Vancomycin detection method

Abstract

The invention discloses a vancomycin detection method, and belongs to the technical field of electrochemical detection. According to the invention, molecular imprinting and polypeptide are combined, specific double recognition of vancomycin detection is realized in plasma, and excellent selectivity and stability are shown. In experimental research, the template volume, the number of electropolymerization turns, the removal time, the rebinding time, the peptide volume and the peptide rebinding time which influence the response of the sensor are optimized. The electrochemical response of the designed electrode to vancomycin is researched by adopting differential pulse voltammetry. Compared with a pure molecular imprinting modified electrode, the molecular imprinting-peptide polymer electrode has higher selectivity and specific recognition capability, and the lowest detection limit is 0.88 mu g / mL. The experiment provides a rapid and sensitive method for detecting the concentration of vancomycin in plasma.

Description

Technical Field

[0001] The invention relates to the technical field of electrochemical detection, and in particular to a method for detecting vancomycin. Background Art

[0002] Vancomycin (VAN) is a widely used glycopeptide antibiotic, but due to its adverse side effects, it can only be used to treat staphylococcal infections (such as pneumonia, wound infections, sepsis) when other antibiotics are ineffective. It is the last choice for the treatment of methicillin-resistant Staphylococcus aureus and is known as the "last line of defense" on the antibiotic road. The main antibacterial mechanism of vancomycin is to specifically bind to the end of the peptidoglycan-Lys-D-Ala-D-Ala sequence on the bacterial wall. This binding inhibits the synthesis of cell wall mucopeptides, ultimately causing the cells to stop growing and lyse, thereby achieving its antibacterial effect. Based on this special interaction, a peptide containing the same terminal sequence (L-Lys-D-Ala-D-Ala) was artificially synthesized and used as a recognition component to recognize VAN. The 2020 revised evidence-based guidelines for vancomycin therapeutic monitoring recommend that the patient's optimal blood peak and trough concentrations be maintained within the range of 5-20 μg / mL. This narrow therapeutic range emphasizes the importance of closely monitoring blood drug concentrations during clinical administration. In recent years, traditional detection methods have been hampered by expensive equipment, long detection time, and complex procedures, which limit their widespread application in challenging environments.

[0003] One of the key challenges in determining clinical blood levels by electrochemical analysis is the accuracy and responsiveness of the created sensors. Currently, there is a strong interest in the development and use of molecular imprinting, which is now recognized as analogous to antigen-antibody due to its strong specific recognition ability. The main distinguishing features of molecular imprinting polymers (MIPs) are their remarkable selectivity for specific target molecules, as well as their good physical and chemical stability. Currently, MIPs have been widely used in the detection field. Molecularly imprinted polymers (MIPs) can be produced by copolymerization of functional monomers and crosslinkers in the presence of specific target molecules, which serve as molecular templates. Functional group monomers play a vital role in providing functional groups that can interact with the template through covalent or non-covalent interactions. After copolymerization, the template molecule is removed from the polymer by chemical decomposition or solvent extraction. Subsequently, the highly cross-linked polymer matrix forms recognition cavities that are complementary to the template molecule in shape, size and chemical functionality, allowing the template molecule to selectively rebind from a closely related mixture. It exhibits a high degree of structural predictability, excellent sensitivity and the ability to accurately recognize specific selective target molecules. Therefore, MIPs have been widely used in food, environment, body fluids, drugs, cytokines, viruses, etc. At present, there are no reports on the detection of vancomycin using MIPs. Summary of the invention

[0004] The purpose of the present invention is to provide a method for detecting vancomycin to solve the problems existing in the above-mentioned prior art. The present invention establishes a method for detecting the blood concentration of antibiotics with fast reaction speed, high sensitivity, low cost and simple operation, which can provide a basis for monitoring the blood concentration of antibiotics in the later clinical practice and adjusting the dosage.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is a method for preparing a Peptide / MIPDA / AuNPs / CS-900 / ITO sensor, comprising the following steps:

[0007] (1) dissolving D-glucose and sodium chloride in water and reacting at high temperature to obtain a carbon precursor;

[0008] (2) heating the carbon precursor in an oil bath, then adding AuCl4H7O3 and stirring for reaction, adding anhydrous ethanol to the reaction system, mixing, centrifuging, collecting the precipitate and annealing, thereby obtaining AuNPs / CS-900;

[0009] (3) dispersing the AuNPs / CS-900 and Nafion solution in anhydrous ethanol, then applying it to the exposed area of ​​the ITO surface and drying it;

[0010] (4) applying a 3-mercaptopropionic acid solution to the surface of AuNPs / CS-900 / ITO to fix AuNPs on the surface of the CS / ITO electrode; then immersing the obtained electrode in a solution containing N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and hydroxysuccinimide, and then dripping a vancomycin solution after drying. After drying, the electrode is placed in a buffer solution for electrochemical polymerization to obtain a polymer film on the surface of the electrode. After drying, the vancomycin molecules embedded in the polymer film are removed, thereby obtaining a molecular imprinting sensor MIPDA / AuNPs / CS-900 / ITO;

[0011] (5) The vancomycin-binding tripeptide is coated on the surface of the molecular imprinting sensor MIPDA / AuNPs / CS-900 / ITO to obtain the Peptide / MIPDA / AuNPs / CS-900 / ITO sensor.

[0012] The second technical solution of the present invention is the Peptide / MIPDA / AuNPs / CS-900 / ITO sensor prepared by the preparation method.

[0013] The third technical solution of the present invention is the use of the Peptide / MIPDA / AuNPs / CS-900 / ITO sensor in the preparation of products for detecting vancomycin.

