A reversible photoelectrochemical microsensor based on optical regulation and its preparation method, and its application in SO2 dynamic monitoring

Abstract

The present invention discloses a reversible photoelectrochemical microsensor based on optical regulation. A reversibly identifiable small molecule probe is introduced into the microsensor as the recognition unit of the photoelectrochemical microsensor and a regulator of fluorescence resonance energy transfer efficiency. The small molecule probe acts as an energy acceptor and combines with energy donor upconversion nanoparticles through the principle of fluorescence resonance energy transfer (FRET) to form a fluorescent probe. The luminescence of the FRET fluorescent probe serves as the excitation light source of the reversible photoelectrochemical microsensor. The reversible response of the small molecule probe to the target SO2 is utilized to regulate the fluorescence emission intensity of the FRET probe, thereby achieving reversible output of the photoelectric signal. The present invention achieves reversible detection, which is currently unattainable in photoelectrochemical sensors due to the limitations of the response mechanism of the recognition element. Furthermore, due to the introduction of a small molecule probe with highly selective chemical recognition as the recognition element, the photoelectrochemical microsensor proposed by the present invention has higher specificity than the recognition element of traditional photoelectrochemical sensors and can detect non-electroactive substances.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical analysis, and in particular to a reversible photoelectrochemical microsensor based on optical regulation and a preparation method thereof, and its application in SO2 dynamic monitoring. Background Art

[0002] In situ dynamic monitoring of the changes in neurochemical molecules during brain physiological and pathological activities will help us gain a deeper understanding of the molecular mechanisms associated with brain diseases, which will provide a strong molecular basis for the clinical diagnosis, treatment, and prevention of brain diseases. SO2, as an endogenous gaseous neurotransmitter, has been reported to be closely related to the development of various brain diseases. For example, increased SO2 levels in the brain can disrupt conditioned place preference in mice; SO2 can improve learning and memory impairments in rats with transient global ischemia-reperfusion; and SO2 has antidepressant and antianxiety effects in mice. However, due to the lack of suitable in situ detection tools, it is impossible to obtain the dynamic changes of SO2 in pathological models.

[0003] Developed based on electrochemical analysis, photoelectrochemical sensing is a new and promising method for in vivo analysis. It inherits the advantages of electrochemical analysis, including high temporal and spatial resolution and high sensitivity. Its separation of excitation and detection signals imparts enhanced sensitivity. More importantly, its use of light as an excitation source makes it possible to incorporate the chemical recognition mode of optical probes into the photoelectrochemical sensing interface, allowing for the detection of non-electroactive substances such as SO2.

[0004] However, SO2 levels in vivo fluctuate dynamically with physiological and pathological processes, which means that there are two possible concentrations: increased and decreased. To achieve in situ dynamic monitoring of SO2 level fluctuations, the sensor must be able to detect both the concentration increase and concentration decrease processes, that is, it must be reversible. However, the existing SO2 photoelectrochemical sensors based on light regulation are designed based on irreversible probes and can only continuously monitor the continuous process of concentration increase, but cannot achieve continuous monitoring of the concentration increase-decrease process, which limits their application in in situ dynamic monitoring. The key to solving this problem is to introduce small molecule probes that can achieve reversible recognition at the photoelectrochemical sensing interface, and use the reversible optical regulation of the probe to achieve reversible output of the photoelectric signal. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a reversible photoelectrochemical microsensor based on optical regulation in response to the deficiencies of the above-mentioned prior art. An organic small molecule probe with good reversible response performance to a single target object is used as the recognition element of the photoelectrochemical microsensor, thereby ensuring the reversible signal output of the photoelectrochemical microsensor and solving the problem that the photoelectrochemical sensors reported so far cannot achieve reversible detection of the target object due to the limitation of the response mechanism of the recognition element.

[0006] The technical solution adopted by the present invention to solve the above-mentioned problems is:

[0007] A reversible photoelectrochemical microsensor based on optical regulation comprises a base electrode, and a photoelectric material, a FRET fluorescent probe, and an anti-biofouling protective layer sequentially modified on the base electrode's surface from the inside out. The photoelectric material is a cadmium telluride quantum dot / carboxylated multi-walled carbon nanotube complex, the excitation light source is a FRET fluorescent probe composed of a reversible energy acceptor small molecule probe and energy donor upconversion nanoparticles, and the anti-biofouling protective layer is a bovine serum albumin-glutaraldehyde cross-linked film (BSA-GA cross-linked film). This reversible photoelectrochemical microsensor uses the FRET fluorescent probe as the excitation light source and utilizes the reversible response of the small molecule probe to the target SO2 to regulate the FRET fluorescent probe's fluorescence emission intensity, thereby achieving reversible output of the photoelectric signal.

[0008] According to the above scheme, the base electrode can be a conductive fibrous material with good biocompatibility and inertness (does not react with biological thiols), such as titanium wire, stainless steel needle, carbon fiber, etc., and the diameter is required to be less than 200 μm.

[0009] According to the above scheme, the cadmium telluride quantum dot / carboxylated multi-walled carbon nanotube composite is a solid product formed by self-assembly of cadmium telluride quantum dots and carboxylated multi-walled carbon nanotubes in a water solvent. The mass ratio of cadmium telluride quantum dots to carboxylated multi-walled carbon nanotubes is preferably in the range of 1:1 to 4:1. The cadmium telluride quantum dots are functionalized with thioglycolic acid and have ultraviolet absorption below 600 nm. The carboxylated multi-walled carbon nanotubes have a length of 10 to 30 μm and a diameter of 20 to 30 nm.

[0010] According to the above scheme, the BSA-GA cross-linked film is obtained by drying a cross-linked solution formed by bovine serum albumin (BSA) and glutaraldehyde (GA) in a buffer solution. The concentration of GA in the buffer solution is 0.5-1.5 wt.%, and the concentration of BSA is 4-6 mg / mL. The buffer solution is preferably PBS with a pH of 7-8.

