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

Abstract

The application provides a molecular imprinting electrochemical sensor for hippuric acid detection, a preparation method and a segmented response technology, and belongs to the technical field of electrochemical sensors.The molecular imprinting electrochemical sensor for detecting hippuric acid is prepared by an electrochemical polymerization method based on a functional monomer aniline and o-phenylenediamine and taking hippuric acid as a template molecule.The sensor combines the advantages of molecular imprinting technology and electrochemical technology, has excellent sensitivity, fast response time and specific recognition ability, has good signal response for hippuric acid detection under different concentrations, generates a good electric signal in a 0-100nM hippuric acid solution, and forms a better linear relationship between the electric signal and the concentration in a hippuric acid solution with a concentration of more than 100nM, so that the signal response and detection range of the molecular imprinting electrochemical sensor under high concentration are widened, and the molecular imprinting electrochemical sensor is suitable for hippuric acid detection under a wide range.

Description

Technical Field

[0001] This invention relates to the field of electrochemical sensor technology, and in particular to a molecularly imprinted electrochemical sensor for hippuric acid detection, its preparation method, and its segmented response technology. Background Technology

[0002] Hippuric acid, with the molecular formula C6H5CONHCH2COOH, also known as N-benzoylglycine, is a white crystalline solid 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, and transparent liquid with a strong aromatic odor at room temperature. When the human body comes into contact with toluene through the respiratory tract, digestive tract, or skin, toluene is metabolized in the body and converted into benzoic acid. Benzoic acid combines with glycine to form hippuric acid. Therefore, hippuric acid is now considered a major metabolite of occupational toluene exposure and is one of the recommended biomonitoring indicators for occupational toluene exposure in my country.

[0004] Currently, the main methods for detecting hippuric acid include high-performance liquid chromatography (HPLC) and UPLC-MS / MS. HPLC offers advantages such as good separation and high sensitivity, but its operation is relatively complex. UPLC-MS / MS requires strict control of experimental conditions and quality standards. Furthermore, these methods typically require complex sample preparation steps and large-scale detection equipment, making them unsuitable for real-time, rapid on-site detection. Therefore, developing a low-cost, portable, and reliable method for rapid on-site detection of hippuric acid in complex samples is essential.

[0005] Electrochemical detection methods are used to study chemical reaction processes and mechanisms by measuring parameters such as current, voltage, potential, or impedance in an electrochemical system. They offer advantages such as high sensitivity, good selectivity, and real-time monitoring. Molecular imprinting technology is often used for targeted synthesis to achieve the targeted recognition of target molecules, and it has attracted widespread attention due to its outstanding sensitivity and specificity, especially in the application of electrochemical biosensors. Therefore, how to utilize electrochemical biosensors to detect hippuric acid is crucial for simplifying hippuric acid detection methods and reducing the amount of components to be detected. Summary of the Invention

[0006] The purpose of this invention is to provide a molecularly imprinted electrochemical sensor, its preparation method, and a segmented response technology for hippuric acid detection. This invention has high specificity for the target molecule hippuric acid, enabling specific detection of hippuric acid, and is convenient to operate and has low cost.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a molecularly imprinted electrochemical sensor for hippuric acid detection. The molecularly imprinted electrochemical sensor includes two basic electrodes, and gold nanoparticles and a molecularly imprinted polymer film sequentially loaded on the surface of the basic electrodes. The template of the molecularly imprinted polymer film is hippuric acid, and the functional monomers are aniline and o-phenylenediamine, respectively.

[0009] Preferably, the aniline is used to detect hippuric acid at concentrations of 0-100 nM, and the o-phenylenediamine is used to detect hippuric acid at concentrations above 100 nM.

[0010] This invention also provides a method for preparing a molecularly imprinted electrochemical sensor for hippuric acid detection, comprising the following steps:

[0011] (1) Rinse the screen-printed electrodes with ethanol and water respectively;

[0012] (2) Mix hippuric acid with the functional monomer and disperse it evenly in PBS solution, then sonicate to form a homogeneous solution;

[0013] (3) Modify the screen-printed electrode after cleaning with gold nanoparticle solution to obtain a gold nanoparticle / screen-printed electrode;

[0014] (4) Place the gold nanoparticle / screen-printed electrode in a homogeneous solution and perform cyclic voltammetric polymerization to obtain a molecularly imprinted polymer.

[0015] (5) Elute with eluent to obtain a molecularly imprinted electrochemical sensor.

[0016] 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 hippuric acid to o-phenylenediamine is 1:5~9; and the molar ratio of hippuric acid to aniline is 1:1~5.

