Support-free hydrogel photoelectrochemical / fluorescent bimodal sensor and application thereof in detection of persistent organic pollutants

Abstract

The invention discloses a support-free hydrogel photoelectrochemical / fluorescent bimodal sensor, which comprises a working electrode and a reference electrode / counter electrode, the working electrode and the reference electrode both comprise a gold wire, a gel layer and a solidification layer, the gel layer is coated on the surface of the gold wire, the solidification layer is coated on the surface of the gel layer, and the gel layer is coated on the surface of the counter electrode. One end of the gold wire extends out of the gel layer and the solidification layer; the solidification layers are calcium alginate layers; the gel layer of the working electrode is a gel layer containing hydrogel, a photoinitiator, a silver-wrapped-in-gold nanowire and a photoelectric material; the gel layer of the reference electrode is a gel layer containing hydrogel, a photoinitiator and a gold-clad silver nanowire. The support-free hydrogel sensor is a hydrogel sensor which is not supported by a rigid substrate, can be used for detecting or continuously monitoring persistent organic pollutants, and is particularly suitable for detecting or continuously monitoring persistent organic pollutants in organisms; the problems of mechanical mismatching, non-specific adsorption, poor biocompatibility and the like of an existing implantable living body photoelectrochemical sensor are solved.

Description

Technical Field

[0001] The present invention belongs to the field of photoelectrochemical sensing, and particularly relates to a supported hydrogel photoelectrochemical / fluorescent dual-mode sensor, a preparation method thereof, and an application in the detection of persistent organic pollutants in organisms. Background Art

[0002] In recent years, long-term in vivo analysis technology has developed vigorously and received extensive attention, which is crucial for the development of environmental biology, analytical chemistry, and life science fields. For example, in vivo glucose testing can achieve long-term non-invasive or minimally invasive monitoring of blood glucose concentration, which helps to strengthen the control of blood glucose concentration in diabetic patients. In vivo detection of important pollutants provides a solution for studying the occurrence, migration, and fate of pollutants in the ecological environment without sacrificing organisms, and can be used to protect wild animals, especially endangered animals, from targeted pollutants.

[0003] Currently, people are mainly committed to developing fluorescence probes, Raman spectroscopy, implantable electrochemistry, and photoelectrochemical sensors to analyze various targets in organisms. Among them, electrochemical and photoelectrochemical methods are favored because of their high spatial resolution, easy miniaturization, and portability.

[0004] However, implantable electrochemical and photoelectrochemical sensors still have some inevitable limitations: 1) Mechanical mismatch. Generally, microelectrodes are mainly prepared with rigid substrates (such as carbon fiber, tungsten wire, titanium wire, and glass) with a high Young's modulus (>60 GPa), which do not match with low-modulus biological tissues (<100 Mpa), and are prone to cause chronic tissue damage and foreign body reactions; 2) Non-specific adsorption. In a complex in vivo environment, electrodes implanted in living bodies often encounter many biological macromolecules, resulting in non-specific adsorption and biological contamination, which not only leads to electrode passivation and performance degradation, but may also trigger foreign body reactions and immune reactions; 3) Biocompatibility. Some nanomaterials or optoelectronic materials modified on the electrode surface are toxic to organisms when they come into direct contact with tissues after implantation; 4) Additional modification of the sensor or electrode (such as anti-pollution materials, low-toxicity films, etc.) is required, which is complex and impractical. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a hydrogel photoelectrochemical / fluorescent dual-mode sensor aiming at the deficiencies of the above-mentioned existing technologies. When it is used to detect persistent organic pollutants in organisms or test solution samples, it has ideal biocompatibility and mechanical matching, and the detection is sensitive and accurate.

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

[0007] An unsupported hydrogel photoelectrochemical / fluorescent dual-mode sensor, including a working electrode and a reference electrode / counter electrode (note: the reference electrode also serves as the counter electrode), both the working electrode and the reference electrode include a gold wire, a gel layer and a solidified layer. The gel layer is coated on the surface of the gold wire, and the solidified layer is coated on the surface of the gel layer, and one end of the gold wire extends out of the gel layer and the solidified layer; the solidified layer is a calcium alginate layer; the gel layer of the working electrode is a gel layer containing hydrogel, photoinitiator, silver-coated gold nanowires and optoelectronic materials; the gel layer of the reference electrode is a gel layer containing hydrogel, photoinitiator and silver-coated gold nanowires.

[0008] According to the above scheme, the diameter of the gold wire is generally in the range of 0.01-2 mm, the thickness of the gel layer is preferably 30-100 μm, and the thickness of the solidified layer is preferably 10-50 μm.

[0009] According to the above scheme, the hydrogel is one of polyethylene glycol diacrylate, hyaluronic acid methacrylate, polyacrylamide, etc.; the photoinitiator is one of 2-hydroxy-2-methylpropiophenone, ruthenium photoinitiator, phenyl-2,4,6-trimethylbenzylformyl phosphite (such as potassium trimethylbenzylformyl phosphite, etc.) or peroxide, etc.

