Molecularly imprinted electrochemical sensor for rapid detection of hydroxyproline as well as preparation and application of molecularly imprinted electrochemical sensor

Abstract

The invention discloses a hydroxyproline rapid detection molecularly imprinted electrochemical sensor as well as preparation and application thereof, and relates to the field of quality safety monitoring of medicinal plants. The preparation method of the molecularly imprinted electrochemical sensor comprises the following steps: preparing the Fc-MWCNTs composite material, preparing the Fc-MWCNTs / GCE, and preparing the molecularly imprinted electrochemical sensor MIP-Fc-MWCNTs (at) GCE. According to the invention, MWCNTs with good conductivity and Fc which has good stability and oxidation-reduction property and is used as an excellent electron transfer medium are used as electrode substrate materials; and the Fc-MWCNTs and the MWCNTs are covalently bonded through an amide reaction to prepare the composite material Fc-MWCNTs which has excellent electron transfer medium characteristics, low potential reproducibility and strong stability and can remarkably improve the electron transfer rate between the electrode surface and an electrolyte. The modified composite material Fc-MWCNTs is modified on the surface of GCE, so that the molecularly imprinted electrochemical sensor MIP-Fc-MWCNTs (at) GCE with high sensitivity, high selective recognition and fast detection is constructed, and the molecularly imprinted electrochemical sensor is used for detecting an index component Hyp of cornu cervi pantotrichum.

Description

Technical Field

[0001] The invention relates to the field of environmental monitoring and medicinal plant quality and safety monitoring, and in particular to a hydroxyproline rapid detection molecular imprinting electrochemical sensor and a preparation method and application thereof. Background Art

[0002] As a traditional Chinese medicine, deer antler has the effects of strengthening kidney yang, benefiting essence and blood, strengthening tendons and bones, regulating Chong and Ren meridians, and supporting sores and toxins. With the continuous deepening of research on the composition and pharmacological effects of deer antler, its market potential and application prospects will be broader. Due to its rarity and powerful medicinal effects, the price of deer antler continues to rise. At present, the quality of deer antler on the market is uneven and the grades are confusing, which seriously affects and restricts the quality of deer antler and the development of its industry. Faced with the problems of confusion in the quality grade of deer antler slices, how to comprehensively judge from the aspects of authenticity, quality, efficacy, etc., screen out a representative ingredient that represents the overall quality of the medicinal material as much as possible, and realize its rapid detection has become the key to the preliminary quick evaluation of the quality of bulk deer antler medicinal materials.

[0003] Hydroxyproline (Hyp) is one of the recognized index components for the quality evaluation of velvet antler in recent years. Its rapid qualitative and quantitative detection is crucial to the quality assurance of bulk velvet antler medicinal materials and the sustainable development of related industries. Trace detection of Hyp can distinguish between natural velvet antler and possible adulterated samples, thereby regulating the market, prompting the entire industry to improve product quality, ensure the quality and safety of velvet antler products on the market, improve the reputation and competitiveness of the entire industry, and promote the healthy development of the velvet antler industry.

[0004] Currently reported Hyp detection methods include spectrophotometry, high performance liquid chromatography, liquid chromatography-mass spectrometry, automatic amino acid analyzer and capillary electrophoresis. However, these existing detection methods have complex pre-treatment and high operation requirements, making it difficult to achieve rapid detection and evaluation of quality indicator components of traditional Chinese medicine.

[0005] Electrochemical methods can achieve rapid detection of single components, simplify the testing process and improve efficiency. Among them, molecular imprinting electrochemical sensing technology plays an important role in the detection of active drug ingredients due to its advantages such as high sensitivity, simple instrumentation and equipment, and easy automation.

[0006] In view of this, we have developed a high-throughput rapid detection molecular imprinting electrochemical sensor for hydroxyproline (Hyp), an indicator component of velvet antler quality control. It can perform qualitative and quantitative rapid detection of velvet antler quality, provide a more objective evaluation, and also provide a forward-looking exploration of electrochemical analysis technology in the quality evaluation and drug safety of traditional Chinese medicine. Summary of the invention

[0007] The purpose of the present invention is to provide a hydroxyproline rapid detection molecular imprinting electrochemical sensor and its preparation and application in view of the defects of the prior art in the above-mentioned background technology.

