A coaxial integrated implantable optofuel sensor and a method for detecting persistent organic pollutants

Abstract

The present invention discloses a coaxial integrated implantable optofuel sensor, which includes a photoanode, a biocathode and a fuel. The photoanode is based on a light guiding optical fiber, and a gold layer, carbon nanotubes and Ag are sequentially modified at one end thereof. 2 S‑Bi 2 S 3 The heterojunction is obtained; the biocathode is based on a glass capillary. After a gold layer is modified at one end thereof, a nucleic acid aptamer for specifically recognizing a target is further modified, and then a proton exchange membrane is formed by curing with a perfluorosulfonic acid solution at the port of the modified end; the fuel is an ascorbic acid solution; the coaxial is formed by inserting the photoanode into the biocathode to form an integrated structure. The present invention can be used for detection applications in persistent organic pollutants, and solves problems such as low sensitivity, poor selectivity, easy contamination and difficulty in realizing in-situ detection caused by separately implanting multiple electrodes and light sources into a complex environment.

Description

Technical Field

[0001] The present invention belongs to the field of photoelectrochemical sensing, and in particular relates to a coaxial integrated implantable optofuel sensor, a preparation method thereof, and a detection application in persistent organic pollutants. Background Art

[0002] Persistent organic pollutants refer to synthetic chemicals with high toxicity, persistence, bioaccumulation, and long-range transport potential. They are ubiquitous in the environment and have become a major environmental pollution issue of global concern. When persistent organic pollutants leak into the environment, they can bioaccumulate and biomagnify through the food chain, ultimately causing serious negative impacts on human health, such as endocrine disruption, immune and reproductive system dysfunctions, developmental neurotoxicity, and certain cancers. Therefore, there is an urgent need for a rapid, sensitive, portable, and on-site analytical method for detecting persistent organic pollutants, which is a crucial step in assessing their environmental risks. As an important part of the human diet, fish can accumulate persistent organic pollutants from dietary sources and gill membranes, resulting in a higher enrichment of pollutants in their tissues than in the surrounding waters. Fish are a major source of human exposure to persistent organic pollutants. Therefore, in-situ quantitative detection of persistent organic pollutants in fish is of great significance for comprehensively evaluating the harm of persistent organic pollutants to the ecological environment and human health.

[0003] Traditional analytical techniques for pollutants in animal samples usually require the destruction or killing of organisms, which is not only immoral to animals and disadvantageous to endangered species, but also cannot reflect the true accumulation and change status of persistent organic pollutants in vivo. Although solid-phase microextraction technology can achieve in vivo in-situ sampling, subsequent complex elution steps are required and large instruments such as high-performance liquid chromatography are used for sample analysis, making it difficult to achieve on-site monitoring and timely feedback of target pollutants. Among various in vivo and in-situ analytical techniques, electrochemical sensing has been widely used due to its advantages such as high spatio-temporal resolution and simple equipment. As an evolved product of electrochemical analysis, photoelectrochemical sensors not only inherit the above advantages, but also have higher sensitivity due to the separation of photoexcitation signals and electrical detection signals. In addition, self-powered photoelectrochemical sensors composed of a fuel cell with only an anode and a cathode can operate without an external power supply, making it easier to achieve portable equipment and low-cost detection. However, current in vivo photoelectrochemical sensors basically require the implantation of a separate three-electrode system, including a photoanode, a reference electrode, and a counter electrode, which easily leads to the following three problems: 1) Biomacromolecules in the complex in vivo environment cause biological contamination to all three electrodes. Electroactive small molecules in the body easily interfere with the photoanode, and at the same time, the photoactive material on the photoanode may also cause biological toxicity to the living body; 2) The distance between implanted electrodes in freely moving animals cannot be precisely controlled, thus affecting the detection accuracy; 3) Implanting multiple electrodes and additional light sources into the living body requires multiple insertion operations, which will cause greater mechanical damage to the organism. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a coaxial integrated implantable photo-fuel sensor, its preparation method, and its detection application in persistent organic pollutants in view of the above-mentioned deficiencies of the existing technology, which solves the problems of low sensitivity, poor selectivity, easy contamination, and difficulty in achieving in-situ detection caused by separately implanting multiple electrodes and light sources into a complex environment.