[0010] According to the above scheme, the silver-coated gold nanowires can be replaced by carbon nanotube fibers, and silver-coated gold nanowires are preferably considered for their electrical and light-conducting properties.

[0011] According to the above scheme, the optoelectronic material is a composite material of titanium dioxide and porphyrin metal-organic framework, preferably PCN-224(Zn)@TiO 2 , where the mass ratio of PCN-224(Zn) to titanium dioxide is 1:1 to 6:1.

[0012] Further preferably, the gel layer of the working electrode is a gel layer containing polyethylene glycol diacrylate, 2-hydroxy-2-methylpropiophenone, silver-coated gold nanowires and optoelectronic materials; the gel layer of the reference electrode is a gel layer containing polyethylene glycol diacrylate, 2-hydroxy-2-methylpropiophenone and silver-coated gold nanowires.

[0013] According to the above scheme, the gel layer of the working electrode is formed by photocuring a gel precursor solution containing polyethylene glycol diacrylate, 2-hydroxy-2-methylpropiophenone, silver-coated gold nanowires, and an optoelectronic material. In the gel precursor solution of this working electrode, the concentration range of polyethylene glycol diacrylate is 30%-90% w / v (Note: w / v (weight / volume) represents the mass of the solute contained in a unit volume of solution, usually expressed as a percentage. For example, a 30% w / v solution means that every 100 milliliters of the solution contains 30 grams of the solute); the concentration range of 2-hydroxy-2-methylpropiophenone is 1%-4% w / v; the content range of silver-coated gold nanowires is 5-25% w / v; the concentration of the optoelectronic material is 1-10 mg / mL.

[0014] According to the above scheme, the gel layer of the reference electrode is formed by photocuring a gel precursor solution containing polyethylene glycol diacrylate, 2-hydroxy-2-methylpropiophenone, and silver-coated gold nanowires. In the gel precursor solution of this reference electrode, the concentration range of polyethylene glycol diacrylate is 30%-90% w / v; the concentration range of 2-hydroxy-2-methylpropiophenone is 1%-4% w / v; the content range of silver-coated gold nanowires is 10-30%.

[0015] The preparation method of the above unsupported hydrogel optoelectrochemical / fluorescent dual-mode sensor includes the following steps:

[0016] S1. Select a silica gel tube and a gold wire, and the inner diameter of the silica gel tube is larger than the diameter of the gold wire;

[0017] S21. Mix polyethylene glycol diacrylate, 2-hydroxy-2-methylpropiophenone, silver-coated gold nanowires, and an optoelectronic material evenly to obtain a gel layer precursor solution for the working electrode; inject the gel layer precursor solution for the working electrode into a silica gel tube (as a mold), then insert one end of the gold wire into the silica gel tube (the other end extends out of the silica gel tube), demold after ultraviolet curing (that is, remove the silica gel tube), and soak in deionized water to remove the unreacted polyethylene glycol diacrylate and 2-hydroxy-2-methylpropiophenone to obtain a first hydrogel electrode;

[0018] S22. Immerse the first hydrogel electrode in sodium alginate solution and calcium chloride solution in sequence to form a calcium alginate coating to obtain a working electrode;

[0019] S31. Mix polyethylene glycol diacrylate, 2-hydroxy-2-methylpropiophenone, and silver-coated gold nanowires evenly to obtain a gel layer precursor solution for the reference electrode; inject the gel layer precursor solution for the reference electrode into the silica gel tube, then take another gold wire and insert one end of it into the silica gel tube, demold after ultraviolet curing, and soak in deionized water to remove the unreacted polyethylene glycol diacrylate and 2-hydroxy-2-methylpropiophenone to obtain a second hydrogel electrode;

[0020] S32. Immerse the second hydrogel electrode into sodium alginate solution and calcium chloride solution in sequence to form a calcium alginate cladding, thereby obtaining a reference electrode.

[0021] S4. The working electrode obtained in S22 and the reference electrode obtained in S32 form the unsupported hydrogel photoelectrochemical / fluorescent dual-mode sensor of the present invention, which can be used for in vivo tracking detection of persistent organic pollutants.

[0022] According to the above solution, in step S1, the inner diameter of the silica gel tube is preferably 100 - 200 μm larger than the diameter of the gold wire, and the diameter of the gold wire is preferably in the range of 0.01 - 2 mm. Among them, the silica gel tube can be replaced by other transparent tubular molds that can achieve photocuring.

[0023] According to the above solution, in step S21, in the gel precursor solution of the working electrode, the concentration range of polyethylene glycol diacrylate is 30% - 90% w / v, the concentration range of 2-hydroxy-2-methylpropiophenone is 1% - 4% w / v, the content range of gold-coated silver nanowires is 10 - 30% w / v, and the concentration of the optoelectronic material is 1 - 10 mg / mL.