[0014] A fourth technical solution of the present invention is a product for detecting vancomycin, comprising the Peptide / MIPDA / AuNPs / CS-900 / ITO sensor.

[0015] A fifth technical solution of the present invention is a method for detecting vancomycin for purposes other than disease diagnosis or treatment, using the Peptide / MIPDA / AuNPs / CS-900 / ITO sensor or the product to detect vancomycin in a sample to be tested.

[0016] Based on the above technical solution, the present invention has the following technical effects:

[0017] The present invention combines molecular imprinting with polypeptides to achieve specific dual recognition of vancomycin detection in plasma, while showing excellent selectivity and stability. In experimental studies, the template volume, number of electropolymerization cycles, removal time, rebinding time, peptide volume and peptide rebinding time that affect the sensor response were optimized. The electrochemical response of the designed electrode to vancomycin was studied by differential pulse voltammetry. Compared with simple molecular imprinting modified electrodes, molecular imprinting-peptide polymer electrodes have higher selectivity and specific recognition capabilities, with a minimum detection limit of 0.88μg / mL. The electrode was used to measure the concentration of vancomycin in plasma, and the deviation from the EMIT method was less than 12%, with good accuracy and applicability. This experiment provides a rapid and sensitive method for detecting the concentration of vancomycin in plasma. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1A This is the SEM image of AuNPs / CS-900 material.

[0020] Figure 1B This is the SEM image of the MIPDA / AuNPs / CS-900 / ITO surface.

[0021] Figure 1C This is the SEM image of the Peptide / MIPDA / AuNPs / CS-900 / ITO surface.

[0022] Figure 1D Carbon element distribution of Peptide / MIPDA / AuNPs / CS-900.

[0023] Figure 1E This is the nitrogen element distribution of Peptide / MIPDA / AuNPs / CS-900.

[0024] Figure 1F Oxygen element distribution of Peptide / MIPDA / AuNPs / CS-900.

[0025] Figure 1G Gold element distribution of Peptide / MIPDA / AuNPs / CS-900.

[0026] Figure 2A Infrared spectra of AuNPs / CS, MIPDA / AuNPs / CS-900, and Peptide / MIPDA / AuNPs / CS-900.

[0027] Figure 2B This is the full X-ray photoelectron spectroscopy (XPS) spectrum of Peptide / MIPDA / AuNPs / CS-900.

[0028] Figure 2C This is the X-ray photoelectron spectroscopy (XPS) carbon element spectrum of Peptide / MIPDA / AuNPs / CS-900.

[0029] Figure 2D This is the nitrogen element spectrum of Peptide / MIPDA / AuNPs / CS-90 by X-ray photoelectron spectroscopy (XPS).

[0030] Figure 2E This is the oxygen element spectrum of Peptide / MIPDA / AuNPs / CS-900 by X-ray photoelectron spectroscopy (XPS).

[0031] Figure 3A Figure 2 is the relationship between the vancomycin drop volume and the related current response.

[0032] Figure 3B is the relationship between the number of electrochemical polymerization cycles and the related current response.

[0033] Figure 3C is the relationship between the elution template time and the associated current response.

[0034] Figure 3D Figure 4 is the relationship between the incubation time of the MIP / AuNPs / CS-900 electrode in 20 μg / mL vancomycin and the related current response.

[0035] Figure 3E is the relationship between the peptide drop volume and the related current response.

[0036] Figure 3F This is the relationship between the incubation time of the Peptide / MIPDA / AuNPs / CS-900 electrode in 20 μg / mL vancomycin and the related current response.

[0037] Figure 4A Cyclic voltammograms of AuNPs / CS-25 and AuNPs / CS-900 modified materials.

[0038] Figure 4B Differential pulse voltammograms of AuNPs / CS-25 and AuNPs / CS-900 modified materials.

[0039] Figure 4C Electrochemical impedance spectroscopy of AuNPs / CS-25 and AuNPs / CS-900 modified materials

[0040] Figure 4D The differential pulse voltammetric response diagram of AuNPs / CS-25 and AuNPs / CS-900 modified materials in PBS solution and 40 μg / mL vancomycin solution.

[0041] Figure 4E Cyclic voltammograms of MIPDA / AuNPs / CS-900 at different modification stages.

[0042] Figure 4F Differential pulse voltammograms of MIPDA / AuNPs / CS-900 at different modification stages.

[0043] Figure 5A The relationship between the current response of the sensor and the concentration of vancomycin spiked in PBS solution for dual signal recognition.

[0044] Figure 5B Standard linear curve of the current response of the sensor and the concentration of vancomycin spiked in PBS solution for dual signal recognition.

[0045] Figure 5C The current response of the sensor for dual signal recognition is related to the concentration of vancomycin spiked in plasma.

[0046] Figure 5D Standard linear curve of the current response of the sensor versus the concentration of vancomycin spiked in plasma for dual signal identification.

[0047] Figure 5E This is an investigation on the selectivity of Peptide / MIPDA / AuNPs / CS-900 sensor.

[0048] Fig. 5F To investigate the stability of Peptide / MIPDA / AuNPs / CS-900 sensor.

[0049] Figure 5G This is an investigation on the repeatability of the Peptide / MIPDA / AuNPs / CS-900 sensor.