[0008] In order to achieve the above object, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing a hydroxyproline rapid detection molecular imprinting electrochemical sensor, comprising the following steps: (1) Preparation of Fc-MWCNTs composites Weigh 3-7 mg of multi-walled carbon nanotubes (MWCNTs) and add them to 3-7 mL of 1 mg / mL ferrocene (Fc) dimethylformamide (DMF) solution, and ultrasonicate for 2 h to make it dispersed evenly to obtain Fc-MWCNTs composite material suspension, which is stored at 4 °C for future use; (2) Preparation of Fc-MWCNTs / GCE The glassy carbon electrode (GCE) was first polished with metallographic sandpaper and then with 0.3 μm and 0.05 μm A 12 The O3 suspension was polished on the suede, and then ultrasonically cleaned with methanol and double distilled water respectively, and then dried at room temperature to obtain a pretreated glassy carbon electrode (GCE) for use; 15 μL of the Fc-MWCNTs composite material suspension was dripped and modified on the treated blank glassy carbon electrode surface with a pipette, and dried in an oven at 40 °C overnight to obtain a composite electrode Fc-MWCNTs@GCE; (3) Preparation of molecular imprinted electrochemical sensors 3 mg of hydroxyproline (Hyp) was weighed and added to a solution containing 50-90 μL of methacrylic acid (MAA) and 0.5 mL of PBS with a pH value of 4. The solution was ultrasonicated at room temperature for 10 min, and then 0.72-1.30 mL of ethylene glycol dimethacrylate (EGDMA) and 0.24-0.43 mg of azobisisobutyronitrile (AIBN) were added. The solution was ultrasonicated for 10 min to obtain a prepolymer solution. 2 μL of the prepolymer solution was transferred with a pipette and dropped onto the surface of the composite electrode Fc-MWCNTs@GCE. The composite electrode Fc-MWCNTs@GCE was then placed in an oven at 40°C and heated and dried for 24 h to obtain a molecular imprinting electrochemical sensor MIP-Fc-MWCNTs@GCE with a polymer layer on the surface.

[0009] In a second aspect, the present invention further provides a hydroxyproline rapid detection molecular imprinting electrochemical sensor, which is prepared by the above-mentioned preparation method.

[0010] In a third aspect, the present invention also provides an application of a hydroxyproline rapid detection molecular imprinting electrochemical sensor in the quality detection of velvet antler in Shenrong Guben Huanshao Pills.

[0011] Compared with the prior art, the beneficial technical effects of the present invention are: The present invention uses MWCNTs with good conductivity and Fc with good stability, redox properties and excellent electron transfer medium as electrode substrate materials. The two are covalently bonded through an amide reaction to prepare a composite material Fc-MWCNTs with excellent electron transfer medium characteristics, low potential regeneration and strong stability, which can significantly improve the electron transfer rate between the electrode surface and the electrolyte. In the process of functional modification of MWCNTs by Fc, the synergistic effect between the two and the strong π bond coupling can improve the catalytic effect of a single material, thereby increasing the catalytic current response to the target molecule. The modified composite material Fc-MWCNTs is modified on the surface of GCE to construct a molecular imprinting electrochemical sensor MIP-Fc-MWCNTs@GCE with high sensitivity and high selectivity for rapid identification, which is used for the detection of Hyp, an indicator component of deer antler.