[0024] According to the above solution, in step S31, in the gel precursor solution of the reference electrode, the concentration range of polyethylene glycol diacrylate is 30% - 90% w / v, the concentration range of 2-hydroxy-2-methylpropiophenone is 1% - 4% w / v, and the content range of gold-coated silver nanowires is 10 - 30% w / v.

[0025] According to the above solution, in steps S22 and S32, the concentration range of the sodium alginate solution is 0.1% - 0.4% w / v, and the concentration of the calcium chloride solution is 0.05 - 0.2 mol / L.

[0026] The application of the above unsupported hydrogel photoelectrochemical / fluorescent dual-mode sensor in the detection or continuous monitoring of persistent organic pollutants, and the specific application method is as follows: The first is to adopt the photoelectrochemical mode. Insert one end (here it is the end coated with the gel layer and the solidified layer) of the working electrode and the reference electrode / counter electrode of the hydrogel photoelectrochemical / fluorescent dual-mode sensor into the analyte, then irradiate the working electrode with a laser source, and connect the gold wires extending from the other ends of the working electrode and the reference electrode / counter electrode out of the gel layer and the solidified layer to an electrochemical workstation, and then measure the photocurrent and / or photopotential; The second is to adopt the fluorescent mode. Immerse the working electrode of the hydrogel photoelectrochemical / fluorescent dual-mode sensor into the analyte, and then measure the fluorescence signal with a fluorometer. Both modes use the standard curve method to measure the content of persistent organic pollutants in the analyte. Among them, the analyte in the photoelectrochemical mode is a biological body or a solution sample, and the analyte in the fluorescent mode is a solution sample.

[0027] On this basis, the present invention further provides a self-supporting hydrogel photoelectrochemical / fluorescent dual-mode sensing system, including the hydrogel photoelectrochemical / fluorescent dual-mode sensor of the present invention, a light source, a potentiometer and / or a fluorometer; wherein, in the photoelectrochemical mode, the hydrogel photoelectrochemical / fluorescent dual-mode sensor is used to be inserted into the analyte, the light source is used to irradiate the working electrode, and the ends of the gold wires of the working electrode and the reference electrode extending out of the gel layer and the solidified layer are respectively connected to the two electrodes of the potentiometer. The potentiometer is used to record the photocurrent and photopotential signals, and the wavelength of the light source matches the absorption peak of the optoelectronic material; in the fluorescent mode, the working electrode of the hydrogel photoelectrochemical / fluorescent dual-mode sensor is used to be immersed in the analyte, and the fluorometer is used to record the fluorescence signal.

[0028] The target substances of the hydrogel photoelectrochemical / fluorescent dual-mode sensor and the sensing system of the present invention are persistent organic pollutants. Specifically, the persistent organic pollutants refer to pentachlorophenol (PCP), dichlorodiphenyltrichloroethane, chlordane, polychlorinated biphenyls, hexachlorobenzene, dioxins, furans, lindane, etc. The optoelectronic material adopted by the present invention for the target substance PCP is PCN-224(Zn)@TiO 2 , and PCP can axially coordinate and bind with the Zn(II) center in PCN-224(Zn). If other pollutants are selected as the analyte targets, corresponding optoelectronic materials that can specifically recognize them can be selected. Taking the target substance PCP as an example, the principle of the present invention is described below.

[0029] Principle of the photoelectrochemical mode: The sensor is inserted into the analyte (including organisms and solutions), and the light source irradiates the working electrode, exciting the PCN-224(Zn)@TiO embedded in the working electrode 2 , generating photo-generated carriers that migrate to the electrode surface to form photocurrent and photopotential; when the target substance PCP contacts the working electrode, PCP can axially coordinate and bind with the Zn(II) center in PCN-224(Zn), hindering the generation of photo-generated carriers of PCN-224(Zn) and inhibiting the transfer of photo-generated electrons of PCN-224(Zn) to TiO 2 , weakening the intensity of the photocurrent and photopotential. This sensor encapsulates flexible conductive substances and optoelectronic materials in the hydrogel, has ideal biocompatibility and excellent anti-pollution ability, and will not cause obvious inflammation and immune reactions in biological tissues during long-term monitoring of organisms. Principle of the fluorescent mode: PCN-224(Zn) is a fluorescent material that can be excited at 449 nm and emit at 660 nm. After the working electrode containing PCN-224(Zn)@TiO 2 is inserted into the analyte solution and undergoes an axial coordination reaction with the target PCP, the molecular structure of PCN-224(Zn) is changed, enhancing the fluorescence signal of the working electrode.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] 1. The unsupported hydrogel photoelectrochemical / fluorescent dual-mode sensor of the present invention has excellent flexibility, biocompatibility and anti-pollution ability, and can be used for the detection or continuous monitoring of persistent organic pollutants, especially suitable for the detection or continuous monitoring of persistent organic pollutants in organisms. It solves the problems of mechanical mismatch, non-specific adsorption and poor biocompatibility existing in current implantable photoelectrochemical in-vivo sensors. It has the advantages of high sensitivity, good selectivity, good stability and good reproducibility in solution detection. Taking the detection of PCP in the fish brain of living fish as an example, the long-term monitoring application of this method is verified.