[0050] Figure 5H Comparison of the experimental detection results of vancomycin in patient plasma using the EMIT method and this method. DETAILED DESCRIPTION

[0051] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0052] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0053] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0054] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0055] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0056] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0057] The embodiment of the present invention provides a method for preparing a Peptide / MIPDA / AuNPs / CS-900 / ITO sensor, comprising the following steps:

[0058] (1) dissolving D-glucose and sodium chloride in water and reacting at high temperature to obtain a carbon precursor;

[0059] (2) heating the carbon precursor in an oil bath, then adding AuCl4H7O3 and stirring for reaction, adding anhydrous ethanol to the reaction system, mixing, centrifuging, collecting the precipitate and annealing to obtain AuNPs / CS-900;

[0060] (3) dispersing the AuNPs / CS-900 and Nafion solution in anhydrous ethanol, then applying it to the exposed area of ​​the ITO surface and drying it;

[0061] (4) applying 3-mercaptopropionic acid solution to the surface of AuNPs / CS-900 / ITO to fix AuNPs on the surface of CS / ITO electrode; then immersing the obtained electrode in a phosphate buffered saline (PBS) solution containing N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and hydroxysuccinimide, and then dropping vancomycin solution on the electrode after drying. After drying, the electrode is placed in a buffer solution for electrochemical polymerization to obtain a polymer film on the surface of the electrode. After drying, the vancomycin molecules embedded in the polymer film are removed, thereby obtaining a molecular imprinting sensor MIPDA / AuNPs / CS-900 / ITO;

[0062] (5) The vancomycin-binding tripeptide is coated on the surface of the molecular imprinting sensor MIPDA / AuNPs / CS-900 / ITO to obtain the Peptide / MIPDA / AuNPs / CS-900 / ITO sensor.

[0063] In some specific embodiments, in step (1), the concentration of D-glucose is 75 mg / mL;

[0064] The concentration of the sodium chloride is 0.01 mM;

[0065] The high temperature reaction condition is 120° C. for 12 h.

[0066] In some specific embodiments, in step (2), the temperature of the oil bath heating is 100°C;

[0067] The addition amount of AuCl4H7O3 is 0.1 mol / L;

[0068] The stirring reaction was carried out at a speed of 500 r / min for 30 min.

[0069] The volume ratio of the anhydrous ethanol to the reaction system is 1:1;

[0070] The centrifugal condition is 10000 rpm / min for 15 min;

[0071] The ultra-high temperature reaction conditions are: heating to 900° C. at a heating rate of 10° C. / min under N 2 flow for 2 hours.

[0072] In some specific embodiments, in step (3), the concentration of AuNPs / CS-900 is 1 mg / mL;

[0073] The mass volume ratio of the AuNPs / CS-900 and Nafion solution is 1 mg:10 μL;

[0074] The drying condition is drying under an infrared lamp.

[0075] In some specific embodiments, in step (4), the concentration of the 3-mercaptopropionic acid solution is 10 mM, 10 μL; the concentration of the N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is 0.2 M (705.6 μL); the concentration of the hydroxysuccinimide is 0.1 M (0.23018 g); the concentration of the phosphate buffer is 0.1 M, and the volume is 20 mL;

[0076] The concentration of the vancomycin solution is 1 mM and the volume is 30 μL;

[0077] The electrochemical polymerization conditions are: in a 10 mM phosphate buffer solution containing 5 mM dopamine, pH 7.4, nitrogen degassing for 10 min, and electrochemical polymerization for 6 cycles at a potential between -0.5 V and 0.5 V at a scan rate of 20 mV / s;

[0078] The method for removing the vancomycin molecules embedded in the polymer film is: using an ethanol / acetic acid mixture with a volume ratio of 95:5 to remove the vancomycin molecules embedded in the polymer film under magnetic stirring.

[0079] In some specific embodiments, in step (5), the vancomycin binding tripeptide is L-Lys-D-Ala-D-Ala, with a concentration of 0.2 mM and a volume of 20 μL.

[0080] The embodiment of the present invention also provides a Peptide / MIPDA / AuNPs / CS-900 / ITO sensor prepared by the preparation method.

[0081] The embodiment of the present invention also provides the use of the Peptide / MIPDA / AuNPs / CS-900 / ITO sensor in the preparation of a product for detecting vancomycin.

[0082] An embodiment of the present invention further provides a product for detecting vancomycin, comprising the Peptide / MIPDA / AuNPs / CS-900 / ITO sensor.

[0083] The embodiment of the present invention also provides a method for detecting vancomycin for non-disease diagnosis or treatment purposes, using the Peptide / MIPDA / AuNPs / CS-900 / ITO sensor or the product to detect vancomycin in a sample to be tested.

[0084] The present invention constructs a molecular imprinting polymer and peptide combined sensor for specific recognition of vancomycin. First, an AuNPs / CS-900 composite material is prepared. On the one hand, gold nanoparticles are fixed on the surface of carbon spheres by introducing Au-S bonds. On the other hand, EDC and NHS reagents are added to carboxylate mercaptopropionic acid, thereby promoting the formation of amide bonds with the amino groups in vancomycin for fixation. The subsequent stages of molecular imprinting synthesis include using dopamine as a monomer and vancomycin as a template, preparing imprinting polymers by electropolymerization, and obtaining MIP recognition cavities after removing vancomycin by chemical reagents. Then, vancomycin-binding tripeptide (Peptide) is bound to the surface of MIPDA / AuNPs / CS-900 to form an electrode. A polypeptide-molecular imprinting hybrid recognition cavity is obtained. In the process of detection using differential pulse voltammetry, K3[Fe(CN)6] 3- / 4- As a redox probe, the rebinding of vancomycin into the cavity produced a sensitive current response. The successful detection of vancomycin content in patient plasma showed the great potential of the hybrid recognition strategy in practical applications.