[0012] 2. The molecular imprinting electrochemical sensor of the present invention exhibits good specificity and stability when used for actual sample detection. -2 ~1.0 ×10 3 It has a good linear relationship in the μg / mL range and the detection limit is 9.0×10 -3 μg / mL, which can achieve highly sensitive and rapid detection of trace amounts of Hyp in actual samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a schematic diagram of the preparation principle of a hydroxyproline rapid detection molecular imprinting electrochemical sensor of the present invention; Figure 2 FT-IR spectra of (a) Fc, (b) MWCNTs and (c) Fc-MWCNTs in a test example of the present invention; Figure 3 The Raman spectra of (a) Fc-MWCNTs, (b) Fc, and (c) MWCNTs in a test example of the present invention; Figure 4 is a SEM image of MWCNTs@GCE in a test example of the present invention; Figure 5 is a SEM image of Fc@GCE in a test example of the present invention; Figure 6 This is a SEM image of Fc-MWCNTs@GCE in a test example of the present invention; Figure 7 is an EDX spectrum of Fc-MWCNTs in a test example of the present invention; Figure 8CV curves of (a) GCE, (b) Fc@GCE, (c) MWCNTs@GCE, and (d) Fc-MWCNTs@GCE detected by cyclic voltammetry (CV) in a 2.0 mM potassium ferrocyanide / potassium ferrocyanide base solution in a test example of the present invention; Fig. 9 The EIS curves of GCE, Fc, Fc-MWCNTs, and MWCNTs detected by electrochemical impedance spectroscopy (EIS) in a 2.0 mM potassium ferrocyanide / potassium ferrocyanide base solution in a test example of the present invention are shown in FIG. Fig.10 This is the CV curve of Fc-MWCNTs@GCE at a scan rate of 10 to 120 mV / s in a test example of the present invention; Fig.11 is a graph showing the relationship between the peak current and the square root of the scan rate in a test example of the present invention; Fig.12 The chronocoulometry (CC) curves of (a) GCE, (b) MIP-Fc-MWCNTs@GCE after elution, (c) MWCNTs@GCE, (d) MIP-Fc-MWCNTs@GCE before elution, and (e) Fc-MWCNTs@GCE are shown in a test example of the present invention; Fig.13 Q-t1 / 2 curves of (a) GCE, (b) Fc-MWCNTs@GCE, (c) MWCNTs@GCE, (d) MIP-Fc-MWCNTs@GCE before elution, and (e) MIP-Fc-MWCNTs@GCE after elution using chronocoulometry (CC) in a test example of the present invention; Fig.14 The results of a test example of the present invention show that different concentrations of Hyp in 0.1M KCl and 2.0 mM [Fe(CN)6] 3- / 4- DPV curve of the probe solution MIP / Fc-MWCNTs@GCE; Fig.15 This is a graph showing the relationship between the peak current and the Hyp concentration of the molecular imprinting electrochemical sensor MIP-Fc-MWCNTs@GCE in a test example of the present invention; Fig.16 This is a diagram showing the selective detection results of the molecular imprinting electrochemical sensor MIP-Fc-MWCNTs@GCE sensor in a test example of the present invention; Fig.17 This is a diagram showing the repeatability test results of the molecular imprinting electrochemical sensor MIP-Fc-MWCNTs@GCE sensor in a test example of the present invention; Fig.18This is a graph showing the reproducibility test results of the molecular imprinting electrochemical sensor MIP-Fc-MWCNTs@GCE sensor in a test example of the present invention; Fig.19 This is a diagram showing the stability test results of the molecular imprinting electrochemical sensor MIP-Fc-MWCNTs@GCE sensor in a test example of the present invention. DETAILED DESCRIPTION

[0014] The technical solution of the present invention will be described clearly and completely in conjunction with the embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0015] Example 1 Please refer to the attached Figure 1 As shown, the preparation method of a hydroxyproline rapid detection molecular imprinting electrochemical sensor provided in this embodiment includes the following steps: (1) Preparation of Fc-MWCNTs composites Weigh 3 mg of multi-walled carbon nanotubes (MWCNTs) and add them to 3 mL of 1 mg / mL ferrocene (Fc) dimethylformamide (DMF) solution. Ultrasonicate for 2 h to make it dispersed evenly to obtain Fc-MWCNTs composite suspension, which was stored at 4 °C for later use. (2) Preparation of Fc-MWCNTs / GCE The glassy carbon electrode (GCE) was first polished with metallographic sandpaper, and then polished with 0.3 μm and 0.05 μm A12O3 suspensions on suede in turn, and then ultrasonically cleaned with methanol and double distilled water respectively, and then dried at room temperature to obtain a pretreated glassy carbon electrode (GCE) for use; 15 μL of the Fc-MWCNTs composite material suspension was drop-coated on the treated blank glassy carbon electrode (GCE) surface with a pipette gun, and dried in an oven at 40 °C overnight to obtain a composite electrode Fc-MWCNTs@GCE; (3) Preparation of molecular imprinted electrochemical sensors 3 mg of hydroxyproline (Hyp) was weighed and added to a solution containing 50 μL of methacrylic acid (MAA) and 0.5 mL of PBS with a pH value of 4. The solution was ultrasonicated at room temperature for 10 min, and then 0.72 mL of ethylene glycol dimethacrylate (EGDMA) and 0.24 mg of azobisisobutyronitrile (AIBN) were added. The solution was ultrasonicated for 10 min to obtain a prepolymer solution. 2 μL of the prepolymer solution was pipetted with a pipette and dropped onto the surface of the composite electrode Fc-MWCNTs@GCE. The composite electrode Fc-MWCNTs@GCE was then placed in an oven at 40°C and heated and dried for 24 h to obtain a composite electrode Fc-MWCNTs@GCE with a polymer layer on the surface, i.e., a molecular imprinting electrochemical sensor MIP-Fc-MWCNTs@GCE was obtained.