[0032] 2. The preparation method and application method of the unsupported hydrogel photoelectrochemical / fluorescent dual-mode sensor of the present invention are simple and easy to operate. It can also perform dual-mode detection by two methods of photoelectrochemistry and fluorescence. The two methods can verify the detection results with each other, making the sensor have higher accuracy than traditional single-mode sensors. Description of the Drawings

[0033] Figure 1 It is a schematic diagram of the preparation process of the unsupported hydrogel photoelectrochemical / fluorescent dual-mode sensor of the present invention.

[0034] Figure 2 It is a micrograph of the unsupported hydrogel photoelectrochemical / fluorescent dual-mode sensor of the present invention.

[0035] Figure 3 It is a size diagram of the Au@Ag NWs used in the unsupported hydrogel photoelectrochemical / fluorescent dual-mode sensor of the present invention.

[0036] Figure 4 In [it], a, b, and c are respectively the linear relationship diagrams between the photopotential change value, photocurrent change value and fluorescence intensity change value in Application Example 1 and the logarithm of the PCP concentration.

[0037] Figure 5 It is a reproducibility diagram of the unsupported hydrogel photoelectrochemical / fluorescent dual-mode sensor in Application Example 2.

[0038] Figure 6 It is a schematic diagram of in-vivo detection of the unsupported hydrogel photoelectrochemical / fluorescent dual-mode sensor in Application Example 2.

[0039] Figure 7 It is a test result diagram of the enrichment of PCP in the fish brain after the solution in Application Example 2 is exposed to different PCP concentrations for different times.

[0040] Figure 8Comparison chart of the photoelectrochemical test results and fluorescence test results of PCP enrichment in fish brains after exposure to solutions with different PCP concentrations in Application Example 2.

[0041] Figure 9 Comparison chart of the photoelectrochemical test results and the results of liquid chromatography of the PCP concentration in the fish brain after 28 days of exposure when using a free-standing hydrogel photoelectrochemical / fluorescent dual-mode sensor to determine PCP in vivo in Application Example 2. Detailed implementation manners

[0042] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0043] In the following embodiments, the preparation process of the PCN-224(Zn)@TiO 2 composite material is as follows: First, ZrCl 4 (70 mg), H 2 TCPP(50 mg), ZnCl 2 (50 mg), benzoic acid (2.7 g) and TiO 2 nanoparticles (10 mg) are added to DMF (8 mL), sonicated for 10 min, and then the resulting mixture is transferred to a round-bottom flask (10 mL) and reacted at 120 °C for 48 h. After cooling to room temperature, the precipitate is accumulated by centrifugation, washed 5 times with DMF, and then placed in acetone to exchange the residual DMF with acetone at 60 °C for 72 h. After drying, pure PCN-224(Zn)@TiO 2 is obtained, and finally a total mass of about 50 mg is obtained. Since when performing this reaction, the mass of the added TiO 2 nanoparticles is 10 mg, the mass ratio of PCN-224(Zn):TiO 2 is about 4:1.

[0044] The synthesis of gold-coated silver nanowires (Au@Ag NWs) is mainly divided into the following two steps:

[0045] (1) Synthesis of silver nanowires (Ag NWs): Add EG (40 mL) to a round-bottom flask (50 mL), heat in an oil bath at 160 °C for 5 min, inject CuCl 2 solution (3 mL of 4 mmol / L), stir for 10 min, add AgNO 3 (0.4 g) and PVP (Mw 1300000, 0.8 g), react at 160 °C for 45 min, and finally, collect the Ag NWs by centrifugal washing with acetone and ethanol.

[0046] (2) Conformally deposit Au atoms on the surface of the synthesized Ag NWs to prepare Au@Ag NWs: First, mix deionized water (40 mL) containing PVP (Mw 55000, 2.2 g), deionized water (10 mL) containing citric acid (210 mg), and deionized water (18 mL) containing NaOH (145 mg) and stir vigorously to obtain an alkaline solution with pH = 11; after adding the Ag NWs solution (300 μL, 24 mg / mL, dissolved in ethanol), inject the HAuCl 4 precursor (8 mL, 0.25 mmol / L) at a rate of 0.8 mL / h using a syringe pump to obtain Au@Ag NWs at this time; the Au@Ag NWs are centrifugally washed 4 times with water at 3000 rpm for 10 min each time and stored in ethanol for later use.

[0047] In the prepared Au@Ag NWs, Au:Ag = 1:2.8 (mass ratio). As Figure 3 shown, the diameter of the Au@Ag NWs is about 200 - 250 nm.