[0011] According to the above scheme, the FRET fluorescent probe can react with the target (such as SO2) of the reversible photoelectrochemical microsensor to cause changes in the intensity of the photocurrent.

[0012] According to the above scheme, the FRET fluorescent probe is formed by the electrostatic interaction between the reversible small molecule probe and the upconversion nanoparticle. The ratio of the mass of the upconversion nanoparticle to the amount of the reversible small molecule probe is (0.5-5.0) g: (20-100) μmol. The upconversion nanoparticle acts as an energy donor and the reversible small molecule probe acts as an energy acceptor. The strong fluorescence emission peak of the upconversion nanoparticle overlaps with the maximum UV-visible absorption peak of the reversible small molecule probe, thereby ensuring effective fluorescence resonance energy transfer between the two to form the fluorescent probe.

[0013] Furthermore, the chemical composition of the upconversion nanoparticles is NaYF4:Yb,Er, the main emission peak under excitation light of around 980 nm is at 540-550 nm, and the surface of the upconversion nanoparticles is functionalized by polyacrylic acid (i.e., polyacrylic acid-modified upconversion nanoparticles).

[0014] Furthermore, when the target of the reversible photoelectrochemical microsensor is SO2, the reversible small molecule probe contains a positively charged benzopyranyl cationic group and the polyacrylic acid-modified upconversion nanoparticles are negatively charged. The two can be combined through electrostatic interaction to form a fluorescent probe. Further preferably, the chemical structure of the reversible small molecule probe is as follows: , R is an electron-donating group with a carbon number not exceeding 5, such as amide, amino, dialkylamino, alkylamino, alkoxy, etc., and can be specifically , formula 1; , formula 2; , formula 3, etc.

[0015] Among them, the benzopyranyl cation is the recognition unit of the target SO2, and electron-donating groups such as p-phenylamino are conjugated to the benzopyranyl unit to increase the electron cloud density of the conjugated system.

[0016] Another object of the present invention is to provide a method for preparing the above-mentioned reversible photoelectrochemical microsensor based on optical regulation, which is to sequentially immerse the base electrode in a cadmium telluride quantum dot / carboxylated multi-walled carbon nanotube composite aqueous solution, a fluorescent probe dispersion, and a BSA-GA crosslinking solution, and sequentially modify the electrode base surface with a cadmium telluride quantum dot / carboxylated multi-walled carbon nanotube composite modified layer, a fluorescent probe modified layer, and a BSA-GA crosslinking film layer; wherein a drying treatment is required between the two immersions. The concentration of the cadmium telluride quantum dot / carboxylated multi-walled carbon nanotube composite aqueous solution is 2.5-10 mg / mL; the concentration of the fluorescent probe dispersion is 0.5-3.0 mg / mL; the concentration of GA in the BSA-GA crosslinking solution is 0.5-1.5wt.%, and the concentration of BSA is 4-6 mg / mL.

[0017] The present invention also provides a method for preparing the above-mentioned reversible photoelectrochemical microsensor based on optical regulation, which specifically comprises the following steps:

[0018] (1) Synthesis of reversible small molecule probe: 4'-aminoacetophenone and 4-(diethylamino) salicylaldehyde are reacted in concentrated sulfuric acid to obtain a crude product, which is purified to obtain a reversible small molecule probe of formula 1;

[0019] (2) Synthesis of fluorescent probe: The reversible small molecule probe and the upconversion nanoparticles modified with polyacrylic acid are mixed and shaken in a dimethyl sulfoxide aqueous solution, and the solid product obtained by centrifugation is the fluorescent probe, which is dispersed in water to obtain a fluorescent probe dispersion with a final concentration of 0.5~3.0 mg / mL; wherein the concentration of the dimethyl sulfoxide aqueous solution is 1%~5%; after mixing, the concentration of the reversible small molecule probe in the dimethyl sulfoxide aqueous solution is 20~100 μM, and the concentration of the upconversion nanoparticles modified with polyacrylic acid is 0.5~5.0 mg / mL;

[0020] (3) Synthesis of CdTe quantum dot / carboxylated multi-walled carbon nanotube complex: The CdTe quantum dot aqueous solution is mixed with the carboxylated multi-walled carbon nanotube aqueous solution, ultrasonically dispersed to make them uniform, and then allowed to stand until the self-assembly process is completed to obtain the CdTe quantum dot / carboxylated multi-walled carbon nanotube complex, which is then dispersed in water to prepare a CdTe quantum dot / carboxylated multi-walled carbon nanotube complex aqueous solution with a concentration of 2.5-10 mg / mL; wherein the mass ratio of CdTe quantum dots to carboxylated multi-walled carbon nanotubes is in the range of 1:1-4:1;

[0021] (4) Preparation of BSA-GA crosslinking solution: GA aqueous solution and BSA solution were mixed in PBS buffer and placed in a refrigerator at 4 °C for 24 h to complete the crosslinking process between GA and BSA; wherein the concentration of GA in the buffer solution was 0.5-1.5 wt.%, and the concentration of BSA was 4-6 mg / mL; the buffer solution was preferably PBS with a pH of 7-8;

[0022] (5) Preparation of a reversible photoelectrochemical microsensor: The base electrode is immersed in the aqueous solution of the cadmium telluride quantum dots / carboxylated multi-walled carbon nanotubes composite for 20 to 30 hours, and the cadmium telluride quantum dots / carboxylated multi-walled carbon nanotubes composite is adsorbed on the surface of the base electrode. The base electrode is then immersed in a fluorescent probe dispersion, dried, and then immersed in the BSA-GA cross-linking solution prepared in step (4) for about 1 minute. The base electrode is then taken out and naturally dried to form the reversible photoelectrochemical microsensor.