[0005] The technical solution adopted by the present invention to solve the above-mentioned problems is as follows:

[0006] A coaxial integrated implantable photo-fuel sensor includes a photoanode, a biocathode, and a fuel. The photoanode is based on a light-conducting optical fiber, and a gold layer, carbon nanotubes, and Ag 2 S-Bi 2 S 3 heterojunction are sequentially modified at one end thereof; the biocathode is based on a glass capillary, a gold layer is modified at one end thereof, and then a nucleic acid aptamer for specifically recognizing a target is further modified, and then a proton exchange membrane is formed by curing with a perfluorosulfonic acid solution at the port of this end; the fuel is an ascorbic acid solution.

[0007] According to the above solution, the fuel is injected into the biocathode, the photoanode is inserted into the biocathode to contact the fuel, and the photoanode and the biocathode are coaxial.

[0008] According to the above solution, the carbon nanotubes can be multi-walled carbon nanotubes or single-walled carbon nanotubes, etc.

[0009] The preparation method of the above coaxially integrated implantable optofuel sensor mainly includes the following steps:

[0010] S1. Select a glass capillary and a light guiding fiber, and the inner diameter of the glass capillary is larger than the diameter of the light guiding fiber;

[0011] S21. Modify the gold layer at one end of the glass capillary described in S1 by the seed-mediated growth method, then modify the nucleic acid aptamer that can specifically recognize the target on the surface of the gold layer through gold-sulfur bonds, and block the active sites with a 6-mercapto-1-hexanol (MCH) solution to obtain a glass capillary / gold layer / nucleic acid aptamer electrode;

[0012] S22. Inject the perfluorinated resin solution into the modified end of the glass capillary / gold layer / nucleic acid aptamer electrode, and form a proton exchange membrane at the port of the modified end of the electrode after drying to obtain a biocathode;

[0013] S31. Modify the gold layer at one end of the light guiding fiber described in S1 by the seed-mediated growth method, and then modify multi-walled carbon nanotubes (MWNT) on the surface of the gold layer by the layer-by-layer self-assembly method to obtain an optical fiber / gold layer / MWNT electrode;

[0014] S32. Use continuous ion layer adsorption to modify Ag with high photoelectric conversion efficiency at the modified end of the optical fiber / gold layer / MWNT electrode 2 S-Bi 2 S 3 heterojunction to construct a photoanode;

[0015] S4. Pour the fuel ascorbic acid solution into the biocathode obtained in S22, and insert the modified end of the photoanode obtained in S32 into the biocathode to contact the fuel, then the coaxially integrated implantable optofuel sensor is obtained.

[0016] According to the above solution, the diameter of the transparent light guiding fiber is preferably 100-200 μm, and the length of the modified end is preferably 2-3 cm; the inner diameter and outer diameter of the glass capillary are preferably 250-350 μm and 350-450 μm respectively, and the length of the modified end is preferably 1.5-2.5 cm. Further, the modified end of the glass capillary includes a straight tube section and a reduced diameter section (formed by a drawing instrument) located at one end of the straight tube section, and the tip diameter of the reduced diameter section is preferably 35-45 μm.

[0017] According to the above scheme, the method for growing the gold layer in S21 is as follows: Immerse one end of the glass capillary in the dopamine solution for 0.5 - 1.5 h, wash with water and dry, then immerse it in the gold seeds for 10 - 13 h, and finally let it stand in the mixed solution of hydroxylamine hydrochloride and chloroauric acid to grow the gold layer, and then wash with water and dry. Among them, the concentration of the dopamine solution is in the range of 1 - 2 mg / mL, the gold seeds refer to the aqueous solution containing gold nanoparticles, and the concentration is preferably in the range of 50 - 100 μg / mL; in the mixed solution of hydroxylamine hydrochloride and chloroauric acid, the respective concentrations of hydroxylamine hydrochloride and chloroauric acid are preferably in the ranges of 0.5 - 1.5 mM and 0.03 - 0.07 wt%, and let it stand and grow for 3 - 5 min.

[0018] According to the above scheme, the method for modifying the aptamer with gold - sulfur bonds in S21 is as follows: First, dissolve the thiolated aptamer in a 5 - 20 mM PBS buffer solution (pH 7.0 - 7.5), and the concentration is in the range of 6 - 10 μM. Then add an aqueous solution of tris(2 - carboxyethyl)phosphine (TCEP) to activate the aptamer for 1 - 3 h (the concentration of TCEP in the aptamer solution is 0.2 - 1 mM) to obtain an aptamer solution; then, place the end of the glass capillary with the grown gold layer into the above - mentioned aptamer solution and incubate at 4 °C for 12 - 20 h, take it out and wash with the PBS buffer solution; finally, place it in a 0.5 - 2 mM aqueous solution of 6 - mercapto - 1 - hexanol (MCH) to block for 1 - 3 h, take it out again and wash with the PBS buffer solution to obtain a glass capillary / gold layer / aptamer electrode.