[0085] Example 1

[0086] 1 Materials and methods

[0087] 1.1 Reagents and Materials

[0088] Potassium ferrocyanide (K3[Fe(CN)6]), potassium dihydrogen phosphate (KH2PO4), sodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), and potassium chloride (KCl) were purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd.; vancomycin (VAN), hydroxysuccinimide (NHS), N-(3-dimethylpropyl)-N'-ethylcarbodiimide hydrochloride (EDC), 3-mercaptopropionic acid (C3H6O2S), and tetrachloroauric acid trihydrate (HAuCl4·3H2O) were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Glucose and urea were purchased from Tianjin Shengao Chemical Reagent Co., Ltd., and potassium bromide was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. l-Ascorbic acid (ASA), anhydrous ethanol (EtOH Ethnol), and sodium chloride (NaCl) were purchased from Tianjin Xinbote Chemical Co., Ltd. A vancomycin-binding tripeptide with the sequence -L-Lys-D-Ala-D-Ala was synthesized by Shanghai Sangong Biotechnology Co., Ltd. Plasma samples were obtained from volunteers of the Xinjiang Military Region General Hospital using an established venous blood collection method. Ultrapure water (18.2 MΩ) was produced by a Milli-Q water purification system, and all chemicals used were analytical grade chemicals.

[0089] 1.2 Equipment

[0090] All electrochemical measurements were performed on a standard three-electrode electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd., CHI760E), with Ag / AgCl as the reference electrode, platinum wire as the control electrode, and indium tin oxide (ITO) electrode modified with molecular imprinting and vancomycin binding peptide as the working electrode. The morphology of the modified electrode was characterized by a German Zeiss sigma300 scanning electron microscope; the elements and content of the material were characterized by a PHI5000 VersaprobeIII XPS (ULVAC-PHI.INC); IR-prestige21 (Shimadzu, Japan) was used to characterize the functional groups in the synthesized material.

[0091] 2 Synthesis of AuNPs / CS-25 and AuNPs / CS-900

[0092] Dissolve D-glucose (75 mg / mL) and sodium chloride (0.01 mM) in 75 mL of deionized water, stir for 5 min, and pour the solution into a Teflon-lined stainless steel autoclave. After 12 h at 120 ° C, the reaction is complete, and the autoclave begins to cool to room temperature. Collect the precipitate. Wash with pure water and ethanol several times until the filtrate becomes colorless, and dry in an oven at 60 ° C to obtain a carbon precursor.

[0093] 0.5 g of carbon precursor was accurately weighed into a 50 mL three-necked flask and heated in an oil bath at 100 ° C. Then 0.1 mol / L of AuCl4H7O3 was added, stirred at 500 r / min for 30 min, and an equal volume of anhydrous ethanol was added, and ultrasonic treatment and mixing were performed for 5 min. Finally, after centrifugation at 10000 rpm / min for 15 min, the obtained precipitate product was divided into two parts; one part was dried in an oven at 60 ° C to obtain AuNPs / CS-25. The other part was placed in a tubular furnace and heated under N2 flow at a heating rate of 10 ° C / min to 900 ° C for 2 hours. The resulting material is called AuNPs / CS-900.

[0094] 3. Electrode Preparation

[0095] ITO conductive glass (0.5 cm × 2.0 cm) was ultrasonicated for 30 minutes in a solution containing pure water and conductive glass cleaning solution in a ratio of 10:1, and then ultrasonicated for 10 minutes in pure water, and repeated three times. AuNPs / CS-900 (1 mg) and Nafion (perfluorosulfonic acid polymer) solution (10 μL) were dispersed in anhydrous ethanol (1 mL) and ultrasonically dispersed for 30 minutes, and then 20 μL of AuNPs / CS-900 material was drop-coated on the exposed area of ​​the ITO surface ([5 (W) × 5 (H) mm]) and dried under an infrared lamp for 5 minutes.

[0096] To prepare the molecular imprinting and peptide dual recognition electrode, 20 μL of 3-mercaptopropionic acid (10 mM) solution was first drop-coated on the AuNPs / CS-900 / ITO surface and stored at 4 °C overnight. This process fixed the AuNPs on the CS / ITO electrode surface through the Au-S bond. Next, the obtained electrode was immersed in a solution containing N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.2 M) and hydroxysuccinimide (0.1 M) for 1 h. After drying at room temperature, 30 μL of vancomycin solution with a concentration of 1 mM was drop-coated, and it was rinsed with purified water and set aside. After it was completely dried, it was placed in a 10mM phosphate buffer solution (PH=7.4, nitrogen degassing for 10min) containing 5mM fresh dopamine, and electrochemical polymerization was performed for 6 cycles at a scanning rate of 20mV / s at a potential between -0.5V and 0.5V, and a dense non-conductive polymer film was obtained on the electrode surface, which was blown dry with nitrogen. Then, ethanol / acetic acid (v / v=95 / 5) was used to remove the vancomycin molecules embedded in the polymer film under magnetic stirring, and the molecular imprinting sensor MIPDA / AuNPs / CS-900 / ITO with specific recognition ability for vancomycin was obtained.

[0097] Subsequently, 20 μL of vancomycin-binding tripeptide (L-Lys-D-Ala-D-Ala, 0.2 mM) solution was drop-coated on the MIPDA / AuNPs / CS-900 / ITO surface, and then washed with purified water and dried with nitrogen to obtain the Peptide / MIPDA / AuNPs / CS-900 sensor.

[0098] 4 Electrochemical detection

[0099] The electrochemical experiments were conducted using a conventional three-electrode system, with the ITO electrode modified with peptide / MIPDA / AuNPs / CS-900 materials as the working electrode, the Ag / AgCl electrode as the reference electrode, and platinum as the counter electrode.

