Molecularly imprinted electrochemical sensor for hippuric acid detection, preparation method and segmented response technology

Abstract

The invention provides a molecularly imprinted electrochemical sensor for hippuric acid detection, a preparation method and a segmented response technology, and belongs to the technical field of electrochemical sensors. Based on functional monomers aniline and o-phenylenediamine, hippuric acid is taken as a template molecule, and the molecularly imprinted electrochemical sensor for detecting hippuric acid is prepared by an electrochemical polymerization method, and combines the advantages of a molecular imprinting technology and an electrochemical technology; the hippuric acid detection sensor has excellent sensitivity, fast response time and specific recognition capability, has good signal response to hippuric acid detection under different concentrations, generates a good electric signal in a hippuric acid solution of 0-100 nM, forms a better linear relationship between the electric signal generated in the hippuric acid solution with the concentration of 100 nM or above and the concentration, and can be applied to the detection of hippuric acid with the concentration of 100 nM or above. The signal response and detection range of the molecularly imprinted electrochemical sensor under high concentration is widened, and the molecularly imprinted electrochemical sensor is suitable for hippuric acid detection in a wide range.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical sensors, and in particular to a molecular imprinting electrochemical sensor that can be used for hippuric acid detection, a preparation method and a segmented response technology. Background Art

[0002] The molecular formula of hippuric acid is C 6 H 5 CONHCH 2 COOH, also known as N-benzoylglycine, is a white crystal that is mainly formed in the human body by the combination of benzoic acid and glycine.

[0003] Toluene is an organic compound that is a flammable, carcinogenic, colorless, transparent liquid at room temperature with a strong aromatic odor. When the human body comes into contact with toluene through the respiratory tract, digestive tract or skin, toluene will be metabolized in the body and converted into benzoic acid, which combines with glycine to form hippuric acid. Therefore, hippuric acid is now considered to be the main metabolite of occupational exposure to toluene and is one of the recommended biological monitoring indicators for occupational toluene exposure in my country.

[0004] At present, the main methods for detecting hippuric acid include high performance liquid chromatography, UPLC-MS / MS, etc. Among them, high performance liquid chromatography has the advantages of good separation effect and high sensitivity, but the operation is relatively complicated; UPLC-MS / MS requires strict control of experimental conditions and quality standards. In addition, these methods usually require complex sample processing steps and large-scale detection equipment, which are not suitable for real-time and rapid detection on site. Therefore, it is very necessary to develop a low-cost, portable and reliable method for rapid on-site detection of hippuric acid in complex samples.

[0005] Electrochemical detection is a method for studying the chemical reaction process and mechanism by measuring parameters such as current, voltage, potential or impedance in an electrochemical system. It has the advantages of high sensitivity, good selectivity, and real-time monitoring. Molecular imprinting technology is often used for directed synthesis to achieve targeted recognition of target molecules. It has attracted widespread attention due to its outstanding sensitivity and specificity, especially in the application of electrochemical biosensors. Therefore, how to use electrochemical biosensors to detect hippuric acid is crucial to simplify the hippuric acid detection method and reduce the detection components. Summary of the invention

[0006] The purpose of the present invention is to provide a molecular imprinting electrochemical sensor, a preparation method and a segmented response technology that can be used for hippuric acid detection. The present invention has high specificity for the target molecule hippuric acid, can specifically detect hippuric acid, and is easy to operate and low in cost.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a molecular imprinting electrochemical sensor that can be used for hippuric acid detection. The molecular imprinting electrochemical sensor comprises two base electrodes, and gold nanoparticles and a molecular imprinting polymer film sequentially loaded on the surfaces of the base electrodes. The template of the molecular imprinting polymer film is hippuric acid, and the functional monomers are aniline and o-phenylenediamine, respectively.

[0008] Preferably, the aniline is used to detect hippuric acid at a concentration of 0 to 100 nM, and the o-phenylenediamine is used to detect hippuric acid at a concentration of more than 100 nM.