[0023] According to the above scheme, the photoelectrochemical microsensor obtained in step (5) can be further packaged with an optical fiber or inserted into a glass capillary, and the end of the photoelectrochemical microsensor protrudes from the end face of the glass capillary, playing a protective and integrated role, making it easier to use, store, and transport. The tail end of the photoelectrochemical microsensor can be connected to an external electrochemical workstation via a copper wire or other conductor. After packaging, the photoelectrochemical microsensor serves as a working electrode, and an optical fiber connected to a laser of about 980 nm serves as the excitation source of the FRET fluorescent probe, and the glass capillary serves as the packaging shell. During packaging, the optical fiber is inserted as far as possible into the front end of the glass capillary and fixed with optical glue for light guidance.

[0024] According to the above scheme, polyacrylic acid modified upconversion nanoparticles were prepared by a solvothermal method. The specific process was as follows: the precursor mixture Y(NO3)3, Yb(NO3)3 and Er(NO3)3 were dispersed in an ethanol / water mixed solvent, and then transferred to a polytetrafluoroethylene reactor for high temperature and high pressure reaction. After centrifugal washing, upconversion nanoparticles NaYF4:Yb,Er were obtained; then the upconversion nanoparticles NaYF4:Yb,Er were dispersed in a polyacrylic acid aqueous solution and vigorously stirred for 20~30 h to obtain polyacrylic acid modified upconversion nanoparticles.

[0025] Building on the above, the present invention further aims to provide a reversible photoelectrochemical microsensor based on optical regulation for dynamic SO2 monitoring. When SO2 is absent or its concentration decreases, the fluorescent probe emits low luminescence intensity due to fluorescence resonance energy transfer (FRET), resulting in a low PEC signal. When SO2 is present in the detection system or its concentration increases, SO2 reacts with the fluorescent probe, reducing its FRET efficiency and increasing its fluorescence emission intensity, resulting in an enhanced PEC signal. Due to the reversible response of the FRET fluorescent probe to SO2 (when SO2 concentration decreases, the sulfite group in the fluorescent probe-SO2 adduct dissociates from the adduct, returning the fluorescent probe to a free state), the reversible photoelectrochemical microsensor can output a PEC signal that fluctuates with SO2 concentration, enabling reversible detection.

[0026] The design principles of the present invention are as follows:

[0027] The reversible photoelectrochemical microsensor based on optical regulation described in the present invention uses a FRET fluorescent probe composed of a reversibly identifiable small molecule probe and upconversion nanoparticles as an excitation light source, which can excite the photoelectric material - cadmium telluride quantum dots / carboxylated multi-walled carbon nanotube complex to generate photocurrent. The reaction of the FRET fluorescent probe with the target SO2 will affect the photocurrent intensity of the reversible photoelectrochemical microsensor.

[0028] The reversible small molecule probe is designed based on the principle of Michael addition reaction. The benzopyranyl cation serves as the recognition site of SO2, ensuring the specific recognition of SO2 by the reversible small molecule probe to form an adduct. Electron-donating groups such as p-phenylamino are conjugated to the benzopyranyl unit, which increases the electron cloud density of the SO2 recognition site, promotes the departure of SO2 from the adduct, and realizes reversible detection of SO2.

[0029] The reversible small molecule probe's maximum UV-visible absorption peak matches the fluorescence emission peak of the upconversion nanoparticles, allowing for fluorescence resonance energy transfer (FRET) between the two probes to form a FRET fluorescent probe, with the upconversion nanoparticles acting as energy donors and the reversible small molecule probe as energy acceptors. The polyacrylic acid-modified upconversion nanoparticles are negatively charged, while the reversible small molecule probe containing a benzopyranyl cation is positively charged, allowing the two to bind through electrostatic interactions. Due to the fluorescence resonance energy transfer effect, the fluorescence emission of the upconversion nanoparticles is quenched by the reversible small molecule probe, and the FRET fluorescent probe has a low fluorescence signal; the addition of SO2 changes the structure of the reversible small molecule probe, resulting in a decrease in the absorption value of the reversible small molecule probe at the maximum ultraviolet-visible absorption peak, resulting in a decrease in the fluorescence resonance energy transfer efficiency and an increase in the emission intensity of the FRET fluorescent probe; on the contrary, due to the reversibility of the reversible small molecule probe, the decrease in SO2 concentration causes the reversible small molecule probe to return to the free probe structure, resulting in an increase in the absorption value of the reversible small molecule probe at the maximum ultraviolet-visible absorption peak, resulting in an increase in the fluorescence resonance energy transfer efficiency and a decrease in the emission intensity of the FRET fluorescent probe.

[0030] The target-regulated fluorescence signal emitted by the FRET fluorescent probe serves as the excitation light source for the reversible photoelectrochemical microsensor, stimulating the cadmium telluride quantum dot / carboxylated multi-walled carbon nanotube composite photoelectric material to generate a photocurrent, thereby achieving reversible photoelectric signal output based on optical regulation. The addition of SO2 results in an increase in the emission intensity of the FRET fluorescent probe, which increases the photocurrent signal of the reversible photoelectrochemical microsensor. The decrease in SO2 concentration results in a decrease in the emission intensity of the fluorescent probe, which reduces the photocurrent signal of the reversible photoelectrochemical microsensor. This process achieves reversible detection of SO2.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] First, the photoelectrochemical sensors based on optical regulation that have been reported cannot achieve reversible detection of the target due to the limitation of the response mechanism of the recognition element. The present invention utilizes a reversible small molecule probe with good reversible response performance to a single target as the reversible recognition element and regulator of fluorescence resonance energy transfer efficiency of the photoelectrochemical microsensor. The reversible small molecule probe and upconversion nanoparticles construct a FRET fluorescent probe, and the luminescence of the fluorescent probe is used as the excitation light source of the reversible photoelectrochemical microsensor to excite the photoelectric material - cadmium telluride quantum dots / carboxylated multi-walled carbon nanotube complex to generate photocurrent; and the reaction of the FRET fluorescent probe with the target SO2 will affect the photocurrent strength of the reversible photoelectrochemical microsensor, thereby ensuring the reversible signal output of the reversible photoelectrochemical microsensor, and realizing the reversible detection that the current photoelectrochemical sensors cannot achieve due to the limitation of the response mechanism of the recognition element.