[0048] Example

[0049] A preparation method of a unsupported hydrogel photoelectrochemical / fluorescent dual - mode sensor, the specific steps are as follows:

[0050] S1. Select a silica gel tube with an inner diameter of 200 μm and an outer diameter of 300 μm, and two gold wires with a diameter of 50 μm;

[0051] S21. Prepare a precursor solution according to 60% w / v polyethylene glycol diacrylate, 2% w / v 2 - hydroxy - 2 - methylpropiophenone, 15% w / v gold - coated silver nanowires, and 2 mg / mL PCN - 224(Zn)@TiO 2 Take 3 μL of the precursor solution and inject it into the silica gel tube mold selected in S1. Insert one end of the gold wire selected in S1 into the silica gel tube, with the insertion length being about 0.5 cm (the part of the gold wire not inserted into the silica gel tube will be used to connect to the electrochemical workstation later), then cure it with ultraviolet light, and then take it out from the silica gel hose to obtain the first hydrogel electrode, and soak it in deionized water for 2 days to remove unreacted precursor substances;

[0052] S22. Soak the first hydrogel electrode prepared in S21 in 0.2% w / v sodium alginate and 0.1 mol / L calcium chloride solution for 1 s respectively to obtain the working electrode;

[0053] S31. Prepare a precursor solution by mixing 60% w / v polyethylene glycol diacrylate, 2% w / v 2-hydroxy-2-methylpropiophenone, and 25% w / v core-shell gold / silver nanowires. Take 3 μL of the precursor solution and inject it into the silicone tube mold selected in S1. Insert one end of another gold wire selected in S1 into the silicone tube, with an insertion length of about 0.5 cm. Then cure it with ultraviolet light, and then take it out from the silicone tube to obtain the second hydrogel electrode. Immerse it in deionized water for 2 days to remove unreacted precursor substances;

[0054] S32. Immerse the second hydrogel electrode prepared in S31 in 0.2% w / v sodium alginate and 0.1 mol / L calcium chloride solution for 1 s respectively to obtain the reference / counter electrode;

[0055] S4. Combine the working electrode obtained in S22 and the reference / counter electrode obtained in S32 to form a self-supporting, high-performance hydrogel photoelectrochemical / fluorescent dual-mode sensor, which can be used for in vivo tracking of persistent organic pollutants.

[0056] As Figure 2 shown, the diameter of the working electrode of the hydrogel photoelectrochemical / fluorescent dual-mode sensor prepared in the example is between 215 - 225 μm. Among them, the thickness of the gel layer is about 60 μm, the thickness of the solidified layer is about 25 μm, and the diameter of the gold wire is 50 μm (not shown) and is wrapped in the middle of the solidified layer. In order to better observe the flexible conductive material and optoelectronic material inside the hydrogel sensor, the part containing the gold wire is not shown.

[0057] Application Example 1

[0058] Use the self-supporting hydrogel photoelectrochemical / fluorescent dual-mode sensor prepared in the example to detect PCP. Use a 650 nm laser pen as the light source, and measure the photopotential with a portable pH meter. Insert the sensor into the PCP test solution with different concentrations (the preparation method of the PCP test solution: add different concentrations of PCP to artificial cerebrospinal fluid (ACSF)), and irradiate the end of the working electrode containing the gel layer and the solidified layer with the laser pen. Connect the working electrode and the reference / counter electrode to the two ends of the pH meter through copper wires respectively, and then measure the photopotential. The response signals of the self-supporting hydrogel photoelectrochemical / fluorescent dual-mode sensor to a series of PCP with different concentrations are shown in Table 1.

[0059] Table 1

[0060]

[0061] Analysis of the data in Table 1 shows that the response of the hydrogel photoelectrochemical / fluorescent dual-mode sensor to the PCP concentration in ACSF decreases with increasing concentration, and the generated photopotential signal gradually decreases; linear fitting reveals a linear relationship between the change in photopotential ΔOCP (the difference between the OCP of the target and the background, and the background OCP is measured from ACSF without added PCP) and the logarithm of the PCP concentration ( Figure 4 ), and the resulting regression equation is: ΔOCP = –42.6 – 11.9lgC (R 2 = 0.990) (ΔOCP: mV; C: pg / mL), with a linear range of 0 to 1000000 pg / mL and a detection sensitivity of 2.8 fg / mL, which can meet the actual detection requirements.

[0062] While measuring the photopotential, the relationships between the changes in photocurrent and fluorescence intensity and the change in PCP concentration under the corresponding conditions were also measured, and linear equations between the change in photocurrent value and the logarithm of the PCP concentration, and between the change in fluorescence intensity value and the logarithm of the PCP concentration were established, as Figure 4 shown.