[0009] The present invention also provides a method for preparing a molecular imprinting electrochemical sensor that can be used for hippuric acid detection, comprising the following steps: (1) Rinse the screen-printed electrode with ethanol and water respectively; (2) Mix hippuric acid and functional monomers and evenly disperse them in a PBS solution, and form a uniform solution by ultrasonication; (3) modifying the cleaned screen-printed electrode with a gold nanoparticle solution to obtain a gold nanoparticle / screen-printed electrode; (4) placing gold nanoparticles / screen-printed electrodes into a uniform solution and using cyclic voltammetry polymerization to obtain molecularly imprinted polymers; (5) Elution with an eluent to obtain a molecular imprinting electrochemical sensor.

[0010] Preferably, in step (2), the functional monomers are aniline and o-phenylenediamine; the final concentration of the hippuric acid molecule is 5 mM, the molar ratio of the hippuric acid to o-phenylenediamine is 1:5-9; the molar ratio of the hippuric acid to aniline is 1:1-5; Preferably, in step (3), the gold nanoparticle solution comprises an aqueous solution of chloroauric acid; and the concentration of the gold nanoparticle solution is 0.1-5%.

[0011] The present invention also provides a hippuric acid segmented response technology of a molecular imprinting electrochemical sensor prepared based on the preparation method of the molecular imprinting electrochemical sensor, comprising the following steps: The prepared molecular imprinting electrochemical sensor was placed in the solution to be tested and adsorbed for 2 to 16 minutes at room temperature. After rinsing, it was placed in potassium ferrocyanide solution containing KCl and connected to an electrochemical workstation. The scanning parameters were set and differential pulse voltammetry detection was performed.

[0012] The beneficial effects of the present invention compared with the prior art are: (1) The present invention is based on functional monomers aniline and o-phenylenediamine, and uses hippuric acid as a template molecule to prepare a molecular imprinting electrochemical sensor for detecting hippuric acid by an electrochemical polymerization method. The molecular imprinting electrochemical sensor combines the advantages of molecular imprinting technology and electrochemical technology, has excellent sensitivity, fast response time and specific recognition ability, and has a good signal response for hippuric acid detection at different concentrations. A good electrical signal is generated in a hippuric acid solution of 0-100 nM. A better linear relationship is formed between the electrical signal generated in a hippuric acid solution with a concentration of more than 100 nM and the concentration, which broadens the signal response and detection range of the molecular imprinting electrochemical sensor at high concentrations, is suitable for hippuric acid detection in a wide range, and realizes the segmented response detection of hippuric acid concentration.

[0013] (2) The molecular imprinting electrochemical sensor provided by the present invention has a simple preparation process, convenient operation and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] 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 labor.

[0015] Figure 1 Cyclic voltammetry scanning curves of different modified electrodes in Example 1 of the present invention, wherein a is SPE; b is AuNPs / SPE; c is MIPs / AuNPs / SPE; d is MIPs / AuNPs / SPE; e is MIPs / AuNPs / SPE; Figure 2 The molecular imprinting electrochemical sensor prepared in Example 2 of the present invention reacts with hippuric acid at different concentrations at K 3 [Fe(CN) 6 ] and DPV response diagrams in KCl electrolyte; Figure 3 A fitting relationship diagram of the logarithmic value of the hippuric acid concentration above 100 nM and the DPV current change value of the molecular imprinting electrochemical sensor in Example 2 of the present invention; Figure 4 This is a fitting relationship diagram of the logarithmic value of the molecular imprinting electrochemical sensor at a concentration of 0-100 nM hippuric acid and the DPV current change value in Example 3 of the present invention; Figure 5 The molecular imprinting electrochemical sensor in Example 3 of the present invention reacts with different concentrations of hippuric acid at K 3 [Fe(CN) 6 ] and DPV response diagrams in KCl electrolyte; Figure 6 This is the selective detection result of hippuric acid analogs by the molecular imprinting electrochemical sensor in Example 4 of the present invention. DETAILED DESCRIPTION

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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 the skilled artisan. The present invention description and examples are exemplary only.

