Anti-pollution electrochemical sensor for rapidly detecting glucose in whole blood as well as preparation method and detection method of anti-pollution electrochemical sensor

Abstract

The invention discloses an anti-pollution electrochemical sensor for rapidly detecting glucose in whole blood as well as a preparation method and a detection method of the anti-pollution electrochemical sensor, and belongs to the technical field of detection. According to the invention, Ni3 (HITP) 2-MOF particles are dispensed on the surface of the working electrode as a modification material, the Ni3 (HITP) 2-MOF particles have good conductivity and can enhance the conductivity of the electrode, and the MOF material also has good catalytic ability and can enhance the recognition of the sensor on glucose. The glucose molecularly imprinted polymer is synthesized through electropolymerization, the molecularly imprinted polymer has the advantages of high sensitivity and strong specificity, and the specificity and interference resistance of the sensor to glucose are greatly improved. After the molecularly imprinted polymer is prepared on the surface of the working electrode, the anti-fouling material phase change bovine serum albumin is modified, and the anti-fouling material can avoid non-specific adsorption of other interference components in human blood. The method can be used for detecting a glucose standard sample, can also be used for detecting glucose in whole blood of a human body, provides a new idea for clinical prevention and control of diabetes, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical fields of nano-functional materials, biomedical analysis and detection, and electrochemical sensing technology, and provides an electrochemical sensor for detecting glucose in human whole blood, a preparation method thereof, and a detection method thereof. Background Art

[0002] The content of glucose in blood is a key indicator for diagnosing diabetes. According to the report of the World Health Organization (WHO), the number of diabetes patients has increased from 108 million in 1980 to 422 million in 2014, and the diabetes-related mortality rate has increased by 3% between 2000 and 2019. The detection of whole blood glucose plays an important role in the diagnosis, treatment, prevention of complications of diabetes, and early screening of other related diseases. By detecting the whole blood glucose concentration, diabetes can be effectively screened and diagnosed, and for diagnosed diabetes patients, regular detection of the whole blood glucose level is an important means to evaluate the blood glucose control status.

[0003] Currently, the detection of glucose mainly uses chromatography, spectroscopy, etc. However, the above methods have the disadvantages of long detection time, complex operation, and expensive equipment. Compared with the above methods, electrochemical sensors have the advantages of fast analysis speed, ultra-sensitivity, high selectivity, low cost, and easy operation, and are considered a potential new method for detecting glucose. However, the currently developed electrochemical sensors cannot directly detect glucose in human whole blood and need to dilute the blood and use the standard addition method for detection. The molecularly imprinted polymer and the anti-fouling material phase-changing bovine serum albumin synthesized in the present invention improve the anti-interference ability of the electrochemical sensor, can specifically recognize glucose molecules, and realize the detection of glucose in human whole blood.

[0004] The present invention fabricates a screen-printed electrode. First, Ni 3 (HITP) 2 -MOF particles are modified on the working electrode, and a molecularly imprinted polymer is prepared by an electropolymerization method. The template molecule glucose is removed by elution, so as to specifically recognize glucose. An anti-fouling material phase-changing bovine serum albumin is modified on the surface of the working electrode modified with the molecularly imprinted polymer, and then glucose in human whole blood is specifically recognized, and a molecularly imprinted electrochemical sensor for detecting glucose in human whole blood with excellent performance, high sensitivity, and strong specificity is prepared. The present invention uses a screen-printed electrode, which is inexpensive, combines electrochemical technology, and has high sensitivity; combines molecular imprinting technology and the anti-fouling material phase-changing bovine serum albumin, improves the specificity and anti-interference ability of the sensor; the sample does not need complex treatment, the detection time is short, and the detection sensitivity is high, which is a fast, inexpensive, and sensitive detection method. Summary of the Invention

