolimus and a schematic diagram of its preparation process, as shown below. Figure 1 As shown, the specific preparation steps are as follows:

[0028] (1) The gold electrode with a diameter of 3 mm was surface polished, cleaned with deionized water, and then ultrasonically treated for 5 minutes. A mixture containing o-phenylenediamine (5 mmol / L), tacrolimus (0.5 mmol / L), and chloroauric acid (0.1 mmol / L) was prepared in an acetate-sodium acetate buffer solution (0.1 mol / L, pH=4); the gold electrode was inserted into this mixture, and electrochemical polymerization was carried out on the electrode surface; the applied cyclic potential was 0.5 V, the scan rate was 50 mV / s, and 30 cyclic voltammetric scans were performed; the copolymerization process was characterized by a curve determined by cyclic voltammetry. Figure 2 As shown, during the electropolymerization of the molecularly imprinted polymer, the first polymerization cycle exhibits a distinct and irreversible anodic peak at 0.61 V. With increasing scan cycles, the oxidation peak current decreases significantly, indicating the formation of a poly(o-phenylene diamine) layer on the electrode surface.

[0029] (2) After the cyclic voltammetry scan, the gold electrode surface was immersed in an ethanol solution and treated with magnetic stirring for 20 minutes to remove the tacrolimus template molecules from the polymer system, resulting in a polymer containing tacrolimus molecular imprints. Selective binding sites for tacrolimus were left in the molecularly imprinted polymer matrix. The response before and after template molecule removal was characterized by curves determined by cyclic voltammetry. Figure 3 As shown, the formation of the molecularly imprinted polymer layer significantly suppressed the peak current and prevented the redox probe from entering the electrode surface. Eluting the tacrolimus template molecules from the polymer with ethanol resulted in a significantly enhanced peak current after template removal due to the increased permeability of the redox probe through the tacrolimus-specific cavity.

[0030] (3) A poly(o-phenylene diamine) hybrid Au / AuNPs / pDAB-MIP coated with gold nanoparticles and containing tacrolimus molecular imprints was prepared on the surface of the gold electrode. The gold electrode surface before and after modification with the AuNPs / pDAB-MIP electrochemical copolymer was characterized by scanning electron microscopy. Figure 4 As shown, compared to the unmodified molecularly imprinted polymer, the modified gold electrode surface exhibits a rough, granular polymer surface. This is attributed to the rapid polymerization of o-phenylenediamine catalyzed by gold ions, and the different active surface areas also reveal distinguishable surface morphologies. Figure 5 As shown, the UV-Vis absorption spectra of the ethanol solution and the eluent (ethanol solution after template removal) were characterized using an absorption spectrometer. The figure clearly shows the characteristic absorption peak of the template molecule tacrolimus eluted from the polymer. Figure 6 As shown, the X-ray diffraction (XRD) pattern of the prepared AuNPs / pDAB-MIP was measured on the conductive glass, where the crystal planes (111)(200)(220) correspond to the prepared AuNPs; the crystal planes (211)(222)(400)(440) are derived from the characteristic diffraction properties of indium oxide in the conductive glass.

[0031] The modified electrode surface was incubated for 8 minutes in tacrolimus-methanol solutions with concentrations of 0, 1, 10, and 100 ng / mL, respectively. The electrode surface was then gently rinsed with methanol and immersed in a substrate containing potassium ferricyanide. Electrochemical cyclic voltammetry was performed at a potential of 0.5 V and a scan rate of 100 mV / s. Figure 7 As shown, increasing tacrolimus concentration hinders electron transport on the electrode surface, leading to a decrease in peak current. This result indicates a correlation between tacrolimus concentration and electrochemical signal, demonstrating that this molecularly imprinted sensor exhibits excellent targeting and binding ability to tacrolimus, inducing significant current changes, and can be used for tacrolimus detection.

[0032] (4) Using Au / AuNPs / pDAB-MIP as the working electrode interface, the differential pulse voltammetry (DPV) curves of the sensing interface at different tacrolimus concentrations are measured, as Figure 8 (a) shows. Then, the relationship between different tacrolimus concentrations and the corresponding current peak intensities of DPV curves is established, and an excellent linear relationship is fitted, thereby constructing a molecularly imprinted electrochemical sensor for tacrolimus detection, as Figure 8 (b) shows. Wherein, the linear detection range of tacrolimus concentration is 0.0001-10 ng / mL, and the detection limit is 0.05 pg / mL.