[0100] The electrochemical determination in this experiment was carried out at 5 mM [Fe(CN)6] 3- / 4- The electrochemical properties of the modified electrodes were evaluated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques in 0.1 M KCl solution. The EIS measurements were performed at open circuit potential with a frequency range of 0.1 Hz-100 kHz and an amplitude of 50 mV. In addition, differential pulse voltammetry (DPV) was used to evaluate the electrochemical response of the peptide / MIPDA / AuNPs / CS-900 modified indium tin oxide (ITO) electrode in the presence of vancomycin samples, which were added to phosphate buffer (PB) with a pH of 7.0, and the scan rate was also 50 mV / s.

[0101] 5 Enzyme-amplified immunoassay

[0102] Sample determination: According to the Viva-E selfEMIT@2000 vancomycin detector of Siemens Medical Diagnostics Co., Ltd., the vancomycin hydrochloride standard solution (5μg / mL, 10μg / mL, 15μg / mL, 20μg / mL, 30μg / mL, 40μg / mL, 50μg / mL) of the dynamic analyzer calibration product series should be used to draw a standard curve. The patient blood sample was centrifuged at a speed of 5000r / min for 5 minutes, and then the supernatant was taken for detection.

[0103] 6 Results and Discussion

[0104] 6.1 SEM Characterization of Peptide / MIPDA / AuNPs / CS-900

[0105] Figure 1A SEM was performed to characterize the morphology and elemental composition of AuNPs / CS. CS was uniformly distributed spheres, and AgNPs were distributed on the surface of CS. Energy dispersive X-ray (EDX) spectroscopy results further confirmed the presence of Au( Figure 1G ). AuNPs not only increase the stability and conductivity of the electrode.

[0106] Next, the polydopamine (PDA) layer was prepared using electropolymerization cyclic voltammetry, and the MIPDA complex was successfully synthesized. Figure 1B The scanning electron microscopy of MIPDA / AuNPs / CS-900 / ITO is shown, and it can be seen that a dense non-conductive polymer film is obtained on the surface. Finally, the scanning electron microscopy ( Figure 1C ) The results showed that the peptide-coated surface was much rougher than the uncoated surface, indicating that the vancomycin-binding peptide was successfully introduced into the MIPDA / AuNPs / CS-900 / ITO surface. In addition, the element distribution EDS spectrum confirmed the presence of C, N, O and gold elements ( Figure 1D , 1E , 1F, 1G).

[0107] 6.2 Infrared Characterization

[0108] In order to determine whether the molecular imprinting and polypeptide layers were modified in the AuNPs / CS-900 composites, e.g. Figure 2A As shown in Figure 2, the presence of functional groups in the material was confirmed by infrared spectroscopy. In the spectrum of carbon spheres, 2953 and 2712 cm -1 The characteristic peaks at 1636 and 1361 cm -1 The characteristic peaks at 3200-3600 and 669 cm-1 correspond to the C=C and C=O vibrations, which are the result of aromatization of glucose during the treatment process. -1 The broad band in the range can be attributed to the OH stretching vibration of the hydroxyl groups, with 2370 cm -1 The peak of 579-1239cm-1 belongs to the C=O stretching vibration, indicating the successful formation of the polydopamine molecular imprinting film. -1 NH stretching vibration, NH, O=CN, and C=O bending vibration appeared in the range, and the results showed that the polypeptide existed and the electrode was successfully synthesized.

[0109] 6.3XPS

[0110] Figure 2B The full XPS spectrum of Peptide / MIPDA / AuNPs / CS-900 is shown. The main elements in the material are C, N, O and Au, and the C1s peaks are located at 284.8, 286, 288.2 and 292.3 eV ( Figure 2C), which can be attributed to the CC, C=C, CN, C=O, COOR, and π-π* functional groups in carbon spheres and molecular imprinting. The N1s signals at 399.78 and 401.39 eV ( Figure 2D ) can be attributed to R-NH-R and R-NH2, indicating the presence of dopamine and peptides. The O1s signals at 531.5 and 532.8 eV ( Figure 2E ) can be attributed to -OH and CO in polydopamine. The peaks of Au 4f are at 87.3 and 90.9 eV, and their peaks can be assigned to two parts: Au4f 7 / 2 ,Au 4f 5 / 2 .

[0111] 7. Optimization of parameters affecting electrode performance

[0112] 7.1 Drop volume (vancomycin)

[0113] By optimizing the electrode performance parameters, a stable and effective MIP polymer can be obtained. As the first step of the optimization process, 10, 20, 30, 40 and 50 μL of vancomycin solution with a concentration of 1 mM was drop-coated on the AuNPs / CS-900 / ITO surface. Figure 3A The relationship between the drop volume and the peak current is shown. The peak current continues to decrease between 10 and 30 minutes and remains constant after 30 μL. The reason for the decrease in current is that vancomycin inhibits the K3[Fe(CN)6] 3- / 4- The redox reaction occurred on the electrode surface. The results showed that 30 μL of vancomycin was used for all experiments.

