Photoelectrochemical biosensor for detecting choline and preparation method and application thereof

Abstract

The application belongs to the technical field of photoelectric chemical biosensing, and particularly relates to a photoelectric chemical biosensor for detecting choline as well as a preparation method and application thereof. The photoelectric chemical biosensor takes indium tin oxide as a conductive substrate, and a zinc oxide nanosheet array is grown on the surface of the conductive substrate. A polydopamine modification layer is formed on the surface of the zinc oxide nanosheet array through a self-polymerization reaction of dopamine. Choline oxidase is covalently fixed on the surface of the polydopamine modification layer. The synergistic effect of the ZnO nanostructure and the PDA photosensitive material in the photoelectric chemical biosensor improves the light absorption and charge separation efficiency, the specific catalysis of the choline oxidase and the amplification effect of H2O2 on the photoelectric signal are combined, the detection sensitivity is significantly improved, and the accurate quantification of trace choline can be realized.

Description

Technical Field

[0001] This invention belongs to the field of photoelectrochemical biosensing technology, specifically relating to a photoelectrochemical biosensor for detecting choline, its preparation method, and its application. Background Technology

[0002] Choline, a core component of lecithin and a precursor to the neurotransmitter acetylcholine, plays a crucial role in human lipid metabolism and nerve signal transduction. Its deficiency is not only associated with memory impairment, liver dysfunction, and the risk of certain cancers, but may also be involved in the pathological processes of neurodegenerative diseases such as Alzheimer's and Parkinson's. Although the human body can synthesize some choline endogenously, dietary supplementation is still necessary.

[0003] Currently, the detection of choline in formula milk mainly relies on traditional methods such as gas chromatography, high-performance liquid chromatography, chromatography-mass spectrometry, and fluorescence colorimetry. While these methods offer high accuracy, the equipment used—gas chromatographs, high-performance liquid chromatographs, and liquid chromatography-tandem mass spectrometers—generally suffers from problems such as large and expensive equipment, high operational expertise requirements, cumbersome and time-consuming sample pretreatment, and high detection costs, making them unsuitable for rapid on-site screening. Furthermore, the complex matrices of formula milk, such as proteins and fats, easily cause interference, traditional spectroscopic methods lack selectivity, and chromatographic methods suffer from co-elution or derivatization biases. Ordinary electrochemical methods, on the other hand, have low sensitivity and high background noise, failing to achieve both accurate quantification of trace choline and interference resistance. Therefore, there is an urgent need to develop a novel detection product that is easy to operate, has a rapid response, and high sensitivity to overcome the limitations of existing methods. Summary of the Invention

[0004] The purpose of this invention is to provide a photoelectrochemical biosensor for detecting choline, which solves the problems existing in the prior art.

[0005] The technical solution adopted in this invention is: This invention provides a photoelectrochemical biosensor for detecting choline. The photoelectrochemical biosensor uses indium tin oxide as a conductive substrate, and a zinc oxide nanosheet array is grown on the surface of the conductive substrate. A polydopamine modification layer is formed on the surface of the zinc oxide nanosheet array through the self-polymerization reaction of dopamine. Choline oxidase is covalently immobilized on the surface of the polydopamine modification layer.

[0006] The present invention also provides a method for preparing the aforementioned photoelectrochemical biosensor, comprising the following steps: Using indium tin oxide as the working electrode, zinc oxide nanosheet arrays were grown on its surface by electrochemical deposition to obtain an ITO / ZnO NSs electrode; Polydopamine was formed by biomimetic self-polymerization of dopamine, and then loaded onto the surface of ITO / ZnO NSs electrode to obtain a polydopamine-modified photosensitive electrode. By utilizing the dialdehyde group of glutaraldehyde, the amino groups on chitosan are cross-linked with choline oxidase, and the choline oxidase is covalently solidified on the surface of a polydopamine-modified photosensitive electrode, thus obtaining a photoelectrochemical biosensor.

[0007] Preferably, the reaction conditions for the electrochemical deposition method are as follows: Deposition was performed in a potential window of -0.2V to -1.2V using cyclic voltammetry under water bath conditions of 30℃ to 70℃, with a scan rate of 50mV / s and a cycle count of 1 to 25.