[0019] According to the above scheme, the specific operation method of S22 is as follows: Inject the perfluorosulfonic acid resin solution (Nafion) into the modified end port of the capillary glass tube / gold layer / aptamer electrode, and then cure it at room temperature to form a proton - exchange membrane at this port. Among them, the concentration of the Nafion solution is in the range of 0.4 - 0.6 wt%, and the injection volume is in the range of 0.04 - 0.06 μL, and it is sufficient to realize the curing at the port of the modified end to form a proton - exchange membrane.

[0020] According to the above scheme, the method for growing the gold layer in S31 is as follows: Immerse the clean optical fiber in a dopamine solution of 1 - 2 mg / mL for 1.5 - 2.5 h, then wash with water and dry. Then, soak it in acetone for 2 - 4 s to remove the polydopamine layer and the fiber cladding at the front end of the optical fiber with a length of 0.7 - 0.9 cm to make it transparent at the front end. After washing with water and drying, soak the 1.5 - 2.5 cm at this end in the gold seed for 3 - 5 h, and then let it stand and grow in a mixed solution of hydroxylamine hydrochloride and chloroauric acid for 3 - 5 min, and then wash with water and dry (note: no gold layer is grown on the 0.7 - 0.9 cm section at the front end of the optical fiber). The gold seed refers to an aqueous solution containing gold nanoparticles, and the concentration is preferably in the range of 50 - 100 μg / mL; in the mixed solution of hydroxylamine hydrochloride and chloroauric acid, the respective concentrations of hydroxylamine hydrochloride and chloroauric acid are preferably in the ranges of 0.5 - 1.5 mM and 0.03 - 0.07 wt%.

[0021] Further, in S31 and S21, after the gold layer is grown on the optical fiber and the glass capillary, the resistance is 10 - 20 Ω.

[0022] According to the above scheme, in S31 and S32, the gold layer does not completely overlap with the carbon nanotubes, but partially overlaps at the modified end. The modified region of the carbon nanotubes is closer to (or extends to) the port direction of the modified end of the optical fiber relative to the modified region of the gold layer, and the modified region of the carbon nanotubes is larger than the modified region of the gold layer, that is, the modified region of the gold layer does not extend to the port of the modified end, and there is a small distance (such as 0.7 - 0.9 cm) from the port of the modified end, while the modified region of the carbon nanotubes extends to the port of the modified end. The modified region of the carbon nanotubes includes the modified region of the gold layer and the region at the front end of the optical fiber where the gold layer is not modified; Ag 2 S-Bi 2 S 3 The modified region of the heterojunction basically overlaps with the modified region of the carbon nanotubes. Specifically, the 0.7 - 0.9 cm part at the very front end of the modified end of the optical fiber is not modified with the gold layer but is modified with carbon nanotubes. The modified region of the carbon nanotubes includes the modified region of the gold layer and the 0.7 - 0.9 cm part at the front end of the modified optical fiber where the gold layer is not modified.

[0023] According to the above scheme, the method for modifying carbon nanotubes by the layer-by-layer self-assembly method in S31 is as follows: Place the optical fiber after growing the gold layer in an aqueous solution of polydiallyldimethylammonium chloride (PDDA) for adsorption, wash with water, and then place it in an aqueous solution of carboxylated carbon nanotubes for adsorption. This is one cycle in S31; after one cycle, wash with water and dry, and then perform the next cycle to achieve the modification of carbon nanotubes. Among them, the number of cycles ranges from 3 to 5 times; the concentration of PDDA is 0.5 - 1.5 wt%, the adsorption time is 4 - 6 min; the concentration of the aqueous solution of carboxylated carbon nanotubes is in the range of 0.4 - 0.6 mg / mL, and the adsorption time is 4 - 6 min each.

[0024] According to the above scheme, in S32, Ag is modified on the optical fiber / gold layer / MWNT electrode by successive ionic layer adsorption 2 S-Bi 2 S 3 The method for the heterojunction is as follows: in a dark environment, the modified end of the optical fiber / gold layer / MWNT electrode is immersed in an aqueous solution of 0.08 - 0.12 M AgNO 3 for 25 - 35 min, washed with water, and then continuously immersed in a solution of 0.1 - 0.14 M Na 2 S for 25 - 35 min, washed with water and dried; then, successively immersed in an 8 - 12 mM Bi(NO 3 ) 3 solution and an 8 - 12 mM Na 2 S solution for 8 - 12 min each, washed with water and dried. This is one cycle of adsorbing Bi 2 S 3 ; the number of cycles of adsorbing Bi 2 S 3 is 1 - 3 times.