[0114] 7.2 Number of aggregation circles

[0115] Polydopamine (PDA) is usually formed by dopamine self-polymerization or dopamine electropolymerization under alkaline conditions. In order to accurately control the thickness of the molecular imprint, a MIPDA / AuNPs / CS-900 / ITO molecular imprinted sensor was prepared using a controlled electropolymerization method. In the first CV cycle, an almost quasi-reversible redox pair was observed on the electrode at 0.35–0.12 V. With the increase in the number of cycles, the thickness of polydopamine increased, the peak current gradually decreased, and the difference in redox peak potential decreased. The effect of the number of cycles on the VAN response sensitivity in DPV measurement was studied. After 6 CV cycles, the MIPDA / AuNPs / CS-900 / ITO ( Figure 3B ) produced the maximum response signal. Too few cycles resulted in a very thin PDA layer that could not completely cover the vancomycin-peptide complex to produce the entire imprinted cavity of vancomycin, resulting in a small change in the recombination current peak. On the other hand, too many cycles produced a thicker PDA layer, increasing the K3[Fe(CN)6] 3- / 4-The impedance of the redox probe to the electrode surface results in poor detection sensitivity.

[0116] 7.3 Elution time

[0117] The removal of template molecules affects the formation / exposure of specific cavities; therefore, the removal process should be carried out without damaging the polymer film structure, and the selection of the most appropriate solution and time in this step can also achieve the rebinding process. The present invention uses an anhydrous ethanol / acetic acid combination, and immerses the prepared sensor in an anhydrous ethanol / acetic acid (95:5) solution for 5, 10, 15, 20 and 30 minutes at room temperature to optimize the template removal time. Figure 3C The removal time of the template and the corresponding current change are shown. Between 5-15 minutes, the current value continues to increase and remains constant after 15 minutes. Based on these results, a removal time of 15 minutes was selected and used in subsequent experiments.

[0118] 7.4MIPDA / AuNPs / CS-900 / ITO incubation time

[0119] Incubation time is the most important parameter that determines the analysis time, economy, and performance. To evaluate the effect of different incubation times, the prepared MIPDA / AuNPs / CS-900 sensor was immersed in 50 μg / mL vancomycin solution at room temperature for 0, 10, 20, and 30 min. Figure 3D It was shown that, compared with the sharp increase between 0 and 10 min, the peak current difference (ΔI) before and after template removal did not change significantly at 10, 20, and 30 min, and even remained almost unchanged at 20 and 30 min. Therefore, before detecting vancomycin, the incubation time of the electrode immersed in 50 μg / mL vancomycin solution was determined to be 20 min, and remained unchanged in the subsequent steps.

[0120] 7.5 drop volume (peptide)

[0121] By optimizing the electrode performance parameters, a stable and effective MIP-Pepide polymer can be obtained. Peptides with a concentration of 0.2 mM and a volume of 0, 10, 20, and 30 μL were drop-coated on MIPDA / AuNPs / CS-900. Figure 3E The relationship between the drop volume and the peak current is shown. The current value continues to decrease between 0 and 20 min, and remains unchanged after 20 μL. The reason for the decrease in current is that the peptide blocks the K3[Fe(CN)6] 3- / 4- The redox reaction occurred on the electrode surface. The results showed that 20 μL of peptide would be used in subsequent experiments.

[0122] 7.6 Peptide / MIPDA / AuNPs / CS-900 / ITO incubation time

[0123] The rebinding time is the most important parameter that determines the analytical time, economy, and performance. To evaluate the effect of different rebinding times, the prepared MIP-peptide-based sensor was immersed in 50 μg / mL vancomycin solution at room temperature for 10, 20, 30, and 40 min. Figure 3F It showed that the peak current (ΔI) before and after rebinding did not change significantly at 20, 30 and 40 min, but decreased sharply between 10 and 20 min, and remained almost unchanged between 20 and 30 min. 20 min was selected as the optimal incubation time for rebinding and remained unchanged.

[0124] 8. Preparation and characterization of modified electrodes

[0125] Previous studies have shown that high-temperature annealing can promote atomic rearrangement of carbon materials and improve structural order, thereby effectively improving the conductivity of carbon materials. Figure 4A The CV curves of electrodes with different modified materials show that the peak current of AuNPs / CS-900 electrode is higher than that of AuNPs / CS-25, which may be the reason why high temperature annealing can effectively increase the conductivity. Differential pulse voltammetry (DPV) was used for voltammetric testing. Figure 4B shown.

[0126] EIS is widely used to study charge transfer in electrode interface recognition events. Figure 4C It shows the K3[Fe(CN)6] 3- / 4- EIS measurement results of the redox probe on AuNPs / CS-900 / ITO. The larger the Rct value, the greater the transfer resistance. Figure 4C As shown in the figure, compared with the EIS graph of AuNPs / CS-25, the resistance of AuNPs / CS-900 is smaller. This is because the increase in material conductivity is conducive to the transfer of redox probes, resulting in a lower Rct, that is, a lower charge transfer resistance. Compared with CV and EIS, differential pulse voltammetry (DPV) has a higher sensitivity to response, so the change of DPV can be selected as the response signal of vancomycin in the sample. Figure 4D The responses of different treated materials in vancomycin and PBS solutions are shown. In vancomycin solution (40 μg / mL), AuNPs / CS-25 / ITO and AuNPs / CS-900 / ITO have peaks at 0.304-0.324 V, but no peaks appear in PBS solution.