[0017] Preferably, the gold nanoparticle solution in step (3) comprises an aqueous solution of chloroauric acid; the concentration of the gold nanoparticle solution is 0.1-5%.

[0018] This invention also provides a hippuric acid segmented response technology for a molecularly imprinted electrochemical sensor prepared by a method for preparing a molecularly imprinted electrochemical sensor, comprising the following steps:

[0019] The prepared molecularly imprinted electrochemical sensor was placed in the test solution and adsorbed at room temperature for 2–16 minutes. After rinsing, it was placed in a potassium ferricyanide solution containing KCl, connected to an electrochemical workstation, and scan parameters were set for differential pulse voltammetry detection.

[0020] The beneficial effects of this invention compared to the prior art are as follows:

[0021] (1) Based on the functional monomers aniline and o-phenylenediamine, this invention uses hippuric acid as a template molecule and prepares a molecularly imprinted electrochemical sensor for detecting hippuric acid by electrochemical polymerization. This molecularly imprinted electrochemical sensor combines the advantages of molecular imprinting technology and electrochemical technology, and has excellent sensitivity, fast response time and specific recognition ability. It has good signal response for the detection of hippuric acid at different concentrations. It generates good electrical signals in hippuric acid solutions of 0~100nM. In hippuric acid solutions with concentrations above 100nM, the generated electrical signals form a better linear relationship with the concentration, which broadens the signal response and detection range of this molecularly imprinted electrochemical sensor at high concentrations. It is suitable for the detection of hippuric acid in a wide range and realizes the segmented response detection of hippuric acid concentration.

[0022] (2) The molecularly imprinted electrochemical sensor provided by the present invention has a simple preparation process, is easy to operate and has a low cost. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The figures show the cyclic voltammetric scan curves of different modified electrodes in Embodiment 1 of the present invention, where a is SPE; b is AuNPs / SPE; c is MIPs / AuNPs / SPE; d is MIPs / AuNPs / SPE; and e is MIPs / AuNPs / SPE.

[0025] Figure 2 The image shows the DPV response of the molecularly imprinted electrochemical sensor prepared in Example 2 of this invention to different concentrations of hippuric acid in K3[Fe(CN)6] and KCl electrolytes.

[0026] Figure 3 The fitting relationship between the logarithm of hippuric acid concentration above 100 nM and the change in DPV current in Example 2 of the present invention.

[0027] Figure 4 This is a graph showing the fitting relationship between the logarithm of the molecularly imprinted electrochemical sensor in the range of 0-100 nM hippuric acid concentration and the change in DPV current in Example 3 of the present invention.

[0028] Figure 5 This is a DPV response diagram of the molecularly imprinted electrochemical sensor in Example 3 of the present invention for different concentrations of hippuric acid in K3[Fe(CN)6] and KCl electrolytes;

[0029] Figure 6 This is the result of selective detection of hippuric acid analogues by the molecularly imprinted electrochemical sensor in Example 4 of the present invention. Detailed Implementation

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0034] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0035] This invention provides a method for preparing a molecularly imprinted electrochemical sensor for hippuric acid detection, comprising the following steps:

[0036] (1) Rinse the screen-printed electrodes with ethanol and water respectively;

[0037] (2) Mix hippuric acid with the functional monomer and disperse it evenly in PBS solution, then sonicate to form a homogeneous solution;

[0038] (3) Modify the screen-printed electrode after cleaning with gold nanoparticle solution to obtain a gold nanoparticle / screen-printed electrode;

[0039] (4) Place the gold nanoparticle / screen-printed electrode in a homogeneous solution and perform cyclic voltammetric polymerization to obtain a molecularly imprinted polymer.

[0040] (5) Elute with eluent to obtain an electrochemical biosensor.

[0041] Preferably, in step (2), the functional monomers are aniline and o-phenylenediamine; the final molecular concentration of hippuric acid is 5 mM, the molar ratio of hippuric acid to o-phenylenediamine is 1:5~9, most preferably 1:7; the molar ratio of hippuric acid to aniline is 1:1~5, most preferably 1:2.

[0042] Preferably, the gold nanoparticle solution in step (3) includes an aqueous solution of chloroauric acid; the concentration of the gold nanoparticle solution is 0.1-5%, most preferably 1%.

[0043] Preferably, the step of modifying the screen-printed electrode with gold nanoparticles in step (3) 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.

[0044] Preferably, the conditions for cyclic voltammetric polymerization in step (4) are 0~1V, the scan rate is 50-100mv / s, and the number of polymerization cycles is 10.