[0025] According to the above scheme, in S4, the concentration of the fuel ascorbic acid solution is 20 - 100 mM, and the perfusion height in the biocathode is higher than the modified end of the photoanode.

[0026] According to the above scheme, the target substance is a persistent organic pollutant, and in this invention, 3,3',4,4'-tetrachlorobiphenyl (PCB77) is taken as an example; the nucleic acid aptamer that can specifically recognize 3,3',4,4'-tetrachlorobiphenyl (PCB77) is SH-Aptamer, and the sequence is 5′-SH-(CH 2 ) 6 -GGC-GGG-GCT-ACG-AAG-TAG-TGA-TTT-TTT-CCG-ATG-GCC-CGT-G-3′. If other pollutants are selected as the target substances to be detected, corresponding molecules that can specifically recognize can be selected, as long as the recognition molecules contain groups such as amino or mercapto that can be modified onto the gold layer.

[0027] The application of the above coaxially integrated implantable optofuel sensor in detecting 3,3',4,4'-tetrachlorobiphenyl (PCB77), the specific application method is: inserting the above coaxially integrated implantable optofuel sensor into the PCB77 solution, and irradiating the unmodified end of the photoanode with a laser source. The light is transmitted along the optical fiber to the modified end of the photoanode to excite the Ag 2 S-Bi 2 S 3Photo-generated electrons are generated, and the electrons are then transmitted through an external circuit to the biocathode to form an open-circuit voltage; when the target substance is recognized on the biocathode through the aptamer, it will hinder electron transmission and reduce the open-circuit potential; both the biocathode and the photoanode are connected to the two ends of the potentiometer through the surface-modified gold layer, and then the change of the open-circuit voltage with the concentration of PCB77 is measured, and the content of PCB77 in the test substance is measured by the standard curve method.

[0028] According to the above scheme, the test sample can be a living body such as a fish, a mouse, a plant, etc., or a solution such as blood, urine, etc.

[0029] According to the above scheme, the potentiometer can be an electrochemical workstation or a portable potentiometer. The portable potentiometer can be replaced by a portable pH meter that can perform potential readings.

[0030] The present invention also provides a coaxial integrated implantable optofuel sensor system, including the coaxial integrated implantable optofuel sensor, a light source and a potentiometer of the present invention; wherein the coaxial integrated implantable optofuel sensor is used to be inserted into the test substance, the light source and the photoanode of the sensor can be connected through the substrate optical fiber of the photoanode, and the gold layers of the biocathode and the photoanode of the sensor are electrically connected to the two ends of the potentiometer respectively by thin copper wires. The wavelength of the light source is within the visible to near-infrared wavelength range that can excite the Ag 2 S-Bi 2 S 3 material.

[0031] The detection principle of the present invention is as follows: the photoactive material Ag 2 S-Bi 2 S 3 modified on the optical fiber surface is excited by the light transmitted along the optical fiber (the laser is connected to the unmodified end of the optical fiber) to generate electron-hole pairs. Due to the energy band matching of Ag 2 S and Bi 2 S 3 , the excited electrons in Bi 2 S 3 are transmitted to Ag 2 S with a relatively negative valence band energy level, then transferred to the carbon nanotubes, then transferred to the optical fiber / gold layer, and finally reach the glass capillary / gold layer / aptamer biocathode through an external circuit, thereby generating electrical energy (open-circuit potential). The ascorbic acid fuel in the glass capillary is used to fill Bi 2 S 3The photo-generated holes thus promote the separation of electron-hole pairs. When the target PCB77 forms a poorly conductive aptamer-PCB77 complex on the surface of the biocathode through interaction with the aptamer, it hinders the separation of hole-electron pairs and reduces the OCP signal, thereby realizing the detection of PCB77. Among them, since the gold layer is opaque, the outermost end of the optical fiber is not coated with a gold layer after acetone treatment, but is modified with carbon nanotubes with better light transmittance to ensure that the light transmitted by the optical fiber can shine on the outermost Ag. 2 S-Bi 2 S 3 And realize material excitation. After the tip of the biocathode is modified with a proton exchange membrane, it does not affect the electron transfer between the cathode and the anode. At the same time, since the proton membrane can only pass protons and electrons, the photoanode hidden in the cathode can avoid contact with the complex biological environment and affect the selectivity of the photoanode.