[0005] The object of the present invention is to provide an anti-pollution electrochemical sensor for rapid detection of glucose in whole blood, and its preparation method and detection method, so as to overcome the problem that existing electrochemical sensors cannot directly detect glucose in human whole blood. First, based on existing literature, the synthesis conditions of Ni 3 (HITP) 2 -MOF particles are optimized, and Ni 3 (HITP) 2 -MOF particles are prepared. The surface of the working electrode is modified with Ni 3 (HITP) 2 -MOF particles, and the concentration of Ni 3 (HITP) 2 -MOF solution is optimized to be 1 mg·mL -1 . Then, a glucose molecularly imprinted electrochemical sensor based on Ni 3 (HITP) 2 -MOF particles is successfully prepared by means of electropolymerization and ethanol polar elution. Then, an anti-pollution material, phase-change bovine serum albumin, is prepared, and the phase-change bovine serum albumin material is modified on the surface of the working electrode to specifically detect glucose in human whole blood. The sensor prepared by the present invention can be directly used for highly sensitive detection of glucose in human whole blood.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a preparation method of an anti-pollution electrochemical sensor for rapid detection of glucose in whole blood. A portable screen-printed electrode is prepared, and a Ni 3 (HITP) 2 -MOF sensitive film is modified on the surface of the working electrode. An electropolymerized poly-o-phenylenediamine molecularly imprinted film is modified outside the Ni 3 (HITP) 2 -MOF layer. The electropolymerized poly-o-phenylenediamine molecularly imprinted film provides imprinting sites for the template molecule glucose. An anti-pollution material, phase-change bovine serum albumin, is modified outside the electropolymerized poly-o-phenylenediamine molecular imprinted layer. The above electrode is placed in a phosphate solution of 5.0 mmol·L -1 K 3 [Fe(CN) 6] and 0.1 mol·L -1 KCl and scanned and detected by cyclic voltammetry until a stable redox peak is obtained, and the electrode is rinsed with ultrapure water and dried;

[0008] The surface of the working electrode is modified with a Ni 3 (HITP) 2 -MOF sensitive film. The specific steps are as follows: 8-12 mg (0.031-0.042 mmol) of nickel chloride hexahydrate (NiCl2 .6H 2 O), 0.25 - 0.35 mL of concentrated ammonia water (13 mol / L), and 5 - 15 mg (0.010 - 0.024 mmol) of 2,3,6,7,10,11 - hexaaminotriphenylene hexahydrochloride (HATP·6HCl) are reacted at 65 °C for 1.5 - 2.5 hours. After the reaction, it is washed with acetone, and finally Ni 3 (HITP) 2 -MOF particles are obtained and formulated into a Ni 3 (HI TP) 2 -MOF solution. Take a Ni -1 solution with a concentration of 0.5 - 3 mg·mL 3 (HITP) 2 -MOF solution. Add 10 - 30 μL of the Ni 3 (HITP) 2 -MOF solution dropwise onto the surface of the working electrode. After drying, an electrode modified with Ni 3 (HITP) 2 -MOF particles is obtained; the thickness of the Ni 3 (HI TP) 2 -MOF particle sensitive film is 1.9 - 3.2 nm. The diameter of the sensitive film Ni 3 (HITP) 2 -MOF particles is 3.7 - 7.8 μm, presenting a granular porous layer structure, arranged loosely. The differential pulse voltammetry of the Ni 3 (HITP) 2 -MOF modified screen - printed electrode shows that the peak value in the potassium ferricyanide electrolyte increases by 2.5 - 3.0 times that of the bare electrode, approximately 180 - 220 μA;

[0009] Add o - phenylenediamine and glucose with a concentration ratio of 2:1 - 6:1 to a phosphate buffer solution with a concentration of 5 mmol·L -1 and a pH of 7.0. Place the modified electrode in the buffer solution for electro - polymerization. The reaction conditions for electro - polymerization are: scanning voltage 0 - 0.8 V, scanning rate 40 - 60 mV / s, and the number of scanning cycles 8 - 12. After obtaining the molecularly imprinted polymer film, perform CV and DPV detections on the electrode. The detection solution is 5.0 mmol·L -1 K 3 [Fe(CN) 6 and 0.1 mol·L -1 KCl. For CV detection: voltage range - 0.2 - 0.5 V, scanning rate 50 - 60 mV / s. For DPV detection: voltage range - 0.4 - 0.6 V, scanning rate 50 - 60 mV / s. The DPV peak value decreases for Ni 3(HITP) 2 2.5 to 2.75 times that of the MOF particle-sensitive film modified electrode. Rinse the electrode with ultrapure water to obtain a molecularly imprinted polymer film;

[0010] Electro-elute the template molecule glucose in a sodium hydroxide solution. Place the working electrode in a 0.8 - 1 mol / L sodium hydroxide solution, with a voltage range of -1 to 1 V and a scan rate of 50 - 60 mV / s. Electro-elute to obtain a molecularly imprinted polymer by cyclic voltammetry. Rinse with a phosphate buffer solution with a pH of 7.0. Perform CV and DPV detections on the eluted electrode. The detection solution is 5.0 mmol·L -1 K 3 [Fe(CN) 6 and 0.1 mol·L -1 KCl. For CV detection: the voltage range is -0.2 to 0.5 V and the scan rate is 50 - 60 mV / s. For DPV detection: the voltage range is -0.4 to 0.6 V and the scan rate is 50 - 60 mV / s. The DPV peak increases to 1.4 - 1.8 times that of the molecularly imprinted membrane modified electrode. Obtain the imprinting sites. The molecularly imprinted membrane has a poly(o-phenylenediamine) molecularly imprinted membrane on the outside of the sensitive membrane, and the poly(o-phenylenediamine) molecularly imprinted membrane provides the imprinting sites for the template molecule glucose.