[0008] Preferably, the reaction conditions for the electrochemical deposition method are: 20 cycles in a 50°C water bath.

[0009] Preferably, the process of loading polydopamine onto the surface of the ITO / ZnO NSs electrode is as follows: The ITO / ZnO NSs electrode was placed in a 0.5 mg / mL dopamine solution and allowed to stand for 20 min to 80 min to obtain a polydopamine-modified photosensitive electrode.

[0010] Preferably, the settling time is 60 minutes.

[0011] Preferably, the covalent curing condition is 1 hour at room temperature.

[0012] Preferably, zinc oxide nanosheet arrays are grown on the surface of the standard three-electrode system using electrochemical deposition. The auxiliary electrode in the standard three-electrode system is a platinum sheet, and the reference electrode is a saturated calomel electrode.

[0013] A third aspect of the present invention also provides an application of the aforementioned photoelectrochemical biosensor, characterized in that the photoelectrochemical biosensor is used to detect choline content.

[0014] Preferably, the method for detecting choline content is as follows: A three-electrode system was obtained by using a photoelectrochemical biosensor as the working electrode, a calomel electrode as the reference electrode, and a platinum sheet electrode as the counter electrode. The three-electrode system was placed in a series of standard solutions to investigate the relationship between photocurrent and choline content, and a standard curve was obtained. The three-electrode system is placed on the sample to obtain the photocurrent value, and the choline content in the sample is calculated using a standard curve.

[0015] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a photoelectrochemical biosensor for detecting choline. The sensor uses indium tin oxide (ITO) as a conductive substrate, on which an array of zinc oxide nanosheets is grown via electrochemical deposition. Further, a polydopamine-modified layer is formed on the surface of the ITO nanosheet array through the self-polymerization reaction of dopamine. Finally, chitosan is used as an enzyme immobilization carrier, and a cross-linking agent is added to covalently immobilize choline oxidase on the surface of the polydopamine-modified layer, thereby obtaining the photoelectrochemical biosensor. This invention provides a photoelectrochemical biosensor based on a ZnO nanosheet array / polydopamine / choline oxidase. This sensor significantly improves light absorption and charge separation efficiency through the synergistic photosensitivity effect of ZnO and PDA, achieving highly sensitive detection of choline. Simultaneously, the preparation process is mild, the raw material cost is low, and only simple dilution or filtration is required before detection, without chromatographic separation or derivatization. A single detection can be completed within minutes, effectively overcoming the interference of complex matrices such as proteins and fats in formula milk, eliminating dependence on large instruments and professional operators, significantly reducing the overall detection cost, and truly meeting the needs for rapid and accurate quantitative analysis on-site.

[0016] Furthermore, this sensor exhibits excellent intra-batch and inter-batch reproducibility. The immobilized enzyme maintains its activity for a considerable period under appropriate storage conditions, and the detection process requires only a small amount of buffer solution, avoiding the use of organic solvents and aligning with green environmental protection principles. More importantly, this sensing platform can be flexibly extended to the detection of other nutrients or harmful substances in formula milk by simply replacing the biorecognition element (such as other oxidases), demonstrating good versatility and promising prospects for industrial application. It provides an economical, efficient, and reliable new technological means for dairy product quality control and food safety monitoring. Attached Figure Description

[0017] Figure 1 Scanning electron microscope images of the products from each step of Example 1; a~c are respectively: ITO / ZnO NSs, ITO / ZnO NSs / PDA and ITO / ZnO NSs / PDA / ChOx.

[0018] Figure 2 Fourier transform infrared spectra of different substances; a~c are the FTIR spectra of PDA, ZnO NSs, and ZnO NSs / PDA, respectively.

[0019] Figure 3 The result is from bias voltage optimization.

[0020] Figure 4 This is the result of optimizing light intensity.

[0021] Figure 5 The results are from the optimization of the electrolyte solution pH.