[0020] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0021] The present invention provides a method for preparing a molecular imprinting electrochemical sensor that can be used for hippuric acid detection, comprising the following steps: (1) Rinse the screen-printed electrode with ethanol and water respectively; (2) Mix hippuric acid and functional monomers and evenly disperse them in a PBS solution, and form a uniform solution by ultrasonication; (3) modifying the cleaned screen-printed electrode with a gold nanoparticle solution to obtain a gold nanoparticle / screen-printed electrode; (4) placing gold nanoparticles / screen-printed electrodes into a uniform solution and using cyclic voltammetry polymerization to obtain molecularly imprinted polymers; (5) Elution with an eluent to obtain an electrochemical biosensor.

[0022] Preferably, in step (2), the functional monomers are aniline and o-phenylenediamine; the final molecular concentration of the hippuric acid is 5 mM, the molar ratio of the hippuric acid to o-phenylenediamine is 1:5-9, most preferably 1:7; the molar ratio of the hippuric acid to aniline is 1:1-5, most preferably 1:2.

[0023] Preferably, in step (3), the gold nanoparticle solution comprises an aqueous solution of chloroauric acid; the concentration of the gold nanoparticle solution is 0.1-5%, most preferably 1%.

[0024] Preferably, in step (3), the step of modifying the screen-printed electrode with gold nanoparticles is as follows: placing the screen-printed electrode in an aqueous solution of chloroauric acid, and obtaining the gold nanoparticle / screen-printed electrode by square wave voltammetry.

[0025] Preferably, in step (4), the conditions of the cyclic voltammetry polymerization are 0-1 V, the scanning speed is 50-100 mv / s, and the number of polymerization cycles is 10 cycles.

[0026] Preferably, in step (5), the eluent comprises one or both of methanol and acetic acid, and the elution time is 1 to 10 minutes.

[0027] Example 1 In Example 1 of the present invention, a molecular imprinting electrochemical sensor for detecting hippuric acid was prepared based on o-phenylenediamine and aniline, and the specific steps were as follows: (1) Preparation of electrodes for detecting hippuric acid at concentrations above 100 nM: A. Clean the screen-printed electrodes with ethanol and water respectively and set them aside.

[0028] B. Hippuric acid and o-phenylenediamine were mixed in a molar ratio of 1:7, and the mixture was evenly dispersed in a 0.01 M PBS solution with a pH of 7.2 to make a final concentration of 5 mM. A uniform solution was formed by ultrasonication.

[0029] C. The screen-printed electrode was placed in a 1% aqueous solution of chloroauric acid and modified using square wave voltammetry. The square wave voltammetry was set to a potential range of -2 V to -0.2 V, an increment of 0.01 V, an amplitude of 0.025 V, a frequency of 30 Hz, and a scanning number of 5 circles. After modification, a gold nanoparticle / screen-printed electrode was obtained.

[0030] D. Place the obtained gold nanoparticles / screen-printed electrode in the uniform solution prepared in step (2), and polymerize using cyclic voltammetry under the conditions of a potential range of 0 V to 1 V, a scan rate of 50 mv / s, and 10 polymerization cycles to obtain a molecularly imprinted polymer.

[0031] E. The hippuric acid molecules were eluted with a mixed solution of V methanol: V acetic acid = 8:2 for 8 minutes, and then rinsed with deionized water to obtain an electrode that can detect hippuric acid at a concentration of more than 100 nM.

[0032] F. Perform cyclic voltammetry scanning on the electrodes prepared in steps A to E. Use the electrode of the molecular imprinting electrochemical sensor prepared in step E to incubate 100 nM hippuric acid for 12 min and perform cyclic voltammetry scanning. The results are as follows: Figure 1 As shown, a is the blank screen-printed electrode (SPE) in step (1); b is the gold nanoparticle / screen-printed electrode (AuNPs / SPE) in step (3); c is the molecular imprinted polymer electrode (MIPs / AuNPs / SPE) in step (4); d is the electrode of the molecular imprinted electrochemical sensor after eluting hippuric acid in step (5) (MIPs / AuNPs / SPE); and e is the electrode of the molecular imprinted electrochemical sensor after incubating hippuric acid (MIPs / AuNPs / SPE).