[0063] After testing 7 hydrogel sensors prepared in different batches in ACSF solution, the photocurrent results were 10.3, 10.02, 9.91, 9.93, 9.65, 10.3, 9.91 nA respectively; after these 7 electrodes recognized a 100 pg / mL PCP solution, the photocurrent results were 5.3, 4.9, 4.9, 4.9, 4.7, 5.3, 4.9 nA respectively ( Figure 5 ); so the detected changes in photocurrent values were: 5, 5.12, 5.01, 5.03, 4.95, 5, 5.01 nA respectively. It was found through calculation that the relative average deviation of these photocurrent change values was 1.07%, indicating that the reproducibility of the sensor was very good.

[0064] Application Example 2

[0065] The unsupported hydrogel photoelectrochemical / fluorescent dual-mode sensor prepared in the Example was used to in-situ monitor the PCP enriched in the fish brain, and the specific process is as follows:

[0066] Before conducting in vivo experiments, live grass carp (body length 12.5 - 14.3 cm, body weight 20.2 - 24.3 g) were raised in an aquarium with continuous oxygen supply and fed with dechlorinated tap water for 2 weeks to adapt to the laboratory breeding environment. Then, 32 grass carp were divided into 4 groups (8 fish in each group in a 50 L aquarium containing 40 L of dechlorinated water, and each fish was numbered), and were respectively exposed to PCP dechlorinated aqueous solutions with concentrations of 0 (control group), 0.01, 0.1, and 1 ng / mL for 70 days. To maintain the stability of the PCP concentration in the water, two-thirds of the contaminated water was replaced with dechlorinated water containing the corresponding concentration of PCP every 24 hours. The water quality (pH 6.8 ± 0.2, dissolved oxygen 7 ± 0.3 mg / L, temperature 25 ± 0.7 °C) was monitored daily.

[0067] After two weeks of diet adaptation, the fish were anesthetized with dechlorinated water containing 0.005% w / v eugenol for about 3 min, and it was observed that the fish lost vertical balance but the gills were still moving. To avoid the slippery fish skin from hindering the subsequent fixation of the plastic tube used to protect the unsupported hydrogel sensor described in the present invention, the epidermis about 1×1 cm in the middle of the fish eye was gently peeled off with a scalpel. Then, two 26-gauge needles were inserted along the axis of the fish into the peeled epidermis area of the fish head to a depth of about 1.5 cm (the insertion spacing between the two needles was 3 mm). At this time, the needles passed through the skull and just touched the fish's brain, leaving only two small wounds in the brain. This not only prevents the hard steel needle from being inserted too deep into the fish brain and causing non-negligible mechanical damage, but also helps to implant the flexible hydrogel sensor (saturated with ACSF) into the fish brain. Thereafter, the working electrode and the reference / counter electrode of the unsupported hydrogel optoelectrochemical / fluorescent dual-mode sensor prepared in the example were respectively inserted into the two auxiliary needles, and then implanted into the fish head to a depth of 1.65 cm (i.e., about 0.15 cm into the brain), leaving 0.5 mm outside the fish head for connecting the detector and the light source (pH meter or electrochemical workstation, and a pH meter was used in this application example). Subsequently, the needles were carefully withdrawn from the head, and then a 200 μL centrifuge tube with the bottom removed was fixed to the head with strong adhesive to ensure that the hydrogel sensor remained moist during long-term monitoring and was not contaminated by the PCP-containing water. After recording the photocurrent and photopotential signals (i.e., background signals) of the implanted hydrogel optoelectrochemical / fluorescent dual-mode sensor, the centrifuge tube cap was closed, and the fish were placed in four tanks containing PCP aqueous solutions with different concentrations (0, 0.01, 0.1, and 1 ng / mL) and fed for 70 days (the hydrogel optoelectrochemical / fluorescent dual-mode sensor was always kept in the fish brain). Photos of the process of implanting the hydrogel optoelectrochemical / fluorescent dual-mode sensor into the fish brain are as Figure 6As shown, by removing the fish from the fish tank, placing it in an 18×8×3 cm acrylic box, and connecting the hydrogel photoelectrochemical / fluorescent dual-mode sensor to a detector for direct signal recording, long-term in vivo PCP monitoring was carried out at 3, 7, 14, 21, 28, 35, 42, 49, 56, 63, and 70 days after implantation.

[0068] According to Figure 4 the photopotential linear equation in a, the PCP concentration in the fish brain was calculated. As Figure 7 shown, when exposed to a 0.01 ng / mL PCP aqueous solution, the measured PCP concentrations in the fish brain at 3 to 70 days (i.e., 3, 7, 14, 21, 28, 35, 42, 49, 56, 63, and 70 days) were 0.05, 0.1, 0.2, 0.23, 1.35, 1.49, 4.5, 102, 310, 650, and 903 ng / mL, respectively; when exposed to a 0.1 ng / mL PCP aqueous solution, the measured PCP concentrations in the fish brain at 3 to 70 days were 0.52, 7.5, 11, 16.7, 18.1, 27.4, 38.1, 950, 1300, 2700, and 4052 ng / mL, respectively; when exposed to a 1 ng / mL PCP aqueous solution, the measured PCP concentrations in the fish brain at 3 to 70 days were 2, 350, 1023, 1405, 9800, 13200, 15780, 43000, 82000, 160800, and 226000 ng / mL, respectively. From the above experimental data, it was found that when exposed to PCP aqueous solutions at different concentrations, the concentration of PCP enriched in the fish brain would increase with the prolongation of the exposure time, indicating that the accumulation of PCP in the fish brain depends on the concentration of PCP in the environment and the exposure time.