[0016] Example 2 Please see attached Figure 1 As shown, the preparation method of a hydroxyproline rapid detection molecular imprinting electrochemical sensor provided in this embodiment includes the following steps: (1) Preparation of Fc-MWCNTs composites Weigh 5 mg of multi-walled carbon nanotubes (MWCNTs) and add them to 5 mL of 1 mg / mL ferrocene (Fc) dimethylformamide (DMF) solution. Ultrasonicate for 2 h to make it dispersed evenly to obtain Fc-MWCNTs composite suspension, which was stored at 4 °C for later use. (2) Preparation of Fc-MWCNTs / GCE The glassy carbon electrode (GCE) was first polished with metallographic sandpaper, and then polished with 0.3 μm and 0.05 μm A12O3 suspensions on suede in turn, and then ultrasonically cleaned with methanol and double distilled water respectively, and then dried at room temperature to obtain a pretreated glassy carbon electrode (GCE) for use; 15 μL of the Fc-MWCNTs composite material suspension was drop-coated on the treated blank glassy carbon electrode (GCE) surface with a pipette gun, and dried in an oven at 40 °C overnight to obtain a composite electrode Fc-MWCNTs@GCE; (3) Preparation of molecular imprinted electrochemical sensors 3 mg of hydroxyproline (Hyp) was weighed and added to a solution containing 70 μL of methacrylic acid (MAA) and 0.5 mL of PBS with a pH value of 4. The solution was ultrasonicated at room temperature for 10 min, and then 1 mL of ethylene glycol dimethacrylate (EGDMA) and 0.33 mg of azobisisobutyronitrile (AIBN) were added. The solution was ultrasonicated for 10 min to obtain a prepolymer solution. 2 μL of the prepolymer solution was pipetted with a pipette and dropped onto the surface of the composite electrode Fc-MWCNTs@GCE. The composite electrode Fc-MWCNTs@GCE was then placed in an oven at 40°C and heated and dried for 24 h to obtain a composite electrode Fc-MWCNTs@GCE with a polymer layer on the surface, i.e., a molecular imprinting electrochemical sensor MIP-Fc-MWCNTs@GCE was obtained.

[0017] Example 3 Please see attached Figure 1 As shown, the preparation method of a hydroxyproline rapid detection molecular imprinting electrochemical sensor provided in this embodiment includes the following steps: (1) Preparation of Fc-MWCNTs composites Weigh 7 mg of multi-walled carbon nanotubes (MWCNTs) and add them to 7 mL of 1 mg / mL ferrocene (Fc) dimethylformamide (DMF) solution, and ultrasonicate for 2 h to make it dispersed evenly to obtain Fc-MWCNTs composite suspension, which was stored at 4 °C for future use; (2) Preparation of Fc-MWCNTs / GCE The glassy carbon electrode (GCE) was first polished with metallographic sandpaper, and then polished with 0.3 μm and 0.05 μm A12O3 suspensions on suede, and then ultrasonically cleaned with methanol and double distilled water, respectively, and then dried at room temperature to obtain a pretreated glassy carbon electrode (GCE) for use; 15 μL of Fc-MWCNTs composite material suspension was drop-coated on the treated blank glassy carbon electrode (GCE) surface with a pipette, and dried in an oven at 40 °C overnight to obtain a composite electrode Fc-MWCNTs@GCE; (3) Preparation of molecular imprinted electrochemical sensors 3 mg of hydroxyproline (Hyp) was weighed and added to a solution containing 90 μL of methacrylic acid (MAA) and 0.5 mL of PBS with a pH value of 4. The solution was ultrasonicated at room temperature for 10 min, and then 1.30 mL of ethylene glycol dimethacrylate (EGDMA) and 0.43 mg of azobisisobutyronitrile (AIBN) were added. The solution was ultrasonicated for 10 min to obtain a prepolymer solution. 2 μL of the prepolymer solution was transferred with a pipette and dropped onto the surface of the composite electrode Fc-MWCNTs@GCE. The composite electrode Fc-MWCNTs@GCE was then placed in an oven at 40°C and heated and dried for 24 h to obtain a composite electrode Fc-MWCNTs@GCE with a polymer layer on the surface, i.e., a molecular imprinting electrochemical sensor MIP-Fc-MWCNTs@GCE was obtained.