[0022] Figure 6 The PEC response and linear relationship of PC; (a): Photocurrent response of PEC biosensor to different concentrations of PC, where a~j represent PC concentrations from 0.03mM to 8mM; (b): Linear relationship of PEC biosensor to different concentrations of PC. Detailed Implementation

[0023] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0024] The inventive concept of this invention is as follows: Traditional methods for detecting choline in formula milk, such as gas chromatography, high-performance liquid chromatography, ion chromatography, chromatography-mass spectrometry, and fluorescence or colorimetric methods, are relatively mature, but they have the following significant limitations in practical applications: 1. High dependence on instruments, making it difficult to achieve rapid on-site detection: These methods generally require large, precise instruments and equipment and specialized laboratory environments. Sample pretreatment is complex and the analysis process is time-consuming, which cannot meet the needs of production sites, distribution links or grassroots units for rapid and real-time screening of choline content.

[0025] 2. High overall cost and high professional threshold for operation: The purchase and maintenance costs of instruments are expensive, the operating consumption is large (such as high-purity reagents and carrier gas), and it requires operation by professionally trained technicians, resulting in high overall testing costs and making it difficult to carry out high-throughput or popular testing.

[0026] 3. Potential interference in complex matrices: Formula milk has a complex composition, containing large amounts of protein, fat, and other nutrients. Some traditional methods (especially certain optical methods) may face severe matrix interference and insufficient selectivity when directly analyzing such complex samples, affecting the accuracy and reliability of the detection results.

[0027] To address the shortcomings of the existing technologies, this invention provides a simple, rapid, sensitive, and low-cost choline analysis method suitable for on-site detection in complex matrices, as detailed below: This invention provides a novel method for detecting choline in formula milk based on a photoelectrochemical biosensor. This method integrates highly selective biorecognition elements with high-performance photoelectroactive materials to construct a miniaturized sensing platform, enabling rapid real-time detection. It overcomes the limitations of large instruments, significantly shortens detection time, and meets the needs of rapid on-site analysis. Simultaneously, the miniaturization and portability of the sensor reduce reliance on expensive equipment and specialized personnel. Furthermore, by utilizing the high specificity of biorecognition and the low background of photoelectrochemical signals, interference from the formula milk matrix is ​​effectively overcome, achieving highly selective and sensitive detection of choline.

[0028] This invention provides a more practical and universally applicable technical tool for the quality control and nutritional safety monitoring of formula milk. This invention constructs a dual-enzyme photoelectrochemical biosensor, the construction process of which is as follows:

[0029] S1. ZnO NSs is grown on the surface of an indium-doped tin oxide electrode by electrodeposition.

[0030] S2. Photosensitive material PDA was attached to the surface of ZnO NSs modified electrode by solution immersion method to develop ITO / ZnONSs / PDA.

[0031] S3. Choline oxidase was immobilized on the photosensitive electrode plane using a cross-linking method, ultimately yielding an ITO / ZnO NSs / PDA / ChOx photoelectrochemical biosensor that is highly sensitive to choline.

[0032] The prepared sensor was used as the working electrode and placed in an electrolytic cell containing phosphate buffer, forming a standard three-electrode system together with the counter electrode and reference electrode. After stabilizing the initial open-circuit potential under dark conditions, the sensor was illuminated with an LED light source, and the stable baseline photocurrent generated was recorded. By measuring the increase in current response caused by the sample solution and comparing it with a standard curve (choline concentration-current response), rapid and quantitative analysis of choline content in formula milk samples can be achieved. The entire process can be completed within minutes, and the sensor exhibits good reproducibility and stability.

[0033] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments. In the description of the present invention, unless otherwise specified, all reagents used are commercially available, and all methods used are conventional techniques in the art.

[0034] The raw materials used in this invention are as follows: Chitosan was purchased from Maclean's, C804729-25g.

[0035] Choline oxidase was purchased from Sigma-Aldrich, with an enzyme activity of 10 units / mg.

[0036] The list of abbreviations for this invention is shown in Table 1.

[0037] Table 1. List of abbreviations for this invention Example 1 The photoelectrochemical biosensor for detecting choline is prepared using the following method: Preparation of S1, ZnO NSs.

[0038] (1) Preparation of ITO electrode.

[0039] A 50mm × 10mm ITO conductive glass was sequentially placed in 2% dilute ammonia, anhydrous ethanol, and ultrapure water, and ultrasonically cleaned for 5 minutes each. A 1cm section was left on one side of the ITO conductive glass as the electrode terminal, and an 8mm diameter circular area was left on the other side as the electrode modification area. The remaining ITO area was covered with insulating varnish, and the ITO electrode was prepared for use.