[0032] Compared with the prior art, the beneficial effects of the present invention are:

[0033] 1. The coaxial integrated implantable optofuel sensor prepared by the present invention has the photoanode hidden in the biocathode, which fixes and shortens the distance between the photoanode and the biocathode, reduces the biological contamination of the photoanode, and improves the sensing sensitivity and accuracy.

[0034] 2. The coaxial integrated implantable optofuel sensor prepared by the present invention can eliminate the interference of electroactive small molecules in the complex in vivo environment or solution environment on the photoanode and overcome the possible biological toxicity of the photoactive material.

[0035] 3. The coaxial integrated implantable optofuel sensor prepared by the present invention reduces the damage caused by the additional insertion of a reference electrode, a counter electrode, and a light source in vivo, and greatly reduces the mechanical damage to living tissues.

[0036] 4. The coaxial integrated implantable optofuel sensor prepared by the present invention can be applied to the in-situ monitoring of the bioaccumulation level of PCB77 in living bodies such as fish brains. The fluctuation of PCB77 in vivo can be directly reflected by a handheld potentiometer on-site, providing an important technical breakthrough for in-situ monitoring technology in living bodies.

[0037] In summary, the coaxial integrated implantable optofuel sensor of the present invention realizes the rapid in-situ detection of persistent organic pollutants in living bodies using a portable potentiometer on-site, with advantages such as high sensitivity, good anti-pollution performance, excellent biocompatibility, low biological toxicity, and small implantation damage; moreover, it is inexpensive, can work without an external voltage, has the performance of being portable and low-cost, and is suitable for on-site detection of various target substances. Description of the Drawings

[0038] Figure 1Schematic diagram of the preparation process of the photoanode of the present invention; wherein, Optical fiber (OF) represents optical fiber, PDA is polydopamine, Acetone is acetone, Remove cladding represents removing the cladding, and AuNPs seeds represents gold seeds.

[0039] Figure 2 Schematic diagram of the preparation process of the biocathode of the present invention; wherein, Glass capillary represents glass capillary, Au - decorated represents gold - decorated layer, PCB77 aptamer represents PCB77 aptamer, and MCH is mercaptohexanol.

[0040] Figure 3 Linear relationship diagram of Application Example 1;

[0041] Figure 4 Schematic diagram of in - vivo detection of the co - axially integrated implantable optofuel sensor in Application Example 2;

[0042] Figure 5 Test result graph of the concentration of pollutants enriched in the fish brain when the fish is exposed to different concentrations of PCB77 for different times in the pollutant solution in Application Example 2.

[0043] Figure 6 Comparison graph of test results of the concentration of PCB77 in the fish brain after 6 - day exposure to different concentrations of PCB77 measured by the sensor we constructed and gas chromatography in Application Example 2. Detailed implementation manners

[0044] To better understand the present invention, the content of the present invention will be further clarified below in conjunction with embodiments, but the present invention is not limited to the following embodiments.

[0045] In the following embodiments, after the gold layer is grown on the light - guiding optical fiber and the glass capillary, the resistance is 10 - 20 Ω.

[0046] Embodiment

[0047] A preparation method of a co - axially integrated implantable optofuel sensor, the specific steps are as follows:

[0048] S11. Use a pulling instrument to pull a capillary with an inner diameter of 300 μm and an outer diameter of 400 μm under the pulling instrument to obtain a conical glass capillary with a tip port diameter of 40 μm; dissolve the nucleic acid aptamer SH - Aptamer that can specifically recognize the target substance PCB77 in a 10 mM PBS (pH = 7.4) solution to obtain an 8 μM aptamer solution, and add 0.5 mM TCEP to activate the aptamer for 2 h;

[0049] S12. Immerse the tip of the capillary glass tube (about 2 cm long) in a dopamine solution of 1.5 mg / mL for 1 h. After washing with water and drying, immerse it in the gold seed for 12 h. Finally, let it stand and grow in a mixed solution of 1 mM hydroxylamine hydrochloride and 0.05 wt% chloroauric acid for 4 min, then wash with water and dry. Then, place it in the aptamer solution obtained in S11 and incubate at 4 °C for 16 h. After taking it out, wash it with 10 mM PBS (pH = 7.4) buffer solution, and then block it in 1 mM MCH solution for 2 h. After taking it out, wash it again with 10 mM PBS (pH = 7.4) buffer solution to obtain the capillary glass tube / gold layer / nucleic acid aptamer electrode;