[0033] Second, the reversible photoelectrochemical microsensor based on optical regulation described in the present invention utilizes the hydrophilicity of carboxylated multi-walled carbon nanotubes and the anti-biological contamination protective layer formed by BSA-GA, which can enable the microsensor to better resist the nonspecific adsorption of biological proteins when detecting in complex biological environments, improve the anti-biological contamination ability, and effectively reduce the possibility of fluorescent probe shedding.

[0034] Third, the reversible photoelectrochemical microsensor described in the present invention uses the long-wavelength (980 nm, NIR) excitation characteristic of upconversion nanoparticles to reduce the limitation of the light source tissue penetration depth during in situ detection in vivo, and can realize in situ detection of the brain in vivo.

[0035] Overall, this reversible photoelectrochemical microsensor can, to a certain extent, realize in situ dynamic detection of SO2 in deep brain regions, and can obtain the changing trend of SO2 over a period of time during the process of brain disease, which helps to gain a deeper understanding of the molecular mechanisms related to brain diseases. This will provide a strong molecular basis for the clinical diagnosis, treatment and prevention of brain diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of the design principle of the reversible photoelectrochemical microsensor based on optical regulation described in the present invention; wherein, UCNPs represent upconversion nanoparticles modified with polyacrylic acid, CdTe QDs represent cadmium telluride quantum dots functionalized with thioglycolic acid, MWCNTs-COOH represent carboxylated carbon nanotubes, rSO2 represents a reversible small molecule probe that specifically recognizes the target SO2, and rSO2-SO2 represents the adduct formed by the reaction of the reversible small molecule probe with SO2.

[0037] Figure 2Schematic diagram of the reversible photoelectrochemical microsensor of the present invention; wherein 1 represents a titanium wire, 2 represents a glass capillary, 3 represents a 980 nm optical fiber, and 4 represents a copper wire.

[0038] Figure 3 The reversible small molecule probe of the present invention is 1 H NMR spectrum (a), 13 C NMR spectrum (b), MS spectrum (c);

[0039] Figure 4 This is a test diagram of the reversible performance of the reversible small molecule probe of the present invention;

[0040] Figure 5 Transmission electron microscopy images of cadmium telluride quantum dots / carboxylated multi-walled carbon nanotube composites, where (a) and (b) are at different magnifications;

[0041] Figure 6 Figure 2 is the fluorescence spectrum of the FRET fluorescent probe, where (a) shows the gradual increase in the content of the reversible small molecule probe loaded on the surface of the upconversion nanoparticles, (b) shows the gradual increase in the SO2 concentration in the fluorescent probe solution at the maximum reversible small molecule probe loading, and (c) shows the gradual decrease in the SO2 concentration in the fluorescent probe solution from (b) at the maximum reversible small molecule probe loading;

[0042] Figure 7 This is the fluorescence emission spectrum of the FRET fluorescent probe in response to SO2;

[0043] Figure 8 The photocurrent response diagram of the reversible photoelectrochemical microsensor; among them, a-titanium wire electrode, b-titanium wire electrode modified with cadmium telluride quantum dots / carboxylated multi-walled carbon nanotubes composite, c-titanium wire electrode modified with cadmium telluride quantum dots / carboxylated multi-walled carbon nanotubes composite + upconversion nanoparticles, d-reversible photoelectrochemical microsensor, e-reversible photoelectrochemical microsensor / SO2 (+), f-reversible photoelectrochemical microsensor / SO2 (-);

[0044] Figure 9 Detection linearity diagram for reversible photoelectrochemical microsensor;

[0045] Figure 10 This is a test diagram of the reversible performance of the reversible photoelectrochemical microsensor. DETAILED DESCRIPTION

[0046] In order to make the technical solutions, process and advantages of the present invention more clearly understood, the present invention is further described in detail with reference to the following embodiments. However, the embodiments of the present invention are not limited thereto. Without departing from the above-mentioned technical ideas of the present invention, various substitutions and modifications can be made according to common technical knowledge and customary means in the art, and all of these should be included within the scope of the present invention. The following specific embodiments are only intended to illustrate the present invention and are not intended to limit the present invention.

[0047] In the following examples, the reversible photoelectrochemical microsensor test conditions are a three-electrode system, the working electrode is the reversible photoelectrochemical microsensor, the reference electrode is a saturated calomel electrode, the counter electrode is a platinum electrode, the electrolyte solution is artificial cerebrospinal fluid (containing 200 μM ascorbic acid), and the photocurrent signal is collected by an electrochemical workstation (Shanghai Chenhua 660E).

[0048] Example 1

[0049] A reversible photoelectrochemical microsensor based on optical regulation uses a FRET fluorescent probe composed of a reversible small molecule probe and upconversion nanoparticles as the light excitation source of the photoelectrochemical microsensor, a cadmium telluride quantum dot / carboxylated multi-walled carbon nanotube complex as the photoelectric material of the photoelectrochemical microsensor, a BSA-GA cross-linked film as the anti-biological contamination layer of the reversible photoelectrochemical microsensor, and a titanium wire electrode as the base electrode of the reversible photoelectrochemical microsensor.

[0050] 1. The chemical structure of the reversible small molecule probe is: , named rSO2, and the specific preparation process is as follows: 0.163 g of 4'-aminoacetophenone and 0.193 g of 4-(diethylamino) salicylaldehyde are dissolved in 3 mL of concentrated sulfuric acid, then stirred at 90°C for 1.5 h. After cooling to room temperature, 0.5 mL of perchloric acid is added, and the resulting mixture is poured into 100 mL of ice water and fully stirred. The crude product is obtained by filtration and washing with water, and then purified by silica gel column chromatography (CH2Cl2:CH3OH=20:1) to obtain the reversible small molecule probe powder.