[0040] (2) ZnO NSs arrays were prepared on the surface of ITO electrode by electrochemical deposition.

[0041] An aqueous solution containing 0.05 M ZnCl2 and 0.05 M KNO3 was used as the electrolyte, and its pH was adjusted to 3 with hydrochloric acid. In a standard three-electrode system, the ITO electrode prepared above was used as the working electrode, a platinum sheet as the auxiliary electrode, and a saturated calomel electrode as the reference electrode. Deposition was performed using cyclic voltammetry within a potential window of -0.2 V to -1.2 V (relative to the reference electrode) under a constant temperature water bath at 50 °C. The scan rate was set to 50 mV / s, and the number of cycles was 20. After deposition, the electrode was thoroughly rinsed with ultrapure water to remove physically adsorbed ions, thus achieving the growth of ZnO NSs on the ITO electrode surface. The product is designated as the ITO / ZnONSs electrode.

[0042] S2. Polydopamine is formed by biomimetic self-polymerization of dopamine, and then loaded onto the surface of ITO / ZnO NSs electrode to obtain a polydopamine-modified photosensitive electrode.

[0043] Weigh out dopamine and dissolve it in 10 mM Tris-HCl buffer at pH 8.5 to obtain a dopamine solution with a concentration of 0.5 mg / mL.

[0044] The ITO / ZnO NSs electrode prepared in S1 was immersed in a dopamine solution with a concentration of 0.5 mg / mL and allowed to react at room temperature for 60 min. After the reaction was completed, the electrode was removed, gently rinsed with ultrapure water to remove any unadsorbed dopamine, and dried at room temperature to obtain the PDA-modified ITO / ZnO NSs electrode, denoted as the ITO / ZnO NSs / PDA electrode.

[0045] S3. Using chitosan as the enzyme immobilization carrier and glutaraldehyde as the crosslinking agent, ChOx is covalently solidified on the surface of the ITO / ZnO NSs / PDA electrode to obtain a photoelectrochemical biosensor.

[0046] Immobilization of ChOx.

[0047] 5 μL of 1% (w / v) chitosan solution was transferred to the surface of the ITO / ZnO NSs / PDA electrode prepared above using a pipette, and dried at room temperature to form a film. The electrode was then washed with PBS at pH 6 and air-dried. 5 μL of 0.55% (w / v) glutaraldehyde solution was coated onto the electrode surface, and after curing for 30 min, the electrode was washed with PBS at pH 6 and dried. Then, 5 μL of 1.8 mg / mL ChOx solution was added, and the immobilization reaction was carried out at room temperature for 1 h. The electrode was rinsed with deionized water to obtain the photoelectrochemical biosensor, denoted as ITO / ZnO NSs / PDA / ChOx.

[0048] The method for preparing the ChOx solution in this invention is as follows: weigh ChOx and dissolve it in 0.1M pH7.5 phosphate buffer to obtain the ChOx solution.

[0049] Example 2 The photoelectrochemical biosensor for detecting choline is prepared using the following method: The difference between this embodiment and Embodiment 1 is that the temperature of the constant temperature water bath in S1 is 30°C. Under this condition, the photoelectrochemical biosensor is prepared. The other steps are the same as in Embodiment 1.

[0050] Example 3 The photoelectrochemical biosensor for detecting choline is prepared using the following method: The difference between this embodiment and Embodiment 1 is that the temperature of the constant temperature water bath in S1 is 70°C. Under this condition, the photoelectrochemical biosensor is prepared. The other steps are the same as in Embodiment 1.

[0051] Example 4 The photoelectrochemical biosensor for detecting choline is prepared using the following method: The difference between this embodiment and Example 1 is that the number of cycles of cyclic voltammetry electropolymerization in S1 is 1. Under this condition, a photoelectrochemical biosensor is prepared. The other steps are the same as in Example 1.

[0052] Example 5 The photoelectrochemical biosensor for detecting choline is prepared using the following method: The difference between this embodiment and Example 1 is that the number of cycles of cyclic voltammetry electropolymerization in S1 is 25. Under this condition, the photoelectrochemical biosensor is prepared. The other steps are the same as in Example 1.