[0011] Modify an anti-fouling material, phase-transition bovine serum albumin, outside the poly(o-phenylenediamine) molecularly imprinted layer. The specific steps are as follows: Mix tris(2-carboxyethyl)phosphine (TCEP) (30 - 50 mM, pH 5.0, adjusted with 3 - 5 M NaOH) and BSA (2 mg·mL -1 ) solutions in a volume ratio of 0.5:1 to 2:1 to prepare a phase-transition bovine serum albumin solution. Native BSA is converted into phase-transition bovine serum albumin oligomer nanoparticles and protofibrils in the solution and forms a phase-transition bovine serum albumin layer at the liquid-solid interface. Take a phase-transition bovine serum albumin solution with a concentration of 1 - 2 mmol·L -1 and drop 20 μL of the phase-transition bovine serum albumin solution onto the surface of the working electrode. After drying, obtain an electrode modified with the phase-transition bovine serum albumin material.

[0012] The present invention provides a method for electrode pretreatment: Pretreat the electrode before modifying Ni 3 (HITP) 2 -MOF particles and the molecularly imprinted polymer; The pretreatment includes: Perform CV scanning in a potassium ferricyanide probe solution, with a scanning potential of: -0.2 to -0.5 V and a scan rate of 50 - 60 mV / s until a stable redox peak is obtained; The electrode is a screen-printed electrode, and the probe solution is a PBS solution of [Fe(CN)6]3- / 4 and KCl; Place Ni 3 (HITP)2 -The working electrode SPCE / Ni modified with MOF particles 3 (HITP) 2 -The MOF was placed in a potassium ferricyanide probe solution for DPV scanning. The scanning potential was -0.4 to 0.6 V, the scanning rate was 50 to 60 mV / s, and the DPV peak increased by 2.5 to 3.0 times that of the bare electrode; the Ni 3 (HITP) 2 -The working electrode Ni 3 (HITP) 2 -The MOF / SPCE was characterized by SEM, and a layered structure of Ni 3 (HITP) 2 -MOF particles were successfully modified on the electrode surface; the MIP / Ni 3 (HITP) 2 -The working electrode MIP / Ni 3 (HITP) 2 -The MOF / SPCE was characterized by SEM, and it was observed that a thin film was covered on the Ni 3 (HITP) 2 -MOF layer, which proved that the molecularly imprinted system formed by electropolymerization was modified on the electrode surface; the working electrode modified with the phase change bovine serum albumin material was characterized by DPV and fluorescence, and it was observed that the electrode was not adsorbed by other components in the blood, which proved that the anti-fouling material phase change bovine serum albumin was successfully modified on the electrode surface and had a good anti-fouling effect.

[0013] The embodiment of the present invention also provides an anti-pollution electrochemical sensor for rapid detection of glucose in whole blood prepared by the foregoing method.

[0014] The embodiment of the present invention also provides an application of the foregoing anti-pollution electrochemical sensor for rapid detection of glucose in whole blood in detecting glucose.

[0015] The embodiment of the present invention also provides a method for rapid detection of glucose in whole blood, which includes:

[0016] Providing the foregoing anti-pollution electrochemical sensor for rapid detection of glucose in human whole blood;

[0017] Placing the anti-pollution electrochemical sensor for rapid detection of glucose in whole blood in glucose solutions with different concentrations and incubating at room temperature for a period of time, then rinsing the electrode with ultrapure water and placing it in a potassium ferricyanide probe solution for differential pulse testing to achieve the detection of the glucose standard sample to be measured and establish a linear equation. Then placing the working electrode of the electrochemical sensor for detecting glucose in human whole blood in a human whole blood sample for differential pulse testing to achieve the detection of glucose in human whole blood.

[0018] The anti-pollution molecularly imprinted electrochemical sensor has a specific recognition function for glucose, and can be combined with differential pulse voltammetry to analyze and determine glucose in samples. When there is no glucose in the detection environment, the working electrode surface is modified with Ni 3 (HITP) 2 -MOF particles. After eluting the template molecules, an imprinted cavity is formed on the surface of the working electrode, which effectively allows electron conduction, and the peak current value of the electrochemical sensor is relatively high. As the glucose concentration in the detection environment increases, more specific imprinted cavities will specifically recognize glucose molecules, and the electron transfer process of the potassium ferricyanide solution on the surface of the working electrode is blocked, and the peak current value of the electrochemical sensor decreases. According to the current change values corresponding to different concentrations of glucose in the detection environment, a linear relationship between the glucose concentration and the peak current change value is established to achieve specific detection of glucose. Drop the anti-pollution material phase change bovine serum albumin on the surface of the working electrode to prevent non-specific adsorption. After adsorbing human whole blood samples, specific detection of glucose in human whole blood is carried out according to the peak signal changes of different samples.