[0033] (2) Preparation of electrodes for detecting hippuric acid at concentrations of 0 nM to 100 nM: An electrode for detecting hippuric acid in a concentration of 0 nM to 100 nM was prepared according to steps A to E in (1), except that the molar ratio of hippuric acid to aniline was 1:2 and the elution time was 2 minutes.

[0034] (3) Preparation of molecular imprinted electrochemical sensors: A molecular imprinting electrochemical sensor comprising two hippuric acid detection electrodes was prepared using the electrodes prepared in (1) and (2).

[0035] Example 2 In Example 2 of the present invention, the molecular imprinting electrochemical sensor prepared in Example 1 was used to adsorb the hippuric acid solution to establish a linear relationship between hippuric acid and the electrochemical signal. The specific steps are as follows: (1) Prepare hippuric acid solutions with concentrations of 100 nM, 1000 nM, and 10000 nM respectively, and place the hippuric acid molecular imprinting sensor in the above solutions for adsorption for 2 minutes; (2) Take out the hippuric acid molecular imprinted electrochemical sensor and rinse it thoroughly with deionized water. After drying, place it in 1mM K 3 [Fe(CN) 6 ] / K 4 [Fe(CN)6 ] containing 1 M KCl solution; (3) The differential pulse voltammetry method of the electrochemical workstation was used to analyze the current peak before and after adsorption, and the relationship between hippuric acid and the electrochemical signal was established. The differential pulse voltammetry method used a potential range of -0.2 V to 0.6 V, where -0.2 V was the starting potential of the differential pulse voltammetry method, 0.6 V was the ending potential of the differential pulse voltammetry method, the pulse amplitude was 0.025 V, the pulse width was 0.05 s, and the pulse period was 0.5 s.

[0036] Depend on Figure 2 It can be seen that as the concentration of hippuric acid increases, the current signal gradually decreases. This is because the target hippuric acid molecules adsorbed on the hippuric acid molecular imprinting electrochemical sensor hinder the charge transfer, resulting in a decrease in the current of the corresponding concentration hippuric acid solution.

[0037] Depend on Figure 3 It can be seen that the detection current value of the o-phenylenediamine monomer molecular imprinting electrochemical sensor for hippuric acid solution shows a good linear relationship with the concentration, and the sensitivity of the sensor is guaranteed even at a high concentration of more than 100 nM.

[0038] Example 3 Example 3 of the present invention uses the molecular imprinting electrochemical sensor prepared in Example 1 to adsorb hippuric acid solution, and adopts the steps of Example 2 to establish a linear relationship between hippuric acid and electrochemical signal. The difference from Example 2 is that Example 3 uses a hippuric acid solution with a concentration of 0 to 100 nM, and the detection results are as follows Figure 4 , 5 shown.

[0039] Depend on Figure 4 It can be seen that as the concentration of hippuric acid increases, the current signal gradually decreases. This is because the target hippuric acid molecules adsorbed on the hippuric acid molecular imprinting electrochemical sensor hinder the charge transfer, resulting in a decrease in the current of the corresponding concentration hippuric acid solution.

[0040] Depend on Figure 5 It can be seen that the molecular imprinting electrochemical sensor has a better signal response to the detection current value of hippuric acid solution between 0 and 100 nM.