[0069] Dual-mode tests of photocurrent and fluorescence were carried out on the PCP enriched in the fish brain exposed to PCP aqueous solutions at different concentrations for 3 days, and then the enriched concentration of PCP was obtained according to Figure 4 the linear equation. When grass carp were exposed to 0.01, 0.1, and 1 ng / mL PCP aqueous solutions respectively, according to Figure 4 the photocurrent linear equation in b, the enriched PCP concentrations in the fish brain were calculated to be 0.7 ng / mL, 12 ng / mL, and 6270 ng / mL respectively; according to Figure 4 the fluorescence linear equation in c, the enriched PCP concentrations in the fish brain were calculated to be 0.58 ng / mL, 13.4 ng / mL, and 6190 ng / mL respectively. By comparing the above experimental data, it can be seen that the results obtained by the fluorescence method are similar to those measured by the photoelectrochemical method (as Figure 8 shown), and the mutual verification of the two modes preliminarily proves the accuracy of the constructed sensor.

[0070] To further verify the reliability of the prepared hydrogel photoelectrochemical / fluorescent bimodal sensor, three fish without implanted sensors were exposed to a 1 ng / mL PCP aqueous solution for 14 days and then killed. The fish brains were removed for sample processing, and the processed samples were detected by liquid chromatography-tandem mass spectrometry. The result was 1.07 ng / mL. This is relatively close to the detection results of 0.83 ng / mL and 1.0 ng / mL obtained by the photocurrent test and photopotential test of the hydrogel photoelectrochemical / fluorescent bimodal sensor prepared by the present invention, combined with the standard curve (as Figure 9 shown). In particular, the PCP content obtained by measuring the photopotential in the photoelectrochemical mode is very close to the detection result obtained by liquid chromatography-tandem mass spectrometry, proving that the hydrogel photoelectrochemical / fluorescent bimodal sensor constructed by the present invention has satisfactory reliability in detecting persistent organic pollutants.

[0071] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope 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 unsupported hydrogel photoelectrochemical / fluorescence dual-modal sensor, comprising a working electrode and a reference electrode, the reference electrode also serving as a counter electrode, characterized in that: The working electrode and the reference electrode both include a gold wire, a gel layer and a coagulation layer, the gel layer is coated on the surface of the gold wire, the coagulation layer is coated on the surface of the gel layer, and one end of the gold wire extends out of the gel layer and the coagulation layer; the coagulation layers are both calcium alginate layers; the gel layer of the working electrode is a gel layer containing hydrogel, photoinitiator, flexible conductive material and photoelectric material; the gel layer of the reference electrode is a gel layer containing hydrogel, photoinitiator and nanowires; the flexible conductive material is gold-coated silver nanowires or carbon nanotube fibers.

2. The unsupported hydrogel photoelectrochemical / fluorescence dual-modal sensor according to claim 1, characterized in that: The diameter of the gold wire is no more than 2 mm, the thickness of the gel layer is 30-100 μm, and the thickness of the solidified layer is 10-50 μm.

3. The unsupported hydrogel photoelectrochemical / fluorescence dual-modal sensor according to claim 1, characterized in that: The hydrogel is polyethylene glycol diacrylate, methacrylic acid hyaluronic acid or polyacrylamide; the photoinitiator is 2-hydroxy-2-methylpropiophenone, ruthenium photoinitiator, phenyl-2,4,6-trimethylbenzyl formyl hypophosphite or peroxide.

4. The unsupported hydrogel photoelectrochemical / fluorescence dual-modal sensor according to claim 1, characterized in that: The gel layer of the working electrode is a gel layer comprising polyethylene glycol diacrylate, 2-hydroxy-2-methylpropiophenone, gold-coated silver nanowires, and a photoelectric material; the gel layer of the reference electrode is a gel layer comprising polyethylene glycol diacrylate, 2-hydroxy-2-methylpropiophenone, and gold-coated silver nanowires; the photoelectric material is a composite material of titanium dioxide and a porphyrin metal organic framework, wherein the mass ratio of the porphyrin metal organic framework to titanium dioxide is 1:1 to 6:

1.