[0051] like Figure 3 As shown, the reversible small molecule probe was characterized by H NMR, C NMR and mass spectrometry. 1HNMR (400 MHz, DMSO) δ 8.58 – 8.48 (m, 1H), 8.15 (dt, J = 12.8, 6.5 Hz, 2H), 7.88 – 7.74 (m, 2H), 7.38 – 7.00 (m, 4H), 6.75 (dd, J = 17.8, 14.9 Hz, 2H), 3.69 – 3.57 (m, 4H), 1.22 (t, J = 7.0 Hz, 6H). HRMS (ESI): calcd. for C 21 H 25 N2O + , [M + H] + , m / z, 293.1648, found: 293.1640, confirming that the reversible small molecule probe was successfully synthesized.

[0052] The reversible small molecule probe powder was dissolved in a 5% dimethyl sulfoxide aqueous solution to prepare a reversible small molecule probe solution with a concentration of 20 μM, and its reversible response performance to SO2 was tested by a 10-fold dilution experiment. Specifically, the absorption curve of the 20 μM reversible small molecule probe solution is curve a, the reversible small molecule probe solution of curve a was diluted 10 times with a PBS solution to obtain curve c, and the adduct solution obtained by the reaction of the reversible small molecule probe solution of curve a with 10 μM SO2 was curve b. The adduct solution of curve b was diluted 10 times with a PBS solution to obtain curve d, and the solution of curve b was diluted 10 times with a PBS solution containing 10 μM SO2 to obtain curve e. As shown in FIG. Figure 4 As shown, the change from curve a to curve b indicates that the small molecule probe responds well to SO2 to form an adduct; when the adduct is diluted with a PBS solution containing 10 μM SO2, the absorbance is significantly reduced from curve b to curve e compared with the small molecule probe diluted only 10 times; and when the adduct is diluted with a PBS solution, the absorbance is not much different from curve b to curve d compared with the small molecule probe diluted only 10 times, which illustrates the good reversible performance of the small molecule probe.

[0053] 2. Preparation method of cadmium telluride quantum dots / carboxylated multi-walled carbon nanotube composites:

[0054] (1) Preparation of cadmium telluride quantum dots. The specific steps are as follows:

[0055] (1.1) Add sodium citrate (100 mg), cadmium nitrate (59 mg), and mercaptopropionic acid (25 μL) to a three-necked flask. Add deionized water (25 mL) and stir to disperse evenly.

[0056] (1.2) Adjust the pH of the mixed solution in the three-necked flask in step (1.1) to 10.5 using NaOH solution (10 M);

[0057] (1.3) Add 18.9 mg of sodium borohydride and 11.1 mg of sodium tellurite to the three-necked flask described in step (1.2), disperse them uniformly by ultrasonication, and then stir and reflux in an oil bath at 120°C.

[0058] (1.4) Monitor the progress of the stirring and reflux reaction in step (1.3) using a UV-visible spectrophotometer: Take an appropriate amount of the reaction solution and measure the UV-visible absorption spectrum until UV absorption is observed at 550 nm, then stop the reaction. After the mixed solution from step (1.3) cools to room temperature, add an equal volume of isopropanol to precipitate the product. Centrifuge at 8000 rpm for 5 minutes, and wash the precipitate with isopropanol. Repeat this process three times. Dry the resulting product in an oven at 60°C for 2 hours to obtain thioglycolic acid-functionalized cadmium telluride quantum dots.

[0059] (2) Preparation of carboxylated multi-walled carbon nanotubes: Commercial multi-walled carbon nanotubes (length 10-30 μm, diameter 20-30 nm) were refluxed in concentrated nitric acid at a concentration of 6.7 mg / mL at 120 °C for 12 h, and then filtered and washed with a large amount of clean water until the filtrate was neutral, thereby forming carboxylated multi-walled carbon nanotubes.

[0060] (3) Preparation of CdTe quantum dots / carboxylated multi-walled carbon nanotubes composite: CdTe quantum dots functionalized with thioglycolic acid were prepared into an aqueous solution with a concentration of 10 mg / mL, and carboxylated multi-walled carbon nanotubes were prepared into an aqueous solution with a concentration of 5 mg / mL; then the aqueous solution of CdTe quantum dots and the aqueous solution of carboxylated multi-walled carbon nanotubes were mixed in a ratio of 1:1 (volume ratio), ultrasonicated for 10 min to disperse them evenly, and allowed to stand for 6 h to complete the self-assembly process to form an aqueous solution of CdTe quantum dots / carboxylated multi-walled carbon nanotubes composite with a total mass concentration of 7.5 mg / mL, of which the concentration of CdTe quantum dots was 5 mg / mL and the concentration of carboxylated multi-walled carbon nanotubes was 2.5 mg / mL. Figure 5 As shown, it can be seen that cadmium telluride quantum dots are successfully adsorbed on the carboxylated multi-walled carbon nanotubes.

[0061] 3. Preparation of Upconversion Nanoparticles: The upconversion nanoparticles, specifically NaYF4: 18% Yb, 2% Er, were prepared via a solvothermal method. Briefly, a mixture of Y(NO3)3·5H2O (4 mmol), Yb(NO3)3·5H2O (0.9 mmol), and Er(NO3)3·5H2O (0.1 mmol) was dissolved in 8 mL of ultrapure water and 18 mL of ethanol at 60°C. A NaF solution (4 mmol in 3 mL of ultrapure water) was added dropwise to the solution under vigorous stirring. After stirring for 30 minutes to allow the solution to clarify, the resulting suspension was carefully transferred to a 50 mL polytetrafluoroethylene reactor and heated at 200°C for 10 hours at a heating rate of 15°C / min to produce the upconversion nanoparticles. Finally, the obtained upconversion nanoparticles were washed three times with ethanol and ultrapure water, and then polyacrylic acid functionalized upconversion nanoparticles (PAA-UCNPs) were obtained by the following steps: the obtained upconversion nanoparticles were mixed with polyacrylic acid dissolved in ultrapure water at a mass ratio of 1:20, and then vigorously stirred at room temperature for 24 h. The polyacrylic acid-coated (modified) upconversion nanoparticles were obtained by centrifugation, washed three times with ethanol and ultrapure water, and then dispersed in 10 mL of ultrapure water for use.