[0041] Example 4 In Example 4 of the present invention, the molecular imprinting electrochemical sensor prepared in Example 1 was used to detect hippuric acid, uric acid, 2'-iodohippuric acid, 4-methylhippuric acid, and 2-aminohippuric acid solutions of the same concentration, and the selectivity of the hippuric acid molecular imprinting was obtained according to the measured current signal intensity. The specific steps are as follows: (1) Prepare 10 nM hippuric acid, uric acid, 2'-iodohippuric acid, 4-methylhippuric acid, and 2-aminohippuric acid solutions respectively as incubation solutions; S2, placing a hippuric acid molecular imprinting electrochemical sensor into the above incubation solution and incubating for 8 minutes, then taking out the molecular imprinting electrochemical sensor and washing it thoroughly with deionized water; S3, placing the hippuric acid molecular imprinting electrochemical sensor treated in step S2 in 1 mM K 3 [Fe(CN) 6 ] / K 4 [Fe(CN) 6 ] The solution contains 1M KCl solution, and the differential pulse voltammetry current of each molecular imprinting electrochemical sensor is measured using an electrochemical workstation. The differential pulse voltammetry current adopts a potential range of -0.2V to 0.6V, wherein -0.2V is the starting potential of the differential pulse voltammetry, 0.6V is the termination potential of the differential pulse voltammetry, the pulse amplitude is 0.025V, the pulse width is 0.05s, and the pulse period is 0.5s. The current response signal intensity of the molecular imprinting electrochemical sensor after incubation with the above incubation solution is recorded; Depend on Figure 6 It can be seen that the sensor can be used to detect hippuric acid, and the current response intensity to uric acid, 2'-iodohippuric acid, 4-methylhippuric acid, and 2-aminohippuric acid is weak, which is significantly different from the signal intensity of the target sample.

[0042] As can be seen from the above, the hippuric acid molecular imprinting electrochemical sensor prepared by the present invention has high specificity for the target molecule hippuric acid and can specifically detect hippuric acid. In addition, the preparation process of the molecular imprinting electrochemical sensor for hippuric acid detection is simple, easy to operate, and low in cost; it has good signal response for hippuric acid detection at different concentrations and is suitable for hippuric acid detection in a wide range.

[0043] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A molecular imprinting electrochemical sensor for hippuric acid detection, characterized in that: The molecular imprinting electrochemical sensor comprises two basic electrodes, and gold nanoparticles and molecular imprinting polymer films sequentially loaded on the surfaces of the basic electrodes. The template of the molecular imprinting polymer film is hippuric acid, and the functional monomers are aniline and o-phenylenediamine respectively.

2. The molecular imprinting electrochemical sensor for hippuric acid detection according to claim 1, characterized in that: The aniline is used to detect hippuric acid at a concentration of 0 to 100 nM, and the o-phenylenediamine is used to detect hippuric acid at a concentration of more than 100 nM.

3. A method for preparing a molecular imprinting electrochemical sensor for hippuric acid detection as claimed in claim 1, characterized in that: The steps include: (1) Rinse the screen-printed electrode with ethanol and water respectively; (2) Mix hippuric acid and functional monomers and evenly disperse them in a PBS solution, and form a uniform solution by ultrasonication; (3) modifying the cleaned screen-printed electrode with a gold nanoparticle solution to obtain a gold nanoparticle / screen-printed electrode; (4) placing gold nanoparticles / screen-printed electrodes into a uniform solution and using cyclic voltammetry polymerization to obtain molecularly imprinted polymers; (5) Elution with an eluent to obtain a molecular imprinting electrochemical sensor.

4. The method for preparing a molecularly imprinted electrochemical sensor for hippuric acid detection according to claim 3, characterized in that: In step (2), the functional monomers are aniline and o-phenylenediamine; the final concentration of the hippuric acid molecule is 5 mM, the molar ratio of the hippuric acid to o-phenylenediamine is 1:5-9; and the molar ratio of the hippuric acid to aniline is 1:1-5.

5. The method for preparing a molecularly imprinted electrochemical sensor for hippuric acid detection according to claim 3, characterized in that: In step (3), the gold nanoparticle solution comprises an aqueous solution of chloroauric acid; the concentration of the gold nanoparticle solution is 0.1-5%.

6. A segmented response technology for hippuric acid of a molecular imprinted electrochemical sensor prepared by the method for preparing the molecular imprinted electrochemical sensor according to any one of claims 1 to 2 or the molecular imprinted electrochemical sensor according to any one of claims 3 to 5, characterized in that: The steps include: The prepared molecular imprinting electrochemical sensor was placed in the solution to be tested and adsorbed for 2 to 16 minutes at room temperature. After rinsing, it was placed in potassium ferrocyanide solution containing KCl and connected to an electrochemical workstation. The scanning parameters were set and differential pulse voltammetry detection was performed.

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