[0050] S13. Inject 0.05 μL of 0.5% Nafion 117 perfluorinated resin solution into the tip of the capillary glass tube / gold layer / nucleic acid aptamer electrode (i.e., the constricted section), and cure the tip port at room temperature to form a Nafion 117 film, thus obtaining a biocathode;

[0051] S21. Immerse the front end of a clean transparent optical fiber with a length of 7 cm and a diameter of 125 μm (the front end is about 2 cm long) in a dopamine solution of 1.5 mg / mL for 2 h. After washing with water and drying, treat it with acetone for 3 s to remove the polydopamine layer and the optical fiber cladding at the front end of the optical fiber (0.8 cm) to make the front end light-transmissive. After washing with water and drying, immerse it in the gold seed for 4 h. Finally, let it stand and grow in a mixed solution of 1 mM hydroxylamine hydrochloride and 0.05% chloroauric acid for 4 min, then wash with water and dry to obtain an optical fiber with a gold layer grown at the front end. Among them, the longitudinal length of the gold layer modification area of the optical fiber is about 1.7 cm, and the distance from the gold layer modification area to the port of the modified end of the optical fiber is about 0.8 cm;

[0052] S22. Adsorb the front end of the optical fiber obtained in S21 in a 1 wt% PDDA solution for 5 min, wash it with water, and then adsorb it in a 0.5 mg / mL carboxylated MWNT solution for 5 min. This is one cycle; a total of 4 cycles, wash with water and dry to obtain the optical fiber / gold layer / MWNT electrode; The frontmost 0.8 cm part of the modified end of the optical fiber is not modified with a gold layer but is modified with carbon nanotubes, and the longitudinal length of the carbon nanotube modification area is about 2.5 cm;

[0053] S23. Place the modified end of the optical fiber / gold layer / MWNT electrode in the dark and immerse it in 0.1 M AgNO 3 solution for 30 min, wash it with water, and then continue to immerse it in 0.12 M Na 2 S solution for 30 min, wash with water and dry; Then soak it in 10 mM Bi(NO 3 ) 3 solution and 10 mM Na 2 S solution for 10 min each. This is the adsorption of Bi 2 S3 One cycle, two cycles, washing with water and drying, thus obtaining a photoanode;

[0054] S3. Infuse the "fuel" ascorbic acid (50 mM) into the biocathode obtained in S13, and insert the modified end of the photoanode prepared in S23 into the modified end of the biocathode. The ascorbic acid solution should be higher than the height of the modified end of the photoanode. This will obtain a coaxially integrated implantable photofuel sensor that can be used to track and detect persistent organic pollutants in vivo.

[0055] Application Example 1

[0056] The coaxial integrated implantable optical fuel sensor prepared in the embodiment was used to detect PCB77 in the buffer solution. A 650nm laser pen was used as the light source, and the open circuit potential was measured with a portable pH meter. The coaxial integrated implantable optical fuel sensor was inserted into a buffer solution containing different concentrations of PCB77, and the laser pen was connected to the non-modified end of the photoanode. The biological cathode and the photoanode were respectively connected to the two ends of the pH meter through copper wires, and then the open circuit voltage was measured. The response signals of the coaxial integrated implantable optical fuel sensor to a series of concentrations of PCB77 were obtained as shown in Table 1.

[0057] Table 1 PCB77 test results

[0058]

[0059] Analysis of the data in Table 1 shows that the open circuit potential signal generated by the implantable optical fuel sensor to the PCB77 series concentration gradually decreases with the increase of concentration; linear fitting finds that there is a linear relationship between the open circuit potential change value ΔOCP (the difference between the OCP of the target and the OCP of the background) and the logarithm of the PCB77 concentration (see Appendix Figure 3 ), the regression equation is: ΔOCP=22.6+12.21lg C(R 2 =0.998)(ΔOCP: mV; C: pg / mL), the linear range is 0-10000pg / mL, and the detection sensitivity is 2.8fg / mL, which can meet the actual detection requirements.

[0060] Application Example 2

[0061] The coaxial integrated implantable optical fuel sensor prepared in the embodiment is used to perform in-situ monitoring of PCB77 enriched in the fish brain. The specific process is as follows:

[0062] Before the in vivo experiment, the fish were placed in an aerated aquarium containing dechlorinated tap water for two weeks to acclimatize to the feed. Thirty-six grass carps were divided into 4 groups (9 fish per 50 L aquarium (40 L water)), and were reared in water containing 0 (control group), 0.1, 1, 10 ng / mL PCB77 for 3, 6, 9 days. To keep the PCB77 concentration in water stable, two-thirds of the contaminated water was replaced every 12 hours, and the water quality (pH 6.8±0.2, dissolved oxygen 7.0±0.3 mg / L, temperature 25±0.7 °C) was monitored daily. At the end of each exposure interval, 3 fish were taken out from each aquarium for photoelectrochemical detection of PCB77 in vivo.