[0062] 4. Preparation of the fluorescent probe: The reversible small molecule probe represented by Formula 1 was dissolved in a 5% aqueous solution of dimethyl sulfoxide to form a 200 μM solution. The solution was then mixed with 10 mg / mL of polyacrylic acid-modified upconversion nanoparticles at a 1:1 ratio (volume ratio). The solution was vortexed for 5 minutes, centrifuged, and the precipitate was washed with water and dispersed in water to form a fluorescent probe dispersion. The final concentration of the fluorescent probe dispersion was 2 mg / mL.

[0063] like Figure 6 As shown in the figure, the strong fluorescence emission peak of the polyacrylic acid modified upconversion nanoparticles is located at a wavelength of 545 nm, and the maximum ultraviolet absorption peak of the reversible small molecule probe is located at a wavelength of 575 nm. The fluorescence emission spectrum of the upconversion nanoparticles is highly consistent with the ultraviolet absorption spectrum of the reversible small molecule probe, which can realize fluorescence resonance energy transfer between the upconversion nanoparticles and the reversible small molecule probe.

[0064] The polyacrylic acid-modified upconversion nanoparticles (PCA) generate 545 nm fluorescence emission under 980 nm excitation, acting as an energy donor. The reversible small molecule probe exhibits strong ultraviolet absorption at 545 nm, acting as an energy acceptor. The reversible small molecule probe is modified onto the surface of the PCA-modified upconversion nanoparticles, and the two bind via electrostatic forces to form a fluorescence resonance energy transfer (FRET)-based fluorescent probe. Due to the spectral match between the two, the fluorescence emission of the PCA-modified upconversion nanoparticles is quenched by the reversible small molecule probe, resulting in a low emission intensity. In the presence of the target SO₂, SO₂ reacts with the reversible small molecule probe to form an adduct, altering the molecular structure of the reversible small molecule probe, resulting in a decrease in FRET efficiency and an increase in the emission intensity of the fluorescent probe. When the concentration of the target SO₂ decreases, the reversible small molecule probe's reversibility causes the sulfite group in the adduct to dissociate from the adduct, returning the reversible small molecule probe to its free state. This increases FRET efficiency and decreases the emission intensity of the fluorescent probe, achieving a reversible regulation process. The reversible regulation effect of the target SO2 on the emission intensity of the fluorescent probe can be Figure 7 As the amount of reversible small molecule probe loaded on the surface of polyacrylic acid-modified upconversion nanoparticles increases, the emission intensity of the fluorescent probe decreases. As the concentration of the target SO₂ increases, the emission intensity of the fluorescent probe increases. As the SO₂ in the solution is removed, the emission intensity of the fluorescent probe decreases again, achieving a reversible optical response of the fluorescent probe to SO₂.

[0065] 4. Preparation of BSA-GA cross-linking solution: Mix 400 μL of 25% GA aqueous solution and 52 mg of BSA solid powder in 10 mL of PBS buffer. Incubate the mixture in a refrigerator at 4°C for 24 h to allow GA and BSA to complete the cross-linking process, forming a BSA-GA cross-linking solution. The GA concentration in the buffer solution is 1% (mass fraction), and the BSA concentration is 5.2 mg / mL. The buffer solution is PBS, pH 7.4.

[0066] 5. Preparation of reversible photoelectrochemical microsensor:

[0067] (5.1) A titanium wire with a length of 1 cm and a diameter of 100 μm was treated in a mixed acid solution (nitric acid: hydrofluoric acid: water = 91:6:3, volume ratio) for 6 min. The titanium wire was then ultrasonicated in acetone, ethanol, and water for 30 min to remove surface impurities. The titanium wire was then connected to a copper wire via conductive silver glue (for easy connection to an electrochemical workstation) to form a titanium wire electrode.

[0068] (5.2) Soaking the titanium wire electrode in the aqueous solution of the cadmium telluride quantum dots / carboxylated multi-walled carbon nanotubes composite for 24 hours until the cadmium telluride quantum dots / carboxylated multi-walled carbon nanotubes composite completes the adsorption process on the surface of the titanium wire electrode.

[0069] (5.3) Immerse the electrode prepared in (5.2) in a 2 mg / mL fluorescent probe dispersion and dry it at 60 °C for 2 h.

[0070] (5.4) Soak the electrode prepared in (5.3) in the BSA-GA cross-linking solution prepared in (4) for 1 min, then take it out and let it dry naturally to form the reversible photoelectrochemical microsensor.

[0071] The reversible photoelectrochemical microsensor prepared in (5.4) was encapsulated together with a microfiber connected to a 980 nm laser into a pre-drawn glass capillary. The microsensor was inserted into the glass capillary, with its end exposed 1 mm from the end face of the glass capillary as the microsensor's recognition and detection portion. The end was then inserted into the electrolyte solution (or test solution), and the tail was connected to the electrochemical workstation via a copper wire. Simultaneously, the optical fiber was inserted as far as possible into the front end of the glass capillary and fixed to the microsensor using light-guiding optical glue. Testing was performed under the following conditions: a 980 nm laser was used as the excitation light for the upconversion nanoparticles; the electrochemical workstation was used to output the photoelectric signal of the reversible photoelectrochemical microsensor; a platinum electrode was used as the counter electrode, a saturated calomel electrode was used as the reference electrode, the reversible photoelectrochemical microsensor was used as the working electrode, and artificial cerebrospinal fluid (pH = 7.4) containing 200 μM ascorbic acid was used as the electrolyte solution. Photoelectrochemical tests were performed on the microelectrodes during the layer-by-layer modification process, wherein the microelectrodes included a titanium wire electrode, an electrode modified with a cadmium telluride quantum dot / carboxylated multi-walled carbon nanotube composite, an electrode modified with a cadmium telluride quantum dot / carboxylated multi-walled carbon nanotube composite and upconversion nanoparticles, and an electrode modified with a cadmium telluride quantum dot / carboxylated multi-walled carbon nanotube composite and a fluorescent probe and a BSA-GA anti-biofouling layer (the reversible photoelectrochemical microsensor).