[0019] The detection linear range of the anti-pollution electrochemical sensor of the embodiment of the present invention for glucose is 1 μmol·L -1 ~100 mmol·L -1 ; the linear regression equation is ΔIp(μA) = 10.921 LgC(mmol·L -1 ) + 81.65, and the correlation coefficient R 2 is 0.997.

[0020] The anti-pollution electrochemical sensor of the embodiment of the present invention has good selectivity and anti-interference performance. The selectivity of the sensor was tested; the sample solutions were 1 mM glucose, uric acid, ascorbic acid, creatine, and lactic acid solutions. The above-mentioned molecularly imprinted electrochemical sensor was placed in the above solutions and incubated for a specific time of 10 - 25 minutes, and the current response was detected by differential pulse voltammetry. The signal response of the sensor to glucose was 65 μA, which was 8, 7, 7.5, and 6.5 times the current response values of uric acid, ascorbic acid, creatine, and lactic acid respectively, indicating that the sensor has excellent selectivity for glucose. The anti-interference performance of the sensor was tested. The interfering substances were 1 mM uric acid, ascorbic acid, creatine, and lactic acid solutions. 1 mM interfering substances were respectively added to 1 mM glucose solution. The above-mentioned molecularly imprinted electrochemical sensor was placed in the above solutions and incubated for a specific time of 10 - 25 minutes, and the current response was detected by differential pulse voltammetry. The signal response of the sensor to glucose was 75 μA., which was 1.05, 1.06, 1.08, and 1.08 times the current response values of uric acid, ascorbic acid, creatine, and lactic acid added respectively, indicating that the sensor has good anti-interference performance for glucose.

[0021] The anti-pollution electrochemical sensor of the embodiment of the present invention has a very high accuracy in detecting glucose in human whole blood. The prepared molecularly imprinted electrochemical biosensor was used to detect 100 whole blood samples of diabetic patients in the hospital. Each whole blood sample was continuously detected three times. The detection results of the sensor were compared with the hospital gold standard detection results, and the accuracy reached 94.2%, and the RSD was less than 3.4%. The sensor had good consistency in whole blood detection.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] (1) The present invention can be used for the detection of glucose in human whole blood, and has the advantages of high sensitivity, rapidity and high accuracy.

[0024] (2) The present invention optimizes the conditions of Ni 3 (HITP) 2 -MOF particles. The synthesized Ni 3 (HITP) 2 -MOF particles can enhance the electrochemical signal by 2.5 to 3.0 times, thereby improving the sensitivity of the sensor of the present invention.

[0025] (3) The molecularly imprinted polymer prepared by the present invention can specifically recognize glucose, thereby improving the specificity of the sensor of the present invention.

[0026] (4) The anti-pollution material phase change bovine serum albumin prepared by the present invention can prevent non-specific adsorption, thereby improving the anti-interference ability of the sensor of the present invention.

[0027] (5) The screen-printed electrode of the present invention is simple to prepare, low in cost, convenient to carry, consumes less samples, is easy to miniaturize and integrate, and has a wide application range.

[0028] (5) Compared with other screen-printed electrode sensors, the present invention can directly detect the glucose content in human whole blood, thereby providing a potential new method for monitoring diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in the present application. For those of ordinary skill in the art, other drawings can be obtained according to these drawings without creative efforts.

[0030] Figure 1 It is the CV control chart for the anti-pollution electrochemical sensor of the present invention in Example 5 to detect glucose. Curve a is the CV chart of the bare electrode, and curve b is the modified Ni 3(HITP) 2 CV diagram of the electrode after -MOF particles, curve c is the CV diagram of the electrode after electro - polymerizing glucose, curve d is the CV diagram of the electrode after eluting glucose, and curve e is the CV diagram of the electrode after adsorbing glucose.

[0031] Figure 2 This is the DPV control diagram for detecting glucose by the anti - fouling electrochemical sensor in Example 5 of the present invention. Curve a is the DPV diagram of the bare electrode, and curve b is the electrode modified with Ni 3 (HITP) 2 DPV diagram of the electrode after -MOF particles, curve c is the DPV diagram of the electrode after electro - polymerizing glucose, curve d is the DPV diagram of the electrode after eluting glucose, and curve e is the DPV diagram of the electrode after adsorbing glucose.