[0063] For in vivo analysis, the fish were first anesthetized with 0.03% eugenol until they lost vertical balance. Then, a 26-gauge acupuncture needle was inserted into the fish head at the midpoint between the two eyes along the fish body axis, inserted about 7 mm to penetrate the skull; after that, the needle was removed, and the integrated sensor was inserted about 1.7 cm into the hole to penetrate the whole brain. At the same time, a laser pen with a wavelength of 650 nm was used as the excitation source, the laser pen was connected to the unmodified end of the photoanode, and the biocathode and the photoanode were respectively connected to both ends of the pH meter with copper wires through the gold layer on their surfaces. Finally, the fish were placed in fresh water to restore vertical balance, and the OCP response was detected by the pH meter.

[0064] The concentrations of PCB77 in the brains of fish exposed to different concentrations of PCB77 and different times were monitored (as Figure 5 ). The results showed that the OCP signal of the sensor in the brains of the control group fish (without PCB77 exposure) remained basically unchanged throughout the experiment, indicating that the sensor background was stable. The accumulation of PCB77 in the fish brains was positively correlated with the exposure dose and exposure time, confirming that the constructed photo-fuel microsensor could directly track the bioaccumulation level of persistent organic pollutants in vivo. Finally, the concentration of PCB77 in the fish brains was detected by gas chromatography, and the obtained results were very close to the detection results of the prepared microsensor (as Figure 6 ), proving that the photo-fuel sensor described in the present invention has high reliability.

[0065] According to the disclosure and teachings of the above specification, those skilled in the art to which the present invention pertains can also make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the invention should also fall within the protection scope of the claims of the present invention. In addition, although some specific terms are used in this specification, these terms are only for convenience of description and do not constitute any limitation to the present invention.

Claims

1. An axially integrated implantable optofuel sensor, characterized in that Comprising a photoanode, a biocathode and a fuel, wherein the photoanode uses a transparent light-conducting optical fiber as a substrate, and a gold layer, carbon nanotubes and Ag are sequentially modified at one end thereof 2 S-Bi 2 S 3 Obtained from a heterojunction; the biocathode uses a glass capillary as a substrate, a gold layer is modified at one end thereof, and then a nucleic acid aptamer for specifically recognizing a target is further modified, and then a proton exchange membrane is formed by curing with a perfluorosulfonic acid solution at the port of the modified end of the glass capillary; the fuel is an ascorbic acid solution; the fuel is injected into the biocathode, the photoanode is inserted into the biocathode to contact the fuel, and the photoanode and the biocathode are coaxial.

2. A method for preparing an axially integrated implantable optofuel sensor, characterized in that it comprises the following steps: S1. Select a glass capillary and a light guiding fiber, the inner diameter of the glass capillary being greater than the diameter of the light guiding fiber; S21. Modify the gold layer at one end of the glass capillary in S1 by the seed-mediated growth method, then modify the nucleic acid aptamer capable of specifically recognizing the target on the surface of the gold layer through gold-sulfur bonds, and block the active sites with 6-mercapto-1-hexanol to obtain a glass capillary / gold layer / nucleic acid aptamer electrode; S22. Inject the perfluorinated resin solution into the modified end of the glass capillary / gold layer / nucleic acid aptamer electrode, and after drying, form a proton exchange membrane at the port of the modified end to obtain a biocathode; S31. Modify the gold layer at one end of the light guiding fiber in S1 by the seed-mediated growth method, and then modify the carbon nanotubes on the surface of the gold layer by the layer-by-layer self-assembly method to obtain an optical fiber / gold layer / carbon nanotube electrode; S32. Modify Ag by continuous ion layer adsorption at the modified end of the optical fiber / gold layer / carbon nanotube electrode 2 S-Bi 2 S 3 heterojunction to construct a photoanode S4. Pour the fuel ascorbic acid solution into the biocathode obtained in S22, and insert the modified end of the photoanode obtained in S32 into the biocathode to contact the fuel, thus obtaining the axially integrated implantable optofuel sensor.

3. The method for preparing an axially integrated implantable optofuel sensor according to claim 2, characterized in that the diameter of the light guiding fiber is 100 - 200 μm, and the length of the modified end is 2 - 3 cm; the inner diameter and outer diameter of the glass capillary are 250 - 350 μm and 350 - 450 μm respectively, and the length of the modified end is 1.5 - 2.5 cm.