[0045] Preferably, the eluent in step (5) includes one or both of methanol and acetic acid, and the elution time is 1 to 10 minutes.

[0046] Example 1

[0047] Example 1 of this invention describes the preparation of a molecularly imprinted electrochemical sensor for detecting hippuric acid based on o-phenylenediamine and aniline. The specific steps are as follows:

[0048] (1) Preparation of an electrode for detecting hippuric acid concentrations above 100 nM:

[0049] A. Clean the screen-printed electrodes with ethanol and water respectively, and then set them aside for use.

[0050] B. After mixing hippuric acid and o-phenylenediamine in a molar ratio of 1:7, they were uniformly dispersed in a 0.01M PBS solution with a pH of 7.2 to achieve a final hippuric acid concentration of 5mM. The solution was then sonicated to form a homogeneous solution.

[0051] C. The screen-printed electrode was placed in a 1% chloroauric acid aqueous solution and modified using square wave voltammetry. The square wave voltammetry was set with a potential range of -2V to -0.2V, an increment of 0.01V, an amplitude of 0.025V, a frequency of 30Hz, and a scanning number of 5 revolutions. After modification, gold nanoparticles / screen-printed electrode was obtained.

[0052] D. Place the obtained gold nanoparticles / screen-printed electrode in the homogeneous solution prepared in step (2), and polymerize it by cyclic voltammetry under the conditions of potential range of 0V~1V, scan rate of 50mv / s, and polymerization cycle of 10 cycles to obtain molecularly imprinted polymer.

[0053] E. Elute hippuric acid molecules with a mixed solution of V methanol:V acetic acid = 8:2 for 8 minutes, then rinse with deionized water to obtain an electrode for detecting hippuric acid concentrations above 100 nM.

[0054] F. Cyclic voltammetry was performed on the electrodes prepared in steps A to E. Using the electrode of the molecularly imprinted electrochemical sensor prepared in step E, 100 nM hippuric acid was incubated for 12 min, and cyclic voltammetry was performed. 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 molecularly imprinted polymer electrode (MIPs / AuNPs / SPE) in step (4); d is the electrode of the molecularly imprinted electrochemical sensor after eluting hippuric acid in step (5) (MIPs / AuNPs / SPE); and e is the electrode of the molecularly imprinted electrochemical sensor after incubation with hippuric acid (MIPs / AuNPs / SPE).

[0055] (2) Preparation of electrodes for detecting hippuric acid concentrations from 0 nM to 100 nM:

[0056] The electrode for detecting hippuric acid concentrations of 0 nM to 100 nM is prepared according to steps A to E in (1). The difference from (1) is that the molar ratio of hippuric acid to aniline is 1:2 and the elution time is 2 minutes.

[0057] (3) Fabrication of molecularly imprinted electrochemical sensors:

[0058] A molecularly imprinted electrochemical sensor containing two hippuric acid detection electrodes was prepared using the electrodes prepared by (1) and (2).

[0059] Example 2

[0060] Example 2 of this invention uses the molecularly imprinted electrochemical sensor prepared in Example 1 to adsorb hippuric acid solution, establishing a linear relationship between hippuric acid and electrochemical signal. The specific steps are as follows:

[0061] (1) Prepare hippuric acid solutions with concentrations of 100 nM, 1000 nM, and 10000 nM respectively, and place the hippuric acid molecular imprint sensor in the above solutions for adsorption. The adsorption time is 2 minutes.

[0062] (2) After removing the hippuric acid molecularly imprinted electrochemical sensor, thoroughly clean it with deionized water, air dry it, and then place it in a 1mM K3[Fe(CN)6] / K4[Fe(CN)6] solution containing 1M KCl.

[0063] (3) The differential pulse voltammetry of the electrochemical workstation was used to analyze the current peak before and after adsorption, and to establish the relationship between hippuric acid and electrochemical signal. The differential pulse voltammetry used a potential range of -0.2V to 0.6V, where -0.2V is the starting potential of the differential pulse voltammetry and 0.6V is the ending 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.

[0064] 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 molecularly imprinted electrochemical sensor hinder charge transfer, resulting in a decrease in the current of the hippuric acid solution at the corresponding concentration.

[0065] Depend on Figure 3 It can be seen that the detection current value of the o-phenylenediamine monomer molecularly imprinted electrochemical sensor for hippuric acid solution has a good linear relationship with the concentration, and the sensitivity of the sensor is guaranteed even at high concentrations above 100 nM.