[0032] Figure 3 This is the optimized diagram of the number of electro - elution cycles for detecting glucose by the anti - fouling molecularly imprinted electrochemical sensor in Example 5 of the present invention

[0033] Figure 4 This is the optimized diagram of the adsorption time for detecting glucose by the anti - fouling molecularly imprinted electrochemical sensor in Example 5 of the present invention

[0034] Figure 5 This is the optimized diagram of the number of electro - polymerization scan cycles for detecting glucose by the anti - fouling molecularly imprinted electrochemical sensor in Example 5 of the present invention

[0035] Figure 6 This is the optimized diagram of the electro - polymerization scan rate for detecting glucose by the anti - fouling molecularly imprinted electrochemical sensor in Example 5 of the present invention

[0036] Figure 7 This is the glucose selectivity detection diagram of the sensor in Example 6 of the present invention

[0037] Figure 8 This is the glucose anti - interference detection diagram of the sensor in Example 6 of the present invention

[0037] Figure 9 This is the sensor stability detection diagram of Example 6 of the present invention

[0038] Figure 10 This is the diagram of the change in the DPV peak value of different concentrations of glucose in Example 6 of the present invention. The concentrations corresponding to the peak currents from high to low are: 1 μmol.L -1 , 10 μmol.L -1 , 100 μmol.L -1 , 1 mmol.L -1 , 10 mmol.L -1 , 100 mmol.L -1

[0039] Figure 11Linear equation graph for detecting glucose by the molecularly imprinted electrochemical sensor in Example 6 of the present invention

[0040] Figure 12 Ni in Example 1 of the present invention 3 (HITP) 2 SEM characterization diagram of Ni(HITP)-MOF particles

[0041] Figure 13 SEM characterization diagram of glucose molecularly imprinted membrane in Example 2 of the present invention

[0042] Figure 14 Fluorescence characterization diagram of anti-fouling material phase change bovine serum albumin in Example 4 of the present invention

[0043] Table 1 is the actual sample detection table for the sensor to detect spiked serum and sweat samples in Example 9 of the present invention

[0044] Table 2 is the actual sample detection table for the sensor to directly detect human whole blood samples in Example 10 of the present invention Detailed implementation manners

[0045] The following specific examples further illustrate the present invention. The following implementations are only used to illustrate the present invention and not to limit the scope of the present invention. All kinds of reagents, reaction conditions, detection methods, etc. used in the following examples, unless otherwise specified, are regarded as reagents, reaction conditions and detection methods commonly used in the art.

[0046] Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0047] The experimental materials used in the following examples can be purchased from conventional biochemical reagent companies without special instructions. The present invention is further illustrated below in conjunction with the embodiments.

[0048] Example 1

[0049] Ni 3 (HITP) 2 Preparation of Ni(HITP)-MOF particles

[0050] Add 6.6 mg (0.028 mmol) of nickel chloride hexahydrate (NiCl 2 .6H 2 O), 0.3 mL of concentrated ammonia water (14 mol·L -1 ) and 10 mg (0.019 mmol) of HATP·6HCl to 10 mL of water, react at 65 °C for two hours, and prepare Ni after washing with acetone3 (HITP) 2 -MOF particles.

[0051] Example 2

[0052] Preparation of molecularly imprinted polymerization system for glucose

[0053] Add o-phenylenediamine and glucose solutions with a concentration ratio of 6:1 to 2:1 to 50 - 60 mL of PBS solution with pH = 7.0 - 7.4, mix well by ultrasonic treatment, then introduce nitrogen for 15 - 20 min in the dark to remove oxygen. Place the modified electrode in the solution for electropolymerization with a voltage range between 0 and 0.8 V to obtain the molecularly imprinted polymerization system.

[0054] Example 3

[0055] Preparation of glucose molecularly imprinted polymer

[0056] On the basis of Example 2, place the obtained electrode in 0.8 - 1 M sodium hydroxide solution, with a voltage range of -1 to 1 V and a scanning rate of 50 - 60 mV / s, and perform elution using cyclic voltammetry. After 8 - 12 cycles of electroelution, obtain the glucose molecularly imprinted polymer.

[0057] Example 4

[0058] Preparation of anti-fouling material phase change bovine serum albumin

[0059] Mix TCEP (50 mM, pH 5.5, adjusted with 5 M NaOH) and BSA (2 mg·mL -1 ) solutions in a volume ratio of 0.5:1 to 2:1 to prepare a phase change bovine serum albumin solution. Native BSA is transformed into phase change bovine serum albumin oligomer nanoparticles and protofibrils in the solution, and a phase change bovine serum albumin layer is formed at the liquid-solid interface.