5. A method for preparing an unsupported hydrogel photoelectrochemical / fluorescence dual-modal sensor according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. Select a transparent tubular mold and a gold wire, wherein the inner diameter of the transparent tubular mold is larger than the diameter of the gold wire; S21. Polyethylene glycol diacrylate, 2-hydroxy-2-methylpropiophenone, gold-coated silver nanowires and optoelectronic materials are uniformly mixed to obtain a gel layer precursor solution of a working electrode; the gel layer precursor solution of the working electrode is injected into a transparent tubular mold, and then a gold wire is inserted into a silicone tube, and after UV curing, the mold is demolded, and the unreacted polyethylene glycol diacrylate and 2-hydroxy-2-methylpropiophenone are removed by soaking in water to obtain a first hydrogel electrode; S22. The first hydrogel electrode is sequentially immersed in a sodium alginate solution and a calcium chloride solution to form a calcium alginate coating to obtain a working electrode; S31. Polyethylene glycol diacrylate, 2-hydroxy-2-methylpropiophenone and gold-coated silver nanowires are mixed evenly to obtain a gel layer precursor solution of a reference electrode; the gel layer precursor solution of the reference electrode is injected into a transparent tubular mold, and another gold wire is inserted into the transparent tubular mold. After UV curing, the mold is demolded, and the unreacted polyethylene glycol diacrylate and 2-hydroxy-2-methylpropiophenone are removed by soaking in water to obtain a second hydrogel electrode; S32. The second hydrogel electrode is sequentially immersed in a sodium alginate solution and a calcium chloride solution to form a calcium alginate coating to obtain a reference electrode; The working electrode obtained in S4.S22 and the reference electrode obtained in S32 constitute the unsupported hydrogel photoelectrochemical / fluorescence dual-modal sensor described in the present invention, which is used to detect persistent organic pollutants.

6. The method for preparing an unsupported hydrogel photoelectrochemical / fluorescence dual-modal sensor according to claim 5, characterized in that: In step S1, the transparent tubular mold is a silicone tube, and the inner diameter of the silicone tube is 100-200 μm larger than the diameter of the gold wire; in steps S22 and S32, the concentration range of the sodium alginate solution is 0.1%-0.4% w / v, and the concentration of the calcium chloride solution is 0.05-0.2 mol / L.

7. The method for preparing an unsupported hydrogel photoelectrochemical / fluorescence dual-modal sensor according to claim 5, characterized in that: In step S21, in the gel precursor solution of the working electrode, the concentration range of polyethylene glycol diacrylate is 30%-90% w / v, the concentration range of 2-hydroxy-2-methylpropiophenone is 1%-4% w / v, the content range of gold-coated silver nanowires is 10-30% w / v, and the concentration of the photoelectric material is 1-10 mg / mL. In step S31, in the gel precursor solution of the reference electrode, the concentration range of polyethylene glycol diacrylate is 30%-90% w / v; the concentration range of 2-hydroxy-2-methylpropiophenone is 1%-4% w / v; and the content range of gold and silver nanowires is 10-30% w / v.

8. The use of the unsupported hydrogel photoelectrochemical / fluorescence dual-modal sensor of claim 1 in detecting or continuously monitoring persistent organic pollutants, characterized in that: The specific application method is as follows: inserting one end of the working electrode and the reference electrode of the hydrogel photoelectrochemical / fluorescence dual-modal sensor into the object to be tested, then irradiating the working electrode with a laser source, extending the gold wires of the gel layer and the solidified layer at the other end of the working electrode and the reference electrode to connect with the electrochemical workstation, and then measuring the photocurrent and / or photopotential; or, immersing the working electrode of the hydrogel photoelectrochemical / fluorescence dual-modal sensor into the object to be tested, and then measuring the fluorescence signal with a fluorescence meter; the photoelectrochemical mode and the fluorescence mode both use the standard curve method to measure the content of persistent organic pollutants in the object to be tested; wherein, the object to be tested in the photoelectrochemical mode is an organism or a solution, and the object to be tested in the fluorescence mode is a solution.

9. The use according to claim 8, characterized in that: The POPs include DDT, chlordane, polychlorinated biphenyls, pentachlorophenol, hexachlorobenzene, dioxins, furans and lindane.

10. An unsupported hydrogel photoelectrochemical / fluorescence dual-modal sensing system, characterized in that: It comprises the hydrogel photoelectrochemical / fluorescence dual-modal sensor and a light source as described in claim 1, as well as a potentiometer and / or a fluorimeter; wherein, in the photoelectrochemical mode, the hydrogel photoelectrochemical / fluorescence dual-modal sensor is used to be inserted into the object to be detected, the light source is used to irradiate the working electrode, the working electrode and the reference electrode are respectively connected to the electrodes at both ends of the potentiometer, the potentiometer is used to record the photocurrent and photopotential signals, and the wavelength of the light source matches the absorption peak of the photoelectric material; in the fluorescence mode, the working electrode of the hydrogel photoelectrochemical / fluorescence dual-modal sensor is used to be immersed in the object to be detected, and the fluorimeter is used to record the fluorescence signal.

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