[0053] Example 6 The photoelectrochemical biosensor for detecting choline is prepared using the following method: The difference between this embodiment and Example 1 is that the static reaction time in S2 is 20 min. Under this condition, the photoelectrochemical biosensor is prepared. The other steps are the same as in Example 1.

[0054] Example 7 The photoelectrochemical biosensor for detecting choline is prepared using the following method: The difference between this embodiment and Example 1 is that the static reaction time in S2 is 80 min. Under this condition, the photoelectrochemical biosensor is prepared. The other steps are the same as in Example 1.

[0055] Example 8 The photoelectrochemical biosensor for detecting choline is prepared using the following method: The difference between this embodiment and Example 1 is that the concentration of the ChOx solution in S3 is 1.2 mg / mL. Under this condition, the other steps for preparing the photoelectrochemical biosensor are the same as in Example 1.

[0056] Example 9 The photoelectrochemical biosensor for detecting choline is prepared using the following method: The difference between this embodiment and Example 1 is that the concentration of the ChOx solution in S3 is 2.4 mg / mL. Under this condition, the other steps for preparing the photoelectrochemical biosensor are the same as in Example 1.

[0057] The photoelectrochemical biosensor prepared in Example 1 is shown below. Figure 1 and Figure 2 The microstructure of the samples before and after modification was characterized by scanning electron microscopy, and the results are as follows: Figure 1 As shown, ITO / ZnO NSs exhibit a typical two-dimensional sheet-like structure, with intersecting and randomly distributed layers forming a loose three-dimensional network. The nanosheets have smooth and flat surfaces with clear edges and no obvious aggregation, indicating good morphological uniformity and dispersibility. After PDA modification, the basic sheet-like framework structure of ITO / ZnO NSs was preserved, without significant sheet collapse or breakage, proving that the modification process did not damage the main structure of the material. Simultaneously, the surface of ITO / ZnO NSs became rougher, with a uniformly distributed PDA layer. The increased surface roughness provides more potential active sites for subsequent functional applications, which is beneficial for improving the interfacial properties and reactivity of the material. Figure 1 As can be seen from c, after immobilizing ChOx, layered proteins are deposited on the electrode surface modified by nanomaterials, indicating that the biological enzyme has been immobilized on the PDA film. Therefore, ChOx has been successfully immobilized on the surface of the photosensitive electrode.

[0058] Figure 2 The FTIR spectra of PDA, ZnO NSs, and ZnO NSs / PDA are shown. Figure 2 As shown in a, the PDA spectrum at 1500 cm⁻¹ -1 and 3000cm -1 Strong absorption is observed at 1000 cm⁻¹, attributed to the C=C / N–H bending vibration and C–H stretching vibration of the aromatic ring, respectively. -1 There is C–O / C–N absorption at that location. For example... Figure 2 As shown in b, ZnO NSs at 500 cm -1 It exhibits characteristic Zn-O absorption peaks at 3500 cm⁻¹, and at 3500 cm⁻¹... -1 The presence of strong OH absorption indicates that its surface is rich in hydroxyl groups. Figure 2 The spectrum of the ZnO NSs / PDA composite material shown in c is at 1500 cm⁻¹. -1 3000cm -1 and 1000cm -1 The absorption characteristics at this location are consistent with those of pure PDA, confirming that PDA has been successfully loaded onto the electrode surface. However, ZnO is located at 500 cm⁻¹. -1 The characteristic peaks of Zn-O did not appear separately in the composite material spectrum, which may be due to the strong background absorption of PDA in this region masking the ZnO signal. It can be inferred that the ZnO nanosheet structure remained intact during the composite process, and PDA formed a coating layer on its surface.

[0059] It has been verified that the photoelectrochemical biosensors prepared in Examples 1 to 9 of this invention can all be used to detect the choline content in formula milk, and there is no significant difference in the detection effect. Therefore, the following text only shows the application effect of the photoelectrochemical biosensor prepared in Example 1.

[0060] Example 10 This embodiment describes the application of a photoelectrochemical biosensor for choline detection. Based on the photoelectrochemical biosensor prepared in Example 1, a photoelectrochemical detection system and an electrochemical workstation are constructed to perform photoelectrochemical detection of choline. The three-electrode system for photoelectrochemical detection is as follows:

[0061] The photoelectrochemical biosensor prepared in Example 1 is used as the working electrode, the calomel electrode as the reference electrode, and the platinum sheet electrode as the counter electrode.