[0060] Example 5

[0061] Preparation method of anti-fouling electrochemical sensor for detecting glucose in human whole blood

[0062] Drop 20 μL of Ni -1 ~3 mg·mL -1 -MOF solution with a concentration of 1 mg·mL 3 (HITP) 2 onto the working electrode surface of the screen-printed electrode, and use it after the electrode is dried.

[0063] Insert the working electrode of the screen-printed electrode into 10 mL of PBS solution containing o-phenylenediamine and glucose solutions with a concentration ratio of 6:1 to 2:1, and perform electropolymerization using cyclic voltammetry in the voltage range of 0 - 0.8 V.

[0064] Place the working electrode of the screen-printed electrode in a 0.8 - 1 M sodium hydroxide solution, with a voltage range of -1 to 1 V and a scanning rate of 50 - 60 mV / s, and electro-elute to obtain the molecularly imprinted polymer by cyclic voltammetry.

[0065] Take the anti-fouling material phase-changing bovine serum albumin with a concentration of 1 mg·mL -1 ~3 mg·mL -1 Drop 10 - 20 μL of the anti-fouling material phase-changing bovine serum albumin on the surface of the working electrode, and obtain the working electrode modified with the anti-fouling material phase-changing bovine serum albumin after drying.

[0066] Place the working electrode in a 6 - 10 μmol / L glucose solution and adsorb it at room temperature for 10 - 20 min to prepare an anti-fouling electrochemical sensor for detecting glucose.

[0067] Example 6

[0068] The detection method of the sensor of the present invention for detecting glucose is as follows:

[0069] Connect the anti-fouling electrochemical sensor obtained in Example 4 to an electrochemical workstation

[0070] Use PBS solutions of 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, and 100 mM glucose as the electrolyte solution

[0071] Set the working mode of the electrochemical workstation to differential pulse voltammetry, with a voltage range of -0.2 to 0.6 V and a scanning rate of 50 - 60 mV / s. Use potassium ferricyanide solution as the detection solution to obtain the relationship between the glucose response current and voltage. As the glucose concentration increases, the current peak response signal decreases accordingly.

[0072] Fit the glucose current response signal and glucose concentration obtained in the steps, and use origin software to draw a standard curve. The standard curve equation is ΔIp(μA) = 10.92 LgC(mmol·L -1 ) + 81.65, and the correlation coefficient R 2 is 0.997. Where the unit of concentration C is mmol / L and the unit of I is μA.

[0073] Example 7

[0074] The detection method of the sensor of the present invention for detecting glucose in whole human blood is as follows:

[0075] Connect the molecularly imprinted electrochemical sensor obtained in Example 4 to an electrochemical workstation

[0076] Using a human whole blood sample as the electrolyte solution

[0077] The working mode of the electrochemical workstation was set to differential pulse voltammetry, with a voltage range of -0.2 to 0.6 V and a scanning rate of 50 to 60 mV / s. Potassium ferricyanide solution was used as the detection solution, and the current response value of glucose in human whole blood was obtained. The content of glucose in human whole blood was calculated by substituting it into the standard curve equation.

[0078] Example 8

[0079] Detection of glucose selectivity and anti-interference ability

[0080] The sample solutions were 1 mM glucose, uric acid, ascorbic acid, creatinine, and lactate solutions. The anti-pollution electrochemical sensor was placed in the above solutions and incubated for a specific time of 10 to 25 minutes. The current response was detected by differential pulse voltammetry. The signal response of the sensor to glucose was 65 μA, which was 8, 7, 7.5, and 6.5 times the current response values of uric acid, ascorbic acid, creatinine, and lactate, respectively, indicating that the sensor had excellent selectivity for glucose. The interfering substances were 1 mM uric acid, ascorbic acid, creatinine, and lactate solutions. 1 mM interfering substances were added to 1 mM glucose solution respectively. The anti-pollution electrochemical sensor was placed in the above solutions and incubated for a specific time of 10 to 25 minutes. The current response was detected by differential pulse voltammetry. The signal response of the sensor to glucose was 75 μA., which was 1.05, 1.06, 1.08, and 1.08 times the current response values of uric acid, ascorbic acid, creatinine, and lactate added, respectively, indicating that the sensor had good anti-interference ability for glucose.

[0081] Example 9

[0082] Actual sample spiking detection of human serum and sweat samples

[0083] On the basis of Example 7, serum and sweat samples of diabetic patients were selected. The serum and sweat samples were stored in the refrigerator overnight and diluted 10 times with deionized water before use. The recovery rate was determined by the standard addition method. The recovery rates of these samples by the sensor were between 98.5% and 103.0%, and the RSD showed that the reproducibility was less than 3.51%.