[0127] During the preparation process, MIPDA / AuNPs / CS-900 / ITO was characterized by electrochemical methods. Figure 4EThe CV curves of the electrode at different modification steps are shown. Compared with bare ITO, the gold nanoparticle carbon nanosphere composite increases the electrode surface area and stability, while improving the peak current. When vancomycin was drop-coated on AuNPs / CS, the peak current dropped significantly. This may be due to the covering effect of vancomycin, which increases the transfer resistance of the redox probe to the electrode surface and reduces the current signal. After DA electropolymerization, the dense PDA film hinders the transfer of K3[Fe(CN)6] 3- / 4- The oxidation peak of MIPDA / AuNPs / CS-900 / ITO decreased sharply due to the electron transfer. However, after the removal of vancomycin by washing with organic reagents, the oxidation peak current increased again because the partially exposed imprinted cavities promoted the electron transfer of K3[Fe(CN)6] 3- / 4- Transfer of the redox probe to the electrode surface. When vancomycin re-binds into the imprinted cavity, the peak current again shows a significant decrease. Figure 4F The DPV curves in the figure show that the peak current variation trend is consistent with the CV measurement results.

[0128] Example 2

[0129] 1Performance Test

[0130] 1.1 Linearity establishment in PBS solution

[0131] Under the optimized conditions, the electrochemical performance of the Peptide / MIPDA / AuNPs / CS-900 / ITO sensor was tested in solutions containing different concentrations of vancomycin (5-50 μg / mL). Figure 5A The DPV graph obtained within the linear concentration range for the determination of vancomycin is given. It can be seen that as the concentration of vancomycin increases, the current trend continues to decrease, indicating that the vancomycin in the solution is continuously bound to the molecular imprint and the peptide. There is a certain linear relationship between the current value and the concentration ( Figure 5B ), the regression equation is I1=-0.01213C1+2.73858(R 2 =0.99736). The LOD (LOD = 3s / m) and LOQ (LOQ = 10s / m) values ​​measured by 10 sets of blank experiments were 0.42μg / mL and 1.4μg / mL, respectively, where S is the response standard deviation and m is the slope of the standard curve. Based on these results, the Peptide / MIPDA / AuNPs / CS-900 / ITO sensor has excellent sensitivity.

[0132] 1.2 Linearity establishment in plasma samples

[0133] The peptide / MIPDA / AuNPs / CS-900 / ITO sensor was applied to the detection of vancomycin concentration in plasma samples to verify the applicability and accuracy of the sensor. First, the DPV linear curve of the peptide / MIPDA / AuNPs / CS-900 / ITO sensor was as follows: Figure 5C As shown, the linear curve of vancomycin was constructed for plasma samples in the linear range of 5 μg / mL to 50 μg / mL ( Figure 5D ), the regression equation is I2=-0.01133C2+2.72448(R 2 =0.99368), the detection limit and detection limit of its electrode in plasma samples were 0.88 μg / mL and 2.9 μg / mL, respectively, indicating that it has certain applicability and accuracy in detection in patient plasma.

[0134] In order to explore the selectivity of MIPDA / AuNPs / CS-900 / ITO and Peptide / MIPDA / AuNPs / CS-900 / ITO sensors for vancomycin, the selectivity of vancomycin (VAN), tigecycline (TIC), meropenem (MER), piperacillin-tazobactam (P / T), fluconazole (FLC), uric acid (UA), imipenem (IPM), polymyxin B (PB), ascorbic acid (ASA) and other drugs combined with vancomycin were tested for the electrodes. Each compound was mixed with vancomycin at a ratio of 1:10 for detection. The concentration of vancomycin was 20μg / mL, and other interfering drugs were added at a concentration of 200μg / mL. Peptide / MIPDA / AuNPs / CS-900 / ITO produced the greatest response to vancomycin and produced very small signals for other compounds (5E). The results showed that the affinity and specificity of Peptide / MIPDA / AuNPs / CS-900 / ITO for vancomycin were higher than those of other compounds. The imprint and peptide dual recognition sensor had higher selectivity than molecular imprint recognition alone because dual recognition had more binding sites than single molecular imprint. Compared with MIPDA / AuNPs / CS-900 / ITO, Peptide / MIPDA / AuNPs / CS-900 / ITO had higher response in the presence of other compounds. This was because the specific recognition ability based on MIPDA was insufficient, resulting in partial binding with other compounds. The excellent selectivity of Peptide / MIPDA / AuNPs / CS-900 / ITO provided excellent anti-interference ability in practical applications. In addition, dual recognition allowed vancomycin to be tightly re-bound to the imprint. The results showed that the selected interfering drugs had no obvious interference effect on the detection of vancomycin.

[0135] The stability of the electrode is an important factor affecting its clinical application effect. Therefore, in order to study the stability of the Peptide / MIPDA / AuNPs / CS-900 / ITO sensor, we stored it in a refrigerator at 4°C. The determination of vancomycin solution was completed every other day for 13 consecutive days. After each measurement, the electrode was washed with anhydrous ethanol / acetic acid (95:5) to expose the imprinted site, and then stored in a refrigerator. After 13 days, the peak current was 91.8% of the initial current value ( Fig. 5F ), the RSD of the peak current was 2.6%, indicating that the Peptide / MIPDA / AuNPs / CS-900 / ITO sensor had good stability.

[0136] The repeatability of the Peptide / MIPDA / AuNPs / CS-900 / ITO sensor was evaluated under comparable conditions. Figure 5G After 10 consecutive measurements using the same electrode, the relative standard deviation (RSD%) was determined to be 2.7%, indicating that this electrode has good reproducibility.

[0137] Finally, the recovery rate of the Peptide / MIPDA / AuNPs / CS-900 / ITO sensor was tested, and the concentration of the sample was selected from the linear range (5, 20, 50 μg / mL) to evaluate the accuracy of the electrode in detecting vancomycin. The average recovery rate was 97.53%-110.4%; the RSD value was 1.25-8.65%, and the results showed the accuracy and applicability of the electrode in detecting vancomycin.