[0062] Experiment 1: Optimization of operating conditions for photoelectrochemical biosensors.

[0063] At room temperature, PBS of different pH values ​​was used as the electrolyte, and LED light source was used, with light intensity controlled at 6 mW / cm². 2 ~18mW / cm 2 A bias voltage of 0.1V to 0.6V is applied to the photoelectric interface. The photoelectric detection system and electrochemical workstation are started, the electrode is inserted into the electrolytic cell, and the chopper is turned on, chopping once every 20 seconds to form a photocurrent-time spectrum, thus determining the optimal operating conditions of the photoelectrochemical biosensor.

[0064] (1) Optimization of bias voltage.

[0065] The bias voltages were set to 0.1V, 0.2V, 0.3V, 0.4V, 0.5V, and 0.6V, respectively. Using PBS at pH 6 as the electrolyte and an LED as the light source at an intensity controlled at 15mW / cm², the above different bias voltages were applied to the photoelectric interface at room temperature. The results are as follows. Figure 3 As shown, the optimal bias voltage is 0.5V.

[0066] (2) Optimization of light intensity.

[0067] Set the light intensity to 6mW / cm² 2 8mW / cm 2 10mW / cm 2 12mW / cm 2 14mW / cm 2 16mW / cm 2 18mW / cm 2 At room temperature, using PBS at pH 6 as the electrolyte and an LED as the light source, a bias voltage of 0.5V was applied at the photoelectric interface. The results are as follows... Figure 4 As shown, the optimal light intensity is 18 mW / cm². 2 .

[0068] (3) pH optimization of electrolyte solution.

[0069] The pH of the electrolyte solution was set to 3, 4, 5, 6, and 7. The photoelectrochemical biosensor was placed in electrolytes of different pH values ​​at room temperature, with an LED light source and an illumination intensity controlled at 15 mW / cm². A bias voltage of 0.5 V was applied at the photoelectric interface. The results are as follows: Figure 5 As shown, the optimal pH of the electrolyte solution is 6.

[0070] In summary, the optimal detection conditions for the photoelectrochemical detection system and electrochemical workstation are: bias voltage 0.5V and light intensity 18mW / cm². 2 The electrolyte solution has a pH of 6, therefore, subsequent experiments will be conducted under these conditions.

[0071] Experiment 2: Acquisition of standard curve and application of photoelectrochemical biosensors in formula milk detection.

[0072] (1) Obtaining the standard curve and regression equation.

[0073] The photoelectrochemical detection of choline was performed using a photoelectrochemical detection system and an electrochemical workstation. The three-electrode system for photoelectrochemical detection was as follows: a photoelectrochemical biosensor as the working electrode, a calomel electrode as the reference electrode, and a platinum sheet electrode as the counter electrode. At room temperature, PBS at pH 6 was used as the electrolyte, and an LED light source with an intensity controlled at 18 mW / cm was employed. A bias voltage of 0.5 V was applied at the photoelectric interface. The photoelectrochemical detection system and electrochemical workstation were activated, and the electrodes were inserted into the electrolytic cell. A chopper was activated, and choppers were applied every 20 s. The photocurrent response at choline concentrations of 0.03 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, and 8 mM was used to investigate the linear relationship between the photocurrent and the choline concentration in the electrolytic cell.

[0074] Under optimal conditions, the photoelectrochemical biosensor prepared in Example 1 was subjected to a PEC reaction with different concentrations of choline PC, and then photocurrent was measured. Figure 6 The photocurrent signal gradually increases with increasing PC concentration. The linear range is 0.03 mM to 8 mM, and the linear regression equation is I = 2.26824C + 0.95287 (R²). 2 =0.99621), and the detection limit is 0.002 mM, indicating that the PEC sensitive interface of the photoelectrochemical biosensor prepared in this invention can achieve a sensitive response to PC.

[0075] (2) Detection of choline in formula milk by photoelectrochemical biosensor.