[0072] like Figure 8As shown, under 980 nm laser irradiation, the titanium wire electrode (a) and the electrode modified with a CdTe quantum dot / carboxylated multi-walled carbon nanotube composite (b) exhibit no significant photocurrent signal. However, the electrode modified with a CdTe quantum dot / carboxylated multi-walled carbon nanotube composite and upconversion nanoparticles (c) exhibits a significant photocurrent signal, demonstrating that the upconversion nanoparticles can stimulate the CdTe quantum dot / carboxylated multi-walled carbon nanotube composite photoelectric material to generate a photocurrent. Simultaneously, due to the quenching of the upconversion nanoparticle fluorescence emission by the reversible small molecule probe, the reversible photoelectrochemical microsensor (d) exhibits a low photocurrent intensity, demonstrating the successful construction of the reversible photoelectrochemical microsensor. When the electrode modified with a CdTe quantum dot / carboxylated multi-walled carbon nanotube composite and upconversion nanoparticles (c, the photoelectrochemical microsensor without the reversible small molecule probe) is incubated with the target SO₂, the photocurrent shows little change, indicating that the effect of the experimental concentration of SO₂ on the photoelectric material performance is negligible. After incubating the reversible photoelectrochemical microsensor (d) with SO₂, the photocurrent signal increased, demonstrating the reversible photoelectrochemical microsensor's ability to recognize SO₂. After incubating the reversible photoelectrochemical microsensor (d) in a solution without SO₂, the photocurrent signal decreased, demonstrating the reversible performance of the reversible photoelectrochemical microsensor. These results demonstrate the feasibility of constructing a reversible photoelectrochemical sensor based on optical regulation. The successful construction of the reversible photoelectrochemical microsensor was confirmed by detecting its photocurrent response.

[0073] Application Examples

[0074] This application example verifies the ability of the constructed reversible photochemical microsensor to detect SO2 based on the above-mentioned embodiment 1.

[0075] The reversible photoelectrochemical microsensor prepared in the example was tested in artificial cerebrospinal fluid (containing 200 μM ascorbic acid). The test conditions were a three-electrode system, with the reversible photoelectrochemical microsensor as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum electrode as the counter electrode. Photoelectric signals were acquired using an electrochemical workstation (Shanghai Chenhua 660E). After immersing the reversible photoelectrochemical microsensor in artificial cerebrospinal fluid supplemented with varying concentrations of SO₂ for 2 minutes, the photocurrent signal was collected to test the reversible photoelectrochemical microsensor's ability to detect SO₂. Figure 9 Shows the photocurrent signals obtained after the reversible photoelectrochemical microsensor was incubated with different concentrations of SO2 (0, 3, 6, 10, 13, 16, 20, 23, and 25 μM) for 2 min.

[0076] Depend on Figure 9It can be seen that the photocurrent signal gradually increases with the increase of SO2 concentration, and has a good linear relationship in the range of 0 μM to 25 μM. The linear equation is y=2.07x+25.6 (R 2 = 0.997), and the detection limit was 0.14 μM. The results show that the reversible photoelectrochemical microsensor proposed in this paper can be used for sensitive detection of SO2.

[0077] In addition, the reversible photoelectrochemical microsensor was immersed in artificial cerebrospinal fluid and artificial cerebrospinal fluid supplemented with 20 μM SO2 for 2 minutes and then its photocurrent signal was tested. The artificial cerebrospinal fluid contains the following components: NaCl (126 mM), KCl (2.4 mM), KH2PO4 (0.5 mM), MgCl2 (0.35 mM), NaHCO3 (27.5 mM), Na2SO4 (0.5 mM), and CaCl2 (1.1 mM). Figure 10 As shown, the reversible photoelectrochemical microsensor can exhibit a reversible photocurrent signal that increases and decreases in sequence with the change of SO2 concentration, and can achieve reversible cycle detection at least five times, which proves the good reversible detection performance of the reversible photoelectrochemical microsensor of the present invention.

[0078] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this field, several improvements and changes can be made without departing from the creative concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. A reversible photoelectrochemical microsensor based on optical regulation, characterized in that: The device comprises a base electrode, and a photoelectric material, an excitation light source, and a protective layer sequentially modified on the surface of the base electrode from the inside out; the photoelectric material is a cadmium telluride quantum dot / carboxylated multi-walled carbon nanotube complex, the excitation light source is a FRET fluorescent probe composed of a reversible small molecule probe and upconversion nanoparticles, and the protective layer is a bovine serum albumin-glutaraldehyde cross-linked film; the fluorescent probe can react with the target of the reversible photoelectrochemical microsensor to cause changes in the intensity of the photocurrent; The target of the reversible photoelectrochemical microsensor is SO2, and the general chemical structure of the reversible small molecule probe is: , R is an electron-donating group; the upconversion nanoparticles are polyacrylic acid functionalized upconversion nanoparticles NaYF4:Yb,Er, which are negatively charged; the reversible small molecule probe and the upconversion nanoparticles are combined through electrostatic interaction to form a FRET fluorescent probe.

2. A reversible photoelectrochemical microsensor based on optical regulation according to claim 1, characterized in that: The base electrode is a conductive fibrous material with a diameter not exceeding 200 μm; the reversible photoelectrochemical microsensor regulates the fluorescence emission intensity of the FRET fluorescent probe through the reversible response of the small molecule probe to the target object, thereby achieving reversible output of the photoelectric signal; the photoelectrochemical microsensor is packaged with the optical fiber or inserted into a glass capillary, and the end of the reversible photoelectrochemical microsensor protrudes from the end face of the glass capillary.