[0033] (5) Add 0.1 mL of real serum sample to 1 mL of acetate-sodium acetate buffer, then add a certain amount of tacrolimus thereto, and form a homogeneous mixture under thorough shaking, wherein the tacrolimus concentration is adjusted to 0, 0.0001, 0.001, 0.01 and 10 ng / mL. Then, using the molecularly imprinted electrochemical sensor method constructed above, the corresponding DPV curves and current peak intensities of different homogeneous mixtures are measured, and according to Figure 8 the linear relationship (i.e., y = 7.22 - 0.85x) between tacrolimus concentration (x) and current peak intensity (y) fitted in (b), the tacrolimus concentration in the sample system corresponding to different current peak intensities is calculated. The consistency between the detected value and the spiked value of tacrolimus concentration (pg / mL) is verified by repeated measurement experiments (detected value / spiked value: 0.105 / 0.1, 0.959 / 1, 1.076 / 1, 10.482 / 10, 101.6 / 100, 102.5 / 100), as well as the high recovery rate (90% < r < 110%) and low relative standard deviation (0 < RSD < 5%) of the detected values, thereby confirming that the molecularly imprinted electrochemical sensor designed in the present invention has the ability to efficiently detect tacrolimus in actual samples.

[0034] Example 2

[0035] The present example relates to a molecularly imprinted electrochemical sensor for detecting tacrolimus and a preparation method thereof, and the specific preparation steps are as follows:

[0036] (1) A gold electrode with a diameter of 3 mm is subjected to surface polishing, cleaned with deionized water, and then sonicated for 5 minutes. A mixed solution containing 5 mmol / L o-phenylenediamine, 0.5 mmol / L tacrolimus and 0.1 mmol / L chloroauric acid is prepared in 0.1 mol / L (pH=4) acetate-sodium acetate buffer; the gold electrode is inserted into this mixed solution, and electrochemical polymerization is carried out on the electrode surface; wherein the applied cycling potential is 0.4 V, the scanning rate is 30 mV / s, and 50 cycles of cyclic voltammetry scanning are performed; the process of the copolymerization reaction is characterized by the curve measured by cyclic voltammetry.

[0037] (2) After the cyclic voltammetry scan is completed, the surface of the gold electrode is immersed in n-butanol solution and soaked for 15 minutes under magnetic stirring to remove the tacrolimus template molecules in the polymer system, and a polymer containing tacrolimus molecular imprint is obtained. The selective binding sites of tacrolimus are left in the molecular imprinted polymer matrix. The response before and after template molecule removal is characterized by the curve determined by cyclic voltammetry.

[0038] (3) On the surface of a gold electrode, a poly(o-phenylenediamine) hybrid Au / AuNPs / pDAB-MIP containing tacrolimus molecular imprints and coated with gold nanoparticles was prepared. The modified electrode surface was incubated for 5 minutes in methanol solutions with concentrations of 0, 0.01, 0.1 and 1 ng / mL tacrolimus, respectively. The electrode surface was then gently rinsed with methanol and then immersed in a substrate containing potassium ferricyanide. Electrochemical cyclic voltammetry was performed at a potential of 0.4 V and a scan rate of 80 mV / s.

[0039] (4) Using Au / AuNPs / pDAB-MIP as the working electrode interface, differential pulse voltammetry (DPV) curves of the sensing interface were measured at different tacrolimus concentrations. Then, the relationship between different tacrolimus concentrations and the corresponding peak current intensities of the DPV curves was established, and an excellent linear relationship was fitted, thereby constructing a molecularly imprinted electrochemical sensor for tacrolimus detection. The linear detection range of tacrolimus concentration was 0.001–1 ng / mL, and the detection limit was 0.1 picogram / mL.

[0040] (5) Add 0.1 mL of real serum sample to 1 mL of acetate-sodium acetate buffer, then add a certain amount of tacrolimus, and shake thoroughly to form a homogeneous mixture. The tacrolimus concentration is adjusted to 0, 0.001, 0.01, 0.1, and 1 ng / mL. Then, using the molecularly imprinted electrochemical sensor method constructed above, the DPV curves and current peak intensities corresponding to different homogeneous mixtures are measured. Based on the fitted linear relationship between tacrolimus concentration and current peak intensity, the concentration of tacrolimus in the sample system corresponding to different current peak intensities is calculated. Repeated measurement experiments are used to verify the consistency between the detected tacrolimus concentration and the spiked value, as well as the high recovery rate and low relative standard deviation of the detected value.

[0041] Example 3

[0042] This embodiment relates to a molecularly imprinted electrochemical sensor for detecting tacrolimus and its preparation method. The specific preparation steps are as follows:

[0043] (1) The gold electrode with a diameter of 3 mm was surface polished, cleaned with deionized water, and then sonicated for 5 minutes. A mixture containing 5 mmol / L o-phenylenediamine, 0.5 mmol / L tacrolimus and 0.1 mmol / L chloroauric acid was prepared in a 0.1 mol / L (pH=4) acetate-sodium acetate buffer solution. The gold electrode was inserted into this mixture, and electrochemical polymerization was carried out on the electrode surface. The applied cyclic potential was 0.8 V, the scan rate was 80 mV / s, and 25 cyclic voltammetric scans were performed. The copolymerization process was characterized by the curves determined by cyclic voltammetry.

[0044] (2) After the cyclic voltammetry scan is completed, the surface of the gold electrode is immersed in the isopropanol solution and soaked for 25 minutes under magnetic stirring to remove the tacrolimus template molecules in the polymer system, and a polymer containing tacrolimus molecular imprint is obtained. The selective binding sites of tacrolimus are left in the molecular imprinted polymer matrix. The response before and after template molecule removal is characterized by the curve determined by cyclic voltammetry.