[0076] The pretreatment of formula milk was performed using the AOAC method. 5.00 g of formula milk was weighed and placed in a test tube, then 30 mL of 1.0 mol / L hydrochloric acid solution was added. The tube was sealed and shaken until completely dispersed. The solution was then placed in a 70°C water bath for 3 hours to hydrolyze and release bound choline ions (Ch⁺), with shaking performed periodically during hydrolysis. After cooling to room temperature, the pH of the hydrolysate was adjusted to 8.0 using a phosphate buffer solution. The solution was transferred to a 50 mL volumetric flask and diluted to volume with water. The solution was filtered through filter paper, and the first 10 mL of filtrate was discarded; subsequent filtrates were collected. The choline content of the filtrate was analyzed using a photoelectrochemical biosensor. Spiked sample solutions with concentrations of 0.02 mM, 0.05 mM, 0.10 mM, 0.5 mM, and 0.8 mM were prepared by adding choline standard to the pretreated formula milk solution.

[0077] The results show that the photoelectrochemical biosensor and HPLC method for detecting choline content in formula milk have comparable accuracy and precision, and can meet the detection requirements of choline in actual samples (results are shown in Table 2).

[0078] Table 2. Results of choline spike recovery in formula milk using photoelectrochemical biosensors. The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0079] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A photoelectrochemical biosensor for detecting choline, characterized in that, The photoelectrochemical biosensor uses indium tin oxide as a conductive substrate, and zinc oxide nanosheet arrays are grown on the surface of the conductive substrate. A polydopamine-modified layer is formed on the surface of the zinc oxide nanosheet array through the self-polymerization reaction of dopamine. Choline oxidase is covalently immobilized on the surface of the polydopamine-modified layer.

2. The method for preparing the photoelectrochemical biosensor as described in claim 1, characterized in that, The steps are as follows: Using indium tin oxide as the working electrode, zinc oxide nanosheet arrays were grown on its surface by electrochemical deposition to obtain an ITO / ZnO NSs electrode; Polydopamine was formed by biomimetic self-polymerization of dopamine, and then loaded onto the surface of ITO / ZnO NSs electrode to obtain a polydopamine-modified photosensitive electrode. By utilizing the dialdehyde group of glutaraldehyde, the amino groups on chitosan are cross-linked with choline oxidase, and the choline oxidase is covalently solidified on the surface of a polydopamine-modified photosensitive electrode, thus obtaining a photoelectrochemical biosensor.

3. The preparation method according to claim 2, characterized in that, The reaction conditions for electrochemical deposition are: Deposition was performed in a potential window of -0.2V to -1.2V using cyclic voltammetry under water bath conditions of 30℃ to 70℃, with a scan rate of 50mV / s and a cycle count of 1 to 25.

4. The preparation method according to claim 3, characterized in that, The reaction conditions for electrochemical deposition are: Under 50℃ water bath conditions, the number of cycles is 20.

5. The preparation method according to claim 2, characterized in that, The process of loading polydopamine onto the surface of the ITO / ZnO NSs electrode is as follows: The ITO / ZnO NSs electrode was placed in a 0.5 mg / mL dopamine solution and allowed to stand for 20 min to 80 min to obtain a polydopamine-modified photosensitive electrode.

6. The preparation method according to claim 5, characterized in that, The settling time is 60 minutes.

7. The preparation method according to claim 2, characterized in that, The covalent curing conditions were 1 hour at room temperature.

8. The preparation method according to claim 2, characterized in that, In a standard three-electrode system, an array of zinc oxide nanosheets was grown on its surface using electrochemical deposition. The auxiliary electrode in the standard three-electrode system was a platinum sheet, and the reference electrode was a saturated calomel electrode.

9. The application of the photoelectrochemical biosensor as described in claim 1, characterized in that, The photoelectrochemical biosensor is used to detect choline levels.

10. The application as described in claim 9, characterized in that, The method for detecting choline levels is as follows: A three-electrode system was obtained by using a photoelectrochemical biosensor as the working electrode, a calomel electrode as the reference electrode, and a platinum sheet electrode as the counter electrode. The three-electrode system was placed in a series of standard solutions to investigate the relationship between photocurrent and choline content, and a standard curve was obtained. The three-electrode system is placed on the sample to obtain the photocurrent value, and the choline content in the sample is calculated using a standard curve.

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