[0066] Example 3

[0067] In Example 3 of this invention, the molecularly imprinted electrochemical sensor prepared in Example 1 was used to adsorb hippuric acid solution. The steps of Example 2 were followed to establish a linear relationship between hippuric acid and the electrochemical signal. The difference from Example 2 is that Example 3 used a hippuric acid solution concentration of 0-100 nM. The detection results are as follows: Figure 4 , 5 As shown.

[0068] 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 molecularly imprinted electrochemical sensor hinder charge transfer, resulting in a decrease in the current of the hippuric acid solution at the corresponding concentration.

[0069] Depend on Figure 5 It can be seen that the molecularly imprinted electrochemical sensor has a better signal response to hippuric acid solution in the range of 0~100 nM.

[0070] Example 4

[0071] In Example 4 of this invention, the molecularly imprinted 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. The selectivity of the hippuric acid molecular imprint was determined based on the measured current signal intensity. The specific steps are as follows:

[0072] (1) Prepare solutions of hippuric acid, uric acid, 2'-iodohippuric acid, 4-methylhippuric acid and 2-aminohippuric acid with a concentration of 10 nM respectively as incubation solutions;

[0073] S2. Place a hippuric acid molecularly imprinted electrochemical sensor into the above incubation solution and incubate for 8 minutes. Then remove the molecularly imprinted electrochemical sensor and wash it thoroughly with deionized water.

[0074] S3. Place the hippuric acid molecularly imprinted electrochemical sensor processed in step S2 into a 1 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution containing 1 M KCl solution. Use an electrochemical workstation to measure the differential pulse voltammetric current of each molecularly imprinted electrochemical sensor. The differential pulse voltammetric current uses a potential range of -0.2V to 0.6V, where -0.2V is the starting potential of the differential pulse voltammetry method, 0.6V is the ending potential of the differential pulse voltammetry method, the pulse amplitude is 0.025V, the pulse width is 0.05s, and the pulse period is 0.5s. Record the current response signal intensity of the molecularly imprinted electrochemical sensor after incubation in the above incubation solution.

[0075] 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, and there is a significant difference in signal intensity compared with the target sample.

[0076] As can be seen from the above, the hippuric acid molecularly imprinted electrochemical sensor prepared by this invention has high specificity for the target molecule hippuric acid and can specifically detect hippuric acid. Furthermore, the preparation process of the molecularly imprinted electrochemical sensor for hippuric acid detection is simple, convenient to operate, and low in cost; it also exhibits good signal response for hippuric acid detection at different concentrations, making it suitable for hippuric acid detection over a wide range.

[0077] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A molecularly imprinted electrochemical sensor for hippuric acid detection, characterized in that, The molecularly imprinted electrochemical sensor includes two basic electrodes, and gold nanoparticles and a molecularly imprinted polymer film sequentially loaded on the surface of the basic electrodes. The template of the molecularly imprinted polymer film is hippuric acid, and the functional monomers are aniline and o-phenylenediamine, respectively. The aniline is used to detect hippuric acid at concentrations of 0-100 nM, and the o-phenylenediamine is used to detect hippuric acid at concentrations above 100 nM. The method for preparing the molecularly imprinted electrochemical sensor for hippuric acid detection includes the following steps: (1) Rinse the screen-printed electrodes with ethanol and water respectively; (2) Mix hippuric acid with the functional monomer and disperse it evenly in PBS solution, then sonicate to form a homogeneous solution; (3) Modify the screen-printed electrode after cleaning with gold nanoparticle solution to obtain a gold nanoparticle / screen-printed electrode; (4) Place the gold nanoparticle / screen-printed electrode in a homogeneous solution and perform cyclic voltammetric polymerization to obtain a molecularly imprinted polymer. (5) Elute with eluent to obtain a molecularly imprinted electrochemical sensor; 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 hippuric acid to o-phenylenediamine is 1:5~9, and the molar ratio of hippuric acid to aniline is 1:1~5.

2. The molecularly imprinted electrochemical sensor for hippuric acid detection according to claim 1, characterized in that, In step (3) of the sensor preparation method, the gold nanoparticle solution includes an aqueous solution of chloroauric acid; the concentration of the gold nanoparticle solution is 0.1-5%.

3. A method for segmental detection of hippuric acid based on the molecularly imprinted electrochemical sensor according to any one of claims 1 to 2, characterized in that, Includes the following steps: The prepared molecularly imprinted electrochemical sensor was placed in the test solution and adsorbed at room temperature for 2–16 minutes. After rinsing, it was placed in a potassium ferricyanide solution containing KCl, connected to an electrochemical workstation, and scan parameters were set for differential pulse voltammetry detection.

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