[0084] Example 10

[0085] Detection of actual samples of glucose in human whole blood samples

[0086] On the basis of Example 7, whole blood samples of diabetic patients were selected, and the prepared anti-pollution electrochemical biosensor was used to detect the whole blood samples of 100 diabetic patients in the hospital. Each whole blood sample was continuously detected three times. The sensor detection results were compared with the hospital gold standard detection results. The accuracy reached 94.2%, and the RSD was less than 3.4%. The sensor had good consistency in whole blood detection;

[0087] The foregoing has shown and described the basic principles, main features and advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-described exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention. Any reference signs in the claims should not be construed as limiting the claimed invention.

[0088] In addition, it should be understood that although this specification is described in terms of embodiments, not every embodiment only contains an independent technical solution. This narrative manner of the specification is only for clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art. Table 1 Actual sample detection table of the sensor for spiked detection of serum and sweat samples Table 2 Actual sample detection table of the sensor for direct detection of human whole blood samples

Claims

1. A method for preparing an anti-pollution electrochemical sensor for rapid detection of glucose in whole blood, characterized in that include: The screen-printed electrode is used as the electrode material, and a Ni3(HITP)2-MOF sensitive film is modified on the surface of the working electrode. A poly(o-phenylenediamine) molecular imprinting membrane is modified outside the Ni3(HITP)2-MOF layer. The poly(o-phenylenediamine) molecular imprinting membrane provides an imprinting site for the template molecule glucose. An anti-fouling material phase-change bovine serum albumin is modified outside the poly(o-phenylenediamine) molecular imprinting layer for detecting the glucose content in human whole blood.

2. The method for preparing the anti-pollution electrochemical sensor for rapid detection of glucose in whole blood according to claim 1, characterized in that: The surface of the working electrode is modified with a Ni3(HITP)2-MOF sensitive film, and the specific steps are as follows: add 8-12 mg (0.031-0.042 mmol) of nickel chloride hexahydrate (NiCl2.6H2O), 0.25-0.35 mL of concentrated ammonia (13 mol / L) and 5-15 mg (0.010-0.024 mmol) of 2,3,6,7,10,11-hexaaminotriphenylhexahydrochloride (HATP.6HCl) to 20 mL of water, react at 65°C for 1.5-2.5 hours, wash with acetone after the reaction, and finally obtain Ni3(HITP)2-MOF particles, and prepare a Ni3(HITP)2-MOF solution with water. Take a concentration of 0.5-3 mg·mL -1 Ni3(HITP)2-MOF solution is added, 10-30 μL of Ni3(HITP)2-MOF solution is dripped on the surface of the working electrode, and after drying, an electrode modified with Ni3(HITP)2-MOF particles is obtained; the thickness of the Ni3(HITP)2-MOF particle sensitive film is 1.9-3.2 nm, the diameter of the sensitive film Ni3(HITP)2-MOF particles is 3.7-7.8 μm, and it presents a porous layered structure of particles, which is loosely arranged. The peak value of the screen-printed electrode modified with the sensitive film Ni3(HITP)2-MOF in potassium ferrocyanide electrolyte increases to 2.5-3.0 times of that of the bare electrode, which is about 180-220 μA, by differential pulse voltammetry; Preferably, the ratio of nickel chloride hexahydrate to 2,3,6,7,10,11-hexaaminotriphenyl hexahydrochloride of the optimal synthetic material is 1:1; the reaction time is 2 hours; the concentration of Ni3(HITP)2-MOF solution is 1 mg·mL -1 , the drop volume is 20mL.

3. The method for preparing the anti-pollution electrochemical sensor for rapid detection of glucose in whole blood according to claim 1, characterized in that: The Ni3(HITP)2-MOF particle layer is modified with a poly(o-phenylenediamine) molecular imprinting membrane. The specific steps are: adding o-phenylenediamine and glucose with a concentration ratio of 2:1 to 6:1 to a concentration of 5 mmol·L -1 , in a phosphate buffer with a pH value of 7.0, the modified electrode is placed in a buffer solution for electropolymerization, and the reaction conditions of the electropolymerization are a scanning voltage of 0 to 0.8 V, a scanning rate of 40 to 60 mV / s, and a scanning number of 8 to 12 circles; then the template molecule glucose is electroeluted in a sodium hydroxide solution, and the working electrode 1 is placed in a 0.8 to 1 mol / L sodium hydroxide solution, the voltage range is -1 to 1 V, the scanning rate is 50 to 60 mV / s, the elution number is 8 to 12 circles, and the molecular imprinting polymer is obtained by electroelution using cyclic voltammetry, and the molecular imprinting polymer is rinsed and dried with a phosphate buffer with a pH value of 7.0 to obtain a molecular imprinting polymer film; the DPV peak value of the molecular imprinting film modified with poly-o-phenylenediamine on the outside of the Ni3(HITP)2-MOF particle layer is reduced to 2.5 to 2.75 times that of the Ni3(HITP)2-MOF particle sensitive film modified electrode, and the electrode is rinsed with ultrapure water to obtain a molecular imprinting polymer film; Preferably, the optimal ratio of o-phenylenediamine to glucose is 5:1; Preferably, the optimal electropolymerization scan rate is 50 mV / s; Preferably, the optimal number of electropolymerization scans is 10; Preferably, the optimal number of electroelution cycles is 9 cycles.