[0018] Test Example 1 In the process of preparing the molecular imprinting electrochemical sensor MIP-Fc-MWCNTs@GCE in Example 2, infrared spectroscopy, Raman spectroscopy, SEM electron microscopy and EDX spectroscopy were performed. The characterization results are as follows Figure 2-7 shown.

[0019] The test results show that Fc-MWCNTs, Fc-MWCNTs@GCE and MIP-Fc-MWCNTs@GCE were all successfully prepared.

[0020] Test Example 2 The molecular imprinting electrochemical sensor MIP-Fc-MWCNTs@GCE of Example 2 was characterized by CV method, EIS method and chronocoulometry. The CV method was carried out in a base solution of 2.0 mM K3[Fe(CN)6] / K4[Fe(CN)6] in 0.01 M KCl at a voltage range of -0.4 V to 0.8 V at a speed of 80 mVs -1 Scan rate, electrochemical characterization results such as Figure 8-13 shown.

[0021] The test results show that Fc-MWCNTs, Fc-MWCNTs@GCE and MIP-Fc-MWCNTs@GCE were all successfully prepared.

[0022] Test Example 3 Differential pulse voltammetry (DPV) was used to investigate the electrochemical response of MIP-Fc-MWCNTs@GCE of Example 2 in a 2.0 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution containing 0.01 M KCl, and the current response was recorded. The MIP-Fc-MWCNTs@GCE was taken out and washed with distilled water, and then immersed in an eluent of methanol / acetic acid = 5:5 (v / v) for 20 min to remove the template molecule (Hyp) in the polymer matrix, and the NIP-Fc-MWCNTs@GCE modified electrode was obtained. The electrochemical response of the NIP-Fc-MWCNTs@GCE modified electrode was investigated, and the current response was recorded. The sensitivity of DPV is 10 -5 , potential range 0.6 V ~ -0.2 V, rest time 2 s, voltage amplitude 0.05 V, pulse period 0.5 s, sampling width 0.0167 s. The test base solution contains 2.0 mM [Fe(CN)6] 3- / 4- and 0.1 M KCl mixed solution. Finally, the linear functional relationship between different Hyp concentrations and electrochemical response values ​​was obtained by mathematical modeling. The test results are shown in Fig.14 and 15 shown.

[0023] The test results show that the logarithm of the peak current (Ipa, μA) of Hyp oxidation and its concentration (μg / mL) is 1×10 -2 ~1×10 3 The linear relationship was found in the range of μg / mL, and the corresponding linear regression equation was: Ip = -0.03295 C + 87.71087 (R2 = 0.98838), and the detection limit was 9.0 × 10 -3 μg / mL (S / N=3).

[0024] Test Example 4 In order to verify the practicality of the molecular imprinting electrochemical sensor MIP-Fc-MWCNTs@GCE of the present invention, the selectivity, repeatability, reproducibility and stability of the MIP-Fc-MWCNTs@GCE prepared in Example 2 were tested. The test results are as follows: Figure 16-19 As shown, when detecting selectivity, the interfering substances selected are histidine (His), glycine (Gly), alanine (Ala), and proline (Pro). Under the optimal experimental conditions, MIP-Fc-MWCNTs@GCE was placed in a solution with a Hyp concentration of 0.03 mg / mL, and interfering substances with 10 times the concentration were added in equal amounts. The results showed that the sensor had the largest current response to Hyp, proving that the sensor has good recognition specificity and selectivity for Hyp.

[0025] In order to investigate the repeatability of the sensor, the same electrode was used to perform five repeated tests on the same concentration of hydroxyproline in PBS (pH=4.0) buffer solution containing 0.3 mg / mL Hyp. After each measurement, the electrode was treated with an eluent to remove the adsorbed template molecules. The results are shown in the figure. The calculated relative standard deviation (RSD) was 1.01%, and the electrode had good repeatability, which also showed that the binding of the template molecule to the cavity on the electrode surface was reversible and the elution method was effective. Five molecular imprinting composite electrodes were prepared using the same method and the same concentration of Hyp was detected under the same conditions to explore the reproducibility of the electrode.

[0026] The prepared MIP-Fc-MWCNTs@GCE was placed at room temperature and the DPV method was used to detect hydroxyproline Hyp every 5 days. The calculated response peak current was 92.2% of the initial peak current, proving that the sensor has good stability.