4. The method for preparing an axially integrated implantable optofuel sensor according to claim 3, characterized in that the glass capillary includes a straight tube section and a reduced diameter section at one end of the straight tube section, the inner diameter of the tip of the reduced diameter section is 35 - 45 μm, and the inner diameter of the straight tube section is 250 - 350 μm.

5. The method for preparing an axially integrated implantable optofuel sensor according to claim 2, characterized in that the method for growing the gold layer in S21 is: soak one end of the glass capillary in dopamine solution for 0.5 - 1.5 h, wash with water and dry, then soak in gold seeds for 10 - 13 h, and finally let it stand in the mixed solution of hydroxylamine hydrochloride and chloroauric acid to grow the gold layer, wash with water and dry; the method for growing the gold layer in S31 is: soak the clean light guiding fiber in dopamine solution for 1.5 - 2.5 h, wash with water and dry, then soak in acetone to remove the polydopamine layer and the fiber cladding at the front end of the fiber to make it transparent at the front end, wash with water and dry, then soak the fiber in gold seeds for 3 - 5 h, and then let it stand in the mixed solution of hydroxylamine hydrochloride and chloroauric acid to grow the gold layer, wash with water and dry.

6. The method for preparing an axially integrated implantable optofuel sensor according to claim 2, characterized in that The method for modifying the nucleic acid aptamer through gold-sulfur bonds in S21 is as follows: First, dissolve the thiolated nucleic acid aptamer in PBS buffer solution with a concentration in the range of 6 - 10 μM, then add an aqueous solution of tris(2-carboxyethyl)phosphine to activate the aptamer to obtain an aptamer solution; then, place the end of the glass capillary with a gold layer grown thereon in the aptamer solution for incubation, take it out and wash it with PBS buffer solution, and then place it in an aqueous solution of 6-mercapto-1-hexanol to block the exposed gold site , take it out again and wash it with PBS buffer solution to obtain a glass capillary / gold layer / nucleic acid aptamer electrode.

7. The preparation method of a coaxial integrated implantable optofuel sensor according to claim 2, characterized in that the specific operation method of S22 is: injecting a perfluorosulfonic acid resin solution into the modified end port of the capillary glass tube / gold layer / nucleic acid aptamer electrode, and then curing it at room temperature at this port to form a proton exchange membrane; the method of modifying carbon nanotubes by layer-by-layer self-assembly in S31 is: placing the optical fiber after growing the gold layer in an aqueous solution of polydiallyldimethylammonium chloride for adsorption, washing with water, and then placing it in an aqueous solution of carboxylated carbon nanotubes for adsorption, and successively circulating the adsorption to achieve the modification of carbon nanotubes.

8. The preparation method of a coaxial integrated implantable optofuel sensor according to claim 2, characterized in that In S32, Ag is modified by successive ionic layer adsorption on the optical fiber / gold layer / carbon nanotube electrode 2 S-Bi 2 S 3 The method for the heterojunction is as follows: In a dark environment, the modified end of the optical fiber / gold layer / carbon nanotube electrode is successively placed in an aqueous solution of AgNO 3 , a Na 2 S solution, a Bi(NO 3 ) 3 solution, and a Na 2 S solution for cyclic soaking. After each soaking, it is washed with water and dried and then placed in the next solution; In S4, the concentration of the fuel ascorbic acid solution is 20 - 100 mM, and the perfusion height in the biocathode should be higher than the modified end of the photoanode.

9. The preparation method of a coaxial integrated implantable optofuel sensor according to claim 2, characterized in that The target substance is the persistent organic pollutant 3,3',4,4'-tetrachlorobiphenyl; the nucleic acid aptamer is SH-Aptamer, and the sequence is 5′-SH-(CH 2 ) 6 -GGC-GGG-GCT-ACG-AAG-TAG-TGA-TTT-TTT-CCG-ATG-GCC-CGT-G-3′.

10. The application of the coaxial integrated implantable optofuel sensor according to claim 1 in detecting persistent organic pollutants, characterized in that the specific application method is: inserting the coaxial integrated implantable optofuel sensor into the analyte, irradiating the unmodified end of the photoanode with a laser source, electrically connecting both the biocathode and the photoanode to both ends of a potentiometer through the surface-modified gold layer, and then measuring the open-circuit voltage, and using the standard curve method to measure the content of persistent organic pollutants in the analyte.

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