3. The reversible photoelectrochemical microsensor based on optical regulation according to claim 1, characterized in that: The cadmium telluride quantum dot / carboxylated multi-walled carbon nanotube composite is a solid product formed by self-assembly of cadmium telluride quantum dots and carboxylated multi-walled carbon nanotubes in solvent water, wherein the mass ratio of the cadmium telluride quantum dots to the carboxylated multi-walled carbon nanotubes is in the range of 1:1 to 4:

1. The cadmium telluride quantum dots are functionalized with thioglycolic acid and have ultraviolet absorption at a wavelength below 600 nm. The carboxylated multi-walled carbon nanotubes have a length of 10 to 30 μm and a diameter of 20 to 30 nm.

4. The reversible photoelectrochemical microsensor based on optical regulation according to claim 1, characterized in that: The bovine serum albumin-glutaraldehyde cross-linked film is obtained by drying a cross-linked solution formed by bovine serum albumin (BSA) and glutaraldehyde (GA) in a buffer solution; wherein the concentration of GA in the buffer solution is 0.5-1.5 wt.%, the concentration of BSA is 4-6 mg / mL; and the buffer solution is PBS with a pH of 7-8.

5. The reversible photoelectrochemical microsensor based on optical regulation according to claim 1, characterized in that: The fluorescent probe is formed by combining a reversible small molecule probe and the upconversion nanoparticles through electrostatic interaction, and the ratio between the mass of the upconversion nanoparticles and the amount of the reversible small molecule probe is (0.5-5.0) g: (20-100) μmol; the strong fluorescence emission peak of the upconversion nanoparticles can overlap with the maximum ultraviolet-visible absorption peak of the reversible small molecule probe.

6. The method for preparing a reversible photoelectrochemical microsensor based on optical regulation according to claim 1, characterized in that: The substrate electrode is sequentially immersed in a cadmium telluride quantum dot / carboxylated multi-walled carbon nanotube composite aqueous solution, a FRET fluorescent probe dispersion, and a BSA-GA crosslinking solution, thereby sequentially modifying the electrode substrate surface with a cadmium telluride quantum dot / carboxylated multi-walled carbon nanotube composite modification layer, a FRET fluorescent probe modification layer, and a BSA-GA crosslinking film layer. Drying is required between soaking; The concentration of the cadmium telluride quantum dot / carboxylated multi-walled carbon nanotube composite aqueous solution is 2.5~10 mg / mL; the concentration of the fluorescent probe dispersion is 0.5~3.0 mg / mL; the concentration of GA in the BSA-GA cross-linking solution is 0.5~1.5wt.%, and the concentration of BSA is 4~6 mg / mL.

7. The method for preparing a reversible photoelectrochemical microsensor based on optical regulation according to claim 1, characterized in that: The specific steps include: (1) Synthesis of reversible small molecule probe: 4'-aminoacetophenone and 4-(diethylamino) salicylaldehyde are reacted in concentrated sulfuric acid to obtain a crude product, which is purified to obtain a reversible small molecule probe of formula 1; Formula 1 (2) Synthesis of FRET fluorescent probe: After the reversible small molecule probe and the upconversion nanoparticles modified with polyacrylic acid are mixed and shaken in a dimethyl sulfoxide aqueous solution, the solid product obtained by centrifugation is the fluorescent probe, which is dispersed in water to obtain a FRET fluorescent probe dispersion with a final concentration of 0.5~3.0 mg / mL; wherein the concentration of the dimethyl sulfoxide aqueous solution is 1%~5%; after mixing, the concentration of the reversible small molecule probe in the dimethyl sulfoxide aqueous solution is 20~100 μM, and the concentration of the upconversion nanoparticles modified with polyacrylic acid is 0.5~5.0 mg / mL; (3) Synthesis of CdTe quantum dot / carboxylated multi-walled carbon nanotube complex: The CdTe quantum dot aqueous solution is mixed with the carboxylated multi-walled carbon nanotube aqueous solution, ultrasonically dispersed to make them uniform, and then allowed to stand until the self-assembly process is completed to obtain the CdTe quantum dot / carboxylated multi-walled carbon nanotube complex, which is then dispersed in water to prepare a CdTe quantum dot / carboxylated multi-walled carbon nanotube complex aqueous solution with a concentration of 2.5-10 mg / mL; wherein the mass ratio of CdTe quantum dots to carboxylated multi-walled carbon nanotubes is in the range of 1:1-4:1; (4) Preparation of BSA-GA crosslinking solution: GA aqueous solution and BSA solution were mixed in PBS buffer and placed in a refrigerator at 4 °C for 24 h to complete the crosslinking process between GA and BSA; wherein the concentration of GA in the buffer solution was 0.5-1.5 wt.%, and the concentration of BSA was 4-6 mg / mL; the buffer solution was PBS, and the pH was 7-8; (5) Preparation of a reversible photoelectrochemical microsensor: The base electrode is immersed in the aqueous solution of the cadmium telluride quantum dots / carboxylated multi-walled carbon nanotubes composite for 20 to 30 hours, and the cadmium telluride quantum dots / carboxylated multi-walled carbon nanotubes composite completes the adsorption process on the surface of the base electrode. The base electrode is then immersed in a FRET fluorescent probe dispersion, dried, and then immersed in the BSA-GA cross-linking solution prepared in step (4). After being taken out and naturally dried, the reversible photoelectrochemical microsensor is formed.

8. The application of a reversible photoelectrochemical microsensor based on optical regulation for dynamic monitoring of SO2 according to claim 1, characterized in that: The application method is as follows: in a three-electrode system, the reversible photoelectrochemical microsensor is used as a working electrode and immersed in SO2 solutions of different concentrations for incubation, and then the photocurrent signal is collected; the sensitive detection of SO2 is achieved through the linear relationship between the photocurrent signal and the SO2 concentration and the reversible response.

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