[0138] Example 3

[0139] Sample measurement and comparison

[0140] Patient plasma samples were analyzed using the Peptide / MIPDA / AuNPs / CS-900 / ITO sensor to demonstrate the applicability of the sensor for the detection of vancomycin in plasma. Figure 5H As shown, the Bias% value of the concentration value of vancomycin detected in plasma using this electrode and EMIT method is less than 12%, which indicates that this electrochemical detection method is very reliable in measuring vancomycin in actual samples.

[0141] Compared with traditional methods, an electrochemical sensor was constructed by introducing vancomycin-binding tripeptide and molecular imprinting to improve the sensitivity, specificity and selectivity of plasma sample analysis. Compared with other detection methods, the limit of quantification (LOD) was as low as 0.88 μg / mL, indicating that this method is reliable and accurate and can be used for the determination of vancomycin in plasma.

[0142] In summary, the present invention combines dopamine molecular imprinting polymer with peptide polymer membrane to successfully construct an electrochemical sensor for selective detection of vancomycin. The sensor was characterized by scanning electron microscopy, and the Peptide / MIPDA / AuNPs / CS-900 / ITO sensor was electrochemically characterized by CV and DPV methods. The electrode has strong specificity for vancomycin, and the established method for detecting plasma samples of vancomycin patients is accurate, with a detection limit as low as 0.88μg / mL. The results show that the combination of molecular imprinting and peptide improves the selectivity and specificity in the detection of vancomycin, which can be used to monitor clinical blood drug concentrations and further promote the transformation of research results into clinical applications.

[0143] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for preparing a peptide / MIPDA / AuNPs / CS-900 / ITO sensor, characterized in that: The following steps are involved: (1) dissolving D-glucose and sodium chloride in water and reacting at high temperature to obtain a carbon precursor; (2) heating the carbon precursor in an oil bath, then adding HAuCl4·3H2O to stir the reaction, adding anhydrous ethanol to the reaction system, mixing, centrifuging, collecting the precipitate and annealing to obtain AuNPs / CS-900; (3) dispersing the AuNPs / CS-900 and Nafion solution in anhydrous ethanol, then applying it to the exposed area of ​​the ITO surface and drying; (4) applying a 3-mercaptopropionic acid solution to the surface of AuNPs / CS-900 / ITO to fix AuNPs on the surface of the CS / ITO electrode; then immersing the obtained electrode in a solution containing N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and hydroxysuccinimide, and then dripping a vancomycin solution after drying. After drying, the electrode is placed in a buffer solution for electrochemical polymerization to obtain a polymer film on the surface of the electrode, and finally the vancomycin molecules embedded in the polymer film are removed to obtain a molecular imprinting sensor MIPDA / AuNPs / CS-900 / ITO; (5) The vancomycin-binding tripeptide is coated on the surface of the molecular imprinting sensor MIPDA / AuNPs / CS-900 / ITO to obtain the Peptide / MIPDA / AuNPs / CS-900 / ITO sensor.

2. The preparation method according to claim 1, characterized in that: In step (1), the concentration of D-glucose is 75 mg / mL; The concentration of sodium chloride is 0.01mM; The high temperature reaction condition is 120° C. for 12 h.

3. The preparation method according to claim 1, characterized in that: In step (2), the temperature of the oil bath heating is 100°C; The addition amount of AuCl4H7O3 is 0.1 mol / L; The stirring reaction was carried out at a speed of 500 r / min for 30 min. The volume ratio of the anhydrous ethanol to the reaction system is 1:1; The centrifugal condition is 10000 rpm / min for 15 min; The ultra-high temperature reaction conditions are: heating at a heating rate of 10°C / min under a N2 flow, the temperature reaches 900°C, and lasts for 2 hours.

4. The preparation method according to claim 1, characterized in that: In step (3), the concentration of AuNPs / CS-900 is 1 mg / mL; The mass volume ratio of the AuNPs / CS-900 and Nafion solution is 1 mg:10 μL; The drying condition is drying under an infrared lamp.

5. The preparation method according to claim 1, characterized in that: In step (4), the concentration of the 3-mercaptopropionic acid solution is 10 mM; The concentration of the N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is 0.2M; the concentration of the hydroxysuccinimide is 0.1M; The concentration of the vancomycin solution is 1 mM and the volume is 30 μL; The electrochemical polymerization conditions are: in a 10 mM phosphate buffer solution containing 5 mM dopamine, pH 7.4, nitrogen degassing for 10 min, and electrochemical polymerization for 6 cycles at a potential between -0.5 V and 0.5 V at a scan rate of 20 mV / s; The method for removing the vancomycin molecules embedded in the polymer film is: eluting with a mixed solution of ethanol / acetic acid with a volume ratio of 95:5 for 20 minutes under magnetic stirring to remove the vancomycin molecules embedded in the polymer film.

6. The preparation method according to claim 1, characterized in that: In step (5), the vancomycin-binding tripeptide is L-Lys-D-Ala-D-Ala, with a concentration of 0.2 mM and a volume of 20 μL.

7. The Peptide / MIPDA / AuNPs / CS-900 / ITO sensor prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the peptide / MIPDA / AuNPs / CS-900 / ITO sensor as claimed in claim 7 in the preparation of a product for detecting vancomycin.

9. A product for detecting vancomycin, characterized in that: Includes the Peptide / MIPDA / AuNPs / CS-900 / ITO sensor as described in claim 7.

10. A method for detecting vancomycin for purposes other than disease diagnosis or treatment, characterized in that: The Peptide / MIPDA / AuNPs / CS-900 / ITO sensor according to claim 7 or the product according to claim 9 is used to detect vancomycin in the sample to be tested.

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