[0027] The test results show that the molecular imprinting electrochemical sensor MIP-Fc-MWCNTs@GCE of the present invention has excellent selectivity, repeatability, reproducibility and stability.

[0028] Application Examples Deer antler is one of the main ingredients of Shenrong Guben Huanshao Pills. If the quality of deer antler is not up to standard, it may weaken the efficacy of Shenrong Guben Huanshao Pills and even cause side effects, thus affecting the therapeutic effect of the entire prescription. How to comprehensively judge the authenticity, quality, efficacy, etc., screen out a representative ingredient that represents the overall quality of the medicinal material as much as possible, and realize its rapid detection has become the key to the preliminary rapid evaluation of the quality of bulk deer antler medicinal materials.

[0029] Since hydroxyproline (Hyp) is one of the indicator components for quality control of pilose antler, the molecular imprinting electrochemical sensor MIP-Fc-MWCNTs@GCE of the present invention can be used for rapid quality detection of pilose antler in Shenrong Guben Huanshao Pills.

[0030] In order to verify the feasibility of the molecular imprinting electrochemical sensor MIP-Fc-MWCNTs@GCE of the present invention in the detection of actual samples, the quality analysis of antler was carried out on the actual samples of Shenrong Guben Huanshao Pills.

[0031] The standard addition method was used to determine the Hyp in the actual Shenrong Guben Huanshao Pills sample. A three-electrode system was used during the determination process. The molecular imprinting electrochemical sensor MIP-Fc-MWCNTs@GCE prepared in Example 2 was used as the working electrode, the saturated calomel electrode (SCE) was used as the reference electrode, and the platinum wire electrode was used as the counter electrode. SWV was used for detection. The detection results are shown in Table 1.

[0032] Table 1 Detection of Hyp in actual samples sample Hyp concentration (μg / mL) Hyp measurement concentration (μg / mL) Recovery rate (%) RSD(%) 1 1 0.98 98.00 1.28 2 10 10.77 107.70 0.53 3 100 97.45 97.45 0.72 The test results show that the molecular imprinting electrochemical sensor MIP-Fc-MWCNTs@GCE of the present invention can realize the detection of the intrinsic microscopic component Hyp of pilose antler in the formula, which plays a vital role in ensuring the high quality and efficacy of the compound preparation and the controllable overall quality.

Claims

1. A method for preparing a hydroxyproline rapid detection molecular imprinting electrochemical sensor, characterized in that: The steps include: (1) Preparation of Fc-MWCNTs composites Weigh 3-7 mg of multi-walled carbon nanotubes and add them to 3-7 mL of 1 mg / mL ferrocene DMF solution, and ultrasonicate for 2 h to make them dispersed evenly to obtain Fc-MWCNTs composite material suspension, which is stored at 4 °C for later use; (2) Preparation of Fc-MWCNTs / GCE The glassy carbon electrode was first polished with metallographic sandpaper and then with 0.3 μm and 0.05 μm A 12 The O3 suspension was polished on the suede, and then ultrasonically cleaned with methanol and double distilled water respectively, and then dried at room temperature to obtain a pretreated glassy carbon electrode for use; 15 μL of the Fc-MWCNTs composite material suspension was dripped and modified on the surface of the treated blank glassy carbon electrode with a pipette, and dried in an oven at 40 °C overnight to obtain a composite electrode Fc-MWCNTs@GCE; (3) Preparation of molecular imprinted electrochemical sensors 3 mg of hydroxyproline was weighed and added to a solution containing 50-90 μL of methacrylic acid and 0.5 mL of PBS with a pH value of 4. The solution was ultrasonicated at room temperature for 10 min, and then 0.72-1.30 mL of ethylene glycol dimethacrylate and 0.24-0.43 mg of azobisisobutyronitrile were added. The solution was ultrasonicated for 10 min to obtain a prepolymer solution. 2 μL of the prepolymer solution was transferred with a pipette and dropped onto the surface of the composite electrode Fc-MWCNTs@GCE. The composite electrode Fc-MWCNTs@GCE was then placed in an oven at 40 °C and heated and dried for 24 h to obtain a molecular imprinting electrochemical sensor MIP-Fc-MWCNTs@GCE with a polymer layer on the surface.

2. A hydroxyproline rapid detection molecular imprinting electrochemical sensor prepared by the preparation method described in claim 1.

3. An application of the hydroxyproline rapid detection molecular imprinting electrochemical sensor as claimed in claim 2 in the quality detection of velvet antler in Shenrong Guben Huanshao Pills.

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