[0045] (3) On the surface of a gold electrode, a poly(o-phenylenediamine) hybrid Au / AuNPs / pDAB-MIP containing tacrolimus molecular imprints and coated with gold nanoparticles was prepared. The modified electrode surface was incubated for 6 minutes in methanol solutions of 0, 0.1, 1 and 10 ng / mL tacrolimus, respectively. The electrode surface was then gently rinsed with methanol and then immersed in a substrate containing potassium ferricyanide. Electrochemical cyclic voltammetry was performed at a potential of 0.5 V and a scan rate of 120 mV / s.

[0046] (4) Using Au / AuNPs / pDAB-MIP as the working electrode interface, differential pulse voltammetry (DPV) curves of the sensing interface were measured at different tacrolimus concentrations. Then, the relationship between different tacrolimus concentrations and the corresponding peak current intensities of the DPV curves was established, and an excellent linear relationship was fitted, thereby constructing a molecularly imprinted electrochemical sensor for tacrolimus detection. The linear detection range of tacrolimus concentration was 0.01–10 nanograms / mL, and the detection limit was 0.5 picograms / mL.

[0047] (5) Add 0.1 mL of real serum sample to 1 mL of acetate-sodium acetate buffer, then add a certain amount of tacrolimus, and mix thoroughly to form a homogeneous mixture. The tacrolimus concentration is adjusted to 0, 0.01, 0.1, 1, and 10 ng / mL. Then, using the molecularly imprinted electrochemical sensor method constructed above, the DPV curves and current peak intensities corresponding to different homogeneous mixtures are measured. Based on the fitted linear relationship between tacrolimus concentration and current peak intensity, the concentration of tacrolimus in the sample system corresponding to different current peak intensities is calculated. Repeated measurement experiments are used to verify the consistency between the detected tacrolimus concentration and the spiked value, as well as the high recovery rate and low relative standard deviation of the detected value.

[0048] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a molecularly imprinted electrochemical sensor for detecting tacrolimus, characterized in that, The method specifically includes the following steps: (1) Electrochemical copolymerization: The gold electrode was polished, washed with water and sonicated. Tacrolimus was used as the template molecule and o-phenylenediamine and chloroauric acid were used as functional monomers. They were added to the substrate containing acidic buffer solution and inserted into the gold electrode. Electrochemical polymerization was carried out on its surface. The applied cyclic potential was 0-1 V, the scan rate was 10-100 mV / s, and 10-50 cyclic voltammetric scans were performed. The copolymerization process was characterized by cyclic voltammetry curves. (2) Elution of template molecules: After the scan, the surface of the gold electrode is immersed in the alcohol solution and treated with magnetic stirring for 10-30 minutes to remove the tacrolimus template molecules in the polymer system, and a polymer containing tacrolimus molecular imprint is obtained. The selective binding sites of tacrolimus are left in the molecular imprinted polymer matrix. The response before and after template molecule removal is characterized by the curve determined by cyclic voltammetry. (3) Electrochemical signal response: An organic / inorganic hybrid of pDAB containing tacrolimus molecularly imprinted MIP and coated with gold nanoparticles AuNPs was prepared on the surface of the gold electrode Au. Its morphology was characterized by scanning electron microscopy. The modified electrode surface Au / AuNPs / pDAB-MIP was inserted into a tacrolimus methanol solution with a concentration of 0-100 ng / mL and incubated for 1-10 minutes. Then, the electrode surface was gently rinsed with methanol and then inserted into a substrate containing potassium ferricyanide. Electrochemical cyclic voltammetry was performed at a potential of 0-0.5 V and the scan rate was adjusted to 50-150 mV / s. (4) Construction of molecularly imprinted electrochemical sensor: Au / AuNPs / pDAB-MIP was used as the working electrode interface. Differential pulse voltammetry (DPV) curves of the sensing interface were measured at different tacrolimus concentrations. The relationship between different tacrolimus concentrations and the corresponding peak current intensity of the DPV curve was established, and an excellent linear relationship was fitted. Thus, a molecularly imprinted electrochemical sensor for tacrolimus detection was constructed. The linear detection range of tacrolimus concentration was 0.0001-100 ng / mL, and the detection limit was 0.05-1 picogram / mL. (5) Detection of tacrolimus in actual samples: Take a certain amount of real serum sample, add a certain amount of tacrolimus to it, and form a homogeneous mixture by shaking it thoroughly. The concentration of tacrolimus in the mixture is adjusted to 0-10 ng / mL. Then, using the molecularly imprinted electrochemical sensor method constructed above, the DPV curve and current peak intensity corresponding to different homogeneous mixtures are measured. Based on the linear relationship between tacrolimus concentration and current peak intensity fitted in step (4), the concentration of tacrolimus in the sample system corresponding to different current peak intensities is calculated. The consistency between the detected value and the spiked value of tacrolimus concentration is verified by repeated measurement experiments, as well as the high recovery rate and low relative standard deviation of the detected value.

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