4. The method for preparing the anti-pollution electrochemical sensor for rapid detection of glucose in whole blood according to claim 1, characterized in that: The antifouling material phase-change bovine serum albumin is modified outside the poly(o-phenylenediamine) molecular imprinting layer. The specific steps are: tri(2-carboxyethyl)phosphine (TCEP) (30-50 mM, pH 5.0, pH adjusted with 3-5 M NaOH) and BSA (2 mg mL -1 ) solution was mixed at a volume ratio of 0.5:1 to 2:1 to prepare a phase-change bovine serum albumin solution, in which natural BSA was converted into phase-change bovine serum albumin oligomer nanoparticles and protofibrils in the solution, and a phase-change bovine serum albumin layer was formed at the liquid-solid interface. The concentration was 1 to 2 mmol·L -1 A phase-change bovine serum albumin solution is prepared, and 20 μL of the phase-change bovine serum albumin solution is dropped onto the surface of the working electrode, and after drying, an electrode modified with a phase-change bovine serum albumin material is obtained; Preferably, the optimal volume ratio of TCEP to BSA is 1:1; the NaOH concentration is 4M.

5. Use of the anti-pollution electrochemical biosensor prepared by the method according to any one of claims 1 to 5 in the detection of glucose in human whole blood.

6. A method for detecting glucose in whole blood by using an anti-pollution electrochemical sensor, characterized in that include: The molecular imprinted electrochemical sensor obtained by the above method was placed in 1 μmol·L -1 , 10 μmol·L -1 , 100 μmol·L -1 , 1mmol·L -1 , 10 μmol·L -1 , 100 μmol·L -1 The molecularly imprinted electrochemical sensor was then placed in human whole blood for glucose detection.

7. The detection method according to claim 6, characterized in that: The linear range of the anti-pollution electrochemical sensor for glucose detection is 1 μmol·L -1 ~100mmol·L -1 ; The linear regression equation is ΔIp(μA)=10.92LgC(mmol·L -1 )+81.65, correlation coefficient R 2 is 0.

997.

8. The detection method according to claim 6, characterized in that: The selectivity of the sensor was tested; the sample solution selected 1mM glucose, uric acid, ascorbic acid, creatine, and lactic acid solutions, the molecular imprinting electrochemical sensor was placed in the above solution for a specific incubation time of 10 to 25 minutes, and the current response was detected by differential pulse voltammetry. The sensor's signal response to glucose was 65μA, which was 8, 7, 7.5, and 6.5 times the current response values ​​of uric acid, ascorbic acid, creatine, and lactic acid, respectively, indicating that the sensor has excellent selectivity for glucose. The anti-interference property of the sensor was tested, and the interfering substances selected 1mM uric acid, ascorbic acid, creatine, and lactic acid solutions, 1mM interfering substances were added to 1mM glucose solution, the molecular imprinting electrochemical sensor was placed in the above solution for a specific incubation time of 10 to 25 minutes, and the current response was detected by differential pulse voltammetry. The sensor's signal response to glucose was 75μA. , which are 1.05, 1.06, 1.08, and 1.08 times the current response values ​​of uric acid, ascorbic acid, creatine, and lactic acid, respectively, indicating that the sensor has good anti-interference ability to glucose.

9. The detection method according to claim 6, characterized in that: The stability of the sensor was tested; the anti-pollution electrochemical sensor was stored for four weeks, and the current response was detected by differential pulse voltammetry. After four weeks, the sensor's electrical signal response to glucose was only reduced by 3%, indicating good stability.

10. The detection method according to claim 6, characterized in that: The prepared anti-pollution electrochemical biosensor was used to test whole blood samples from 30 diabetic patients in the hospital. Each whole blood sample was tested three times in a row. The sensor test results were compared with the hospital gold standard test results. The accuracy reached 94.2% and the RSD was less than 3.4%. The sensor has good accuracy and practical application capabilities in whole blood testing.

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