Preparation method of photoelectric biosensor and photoelectric biosensor

Abstract

The embodiment of the invention provides a preparation method of a photoelectric biosensor and the photoelectric biosensor, and relates to the technical field of photoelectric sensing, and the preparation method comprises the following steps: preparing a biomass derived hierarchical porous carbon PBAC material, preparing PBAC interface blending adsorption layer carbon slurry according to the biomass derived hierarchical porous carbon PBAC material, and preparing working electrode slurry; performing chemical etching on ITO conductive glass to prepare an electrode substrate, printing the working electrode slurry on the electrode substrate by using a screen printing technology, and printing PBAC interface blending adsorption layer carbon slurry on the electrode substrate to obtain a working electrode; a colloidal gold solution is dropwise added to the working electrode, after drying, a probe solution is dropwise added to the surface of the working electrode again, incubation is conducted in a constant-temperature box, and the photoelectrochemical biosensor is obtained after the reaction is completed. According to the invention, the biocompatibility of the photoelectric biosensor is enhanced, and the stability and sensitivity of the sensor are improved.

Description

Technical Field

[0001] The embodiments of the present disclosure relate to the field of photoelectric sensing technology, and more specifically, to a method for preparing a photoelectric biosensor and a photoelectric biosensor. Background Art

[0002] In actual use, high performance liquid chromatography, liquid chromatography-mass spectrometry, immunoassay and fluorescence spectrophotometry can be used to detect neurotransmitter markers such as 5-HT and dopamine. The above methods usually have high requirements for equipment cost, technology and experimental conditions, and may also have problems such as complex operation, large errors in measurement results and possible cross-reactions, and have poor universality. Photoelectrochemical biosensors have the advantages of simple device, convenient operation, low cost and easy miniaturization. At the same time, combined with the photoelectric reaction of semiconductor materials, they also have the characteristics of low background signal and higher sensitivity, which can realize the detection of neurotransmitter markers such as 5-HT and dopamine.

[0003] In the related art, the fixation methods for photoelectrochemical biosensor probes include: physical adsorption of two-dimensional materials, chemical cross-linking and covalent bonding, but they all have their own stability and reliability problems. For example, physical adsorption may be easily affected by environmental factors and fall off, chemical cross-linking may introduce new chemical reactions, and covalent bonding may change the active site of the probe. Therefore, the stability of the probe is poor and the performance of the sensor is poor. Summary of the invention

[0004] The purpose of the present disclosure is to provide a method for preparing a photoelectric biosensor and a photoelectric biosensor, thereby overcoming the problem of poor probe stability caused by the limitations and defects of related technologies to at least a certain extent.

[0005] According to one aspect of the present disclosure, there is provided a method for preparing a photoelectric biosensor, comprising:

[0006] Prepare biomass-derived hierarchical porous carbon PBAC material, prepare PBAC interface blending adsorption layer carbon slurry according to the biomass-derived hierarchical porous carbon PBAC material, and prepare working electrode slurry;

[0007] Chemically etching the ITO conductive glass to prepare an electrode substrate, printing the working electrode slurry on the electrode substrate using a screen printing technique, and printing the PBAC interface blending adsorption layer carbon slurry on the electrode substrate to obtain a working electrode;

[0008] A colloidal gold solution is dripped onto the working electrode, and after drying, a probe solution is dripped onto the surface of the working electrode again, and the mixture is incubated in a constant temperature box. After the reaction is complete, a photoelectrochemical biosensor is obtained.

[0009] In an exemplary embodiment of the present disclosure, the preparation of a biomass-derived hierarchical porous carbon PBAC material comprises:

[0010] The persimmon branches are crushed and dried by a high-speed crusher and then sieved for pretreatment to obtain a biomass precursor powder;

[0011] Carbonizing the biomass precursor powder by heating in a protective atmosphere or a vacuum environment to obtain one-step carbonized porous carbon;

[0012] The one-step carbonized porous carbon is mixed with an activator, and the mixture is activated by heating in the protective atmosphere to obtain the two-step carbonized porous carbon, so as to prepare the biomass-derived hierarchical pore porous carbon PBAC material.

[0013] In an exemplary embodiment of the present disclosure, the mass ratio of the one-step carbonized porous carbon to the activator is 1-1.5:1-4; the specific surface area of ​​the one-step carbonized porous carbon is 400-870 m 2 / g, the total volume of the pores is 0.8 to 1.2 cm 3 / g; the specific surface area of ​​the two-step carbonized porous carbon is 1057-2483m 2 / g, the total volume of the pores is 0.93~1.48cm 3 / g.

[0014] In an exemplary embodiment of the present disclosure, the ITO conductive glass is chemically etched to prepare an electrode substrate, the working electrode slurry is printed on the electrode substrate using a screen printing technique, and the PBAC interface blending adsorption layer carbon slurry is printed on the electrode substrate to obtain a working electrode, comprising:

[0015] Based on the screen printing technology, the ITO conductive glass is partially etched with etching paste to retain the conductive electrode shape, and chemical etching is performed. After removal, the etching paste on the surface of the ITO glass is cleaned to obtain an electrode substrate;

[0016] Printing the working electrode slurry on the working electrode position of the electrode substrate by screen printing, and calcining to obtain a working electrode;

[0017] The PBAC interface harmonizing adsorption layer carbon slurry is printed on the working electrode position through a coating process to prepare the working electrode; the PBAC interface harmonizing adsorption layer carbon slurry is prepared by mixing PBAC porous carbon material with an organic phase.

[0018] In an exemplary embodiment of the present disclosure, the process of preparing the working electrode slurry includes:

[0019] Mixing diethylene glycol butyl ether acetate, diethylene glycol butyl ether, sorbitan fatty acid ester, and ethyl cellulose to prepare a slurry liquid phase component;

[0020] The TiO2 powder and the liquid phase component are mixed in accordance with the above, and the TiO2 slurry is prepared by using a rotation and revolution stirring degassing machine;

[0021] Among them, the ratio of diethylene glycol butyl ether acetate, diethylene glycol butyl ether, anhydrous sorbitan fatty acid ester and ethyl cellulose is 1-1.2:1.1-1.2:0.02-0.03:0.6-0.4, and the viscosity range of the ethyl cellulose is 50-220 mPa.s; the mass ratio of TiO2 powder to the liquid phase component is 0.9-1.2:2.4-2.8.

[0022] In an exemplary embodiment of the present disclosure, the colloidal gold solution is dripped onto the working electrode, and after drying, a probe solution is dripped onto the surface of the working electrode again, and incubated in a constant temperature box, and a photoelectrochemical biosensor is prepared after the reaction is complete, comprising:

[0023] The colloidal gold solution is dripped onto the surface of the working electrode and then subjected to heat preservation treatment; the amount of the colloidal gold solution dripped is 10-15 μL / cm 2 ;

[0024] After preparing the probe solution and activating the probe solution, the activated probe solution is dripped onto the surface of the working electrode and incubated in a constant temperature box. After the probe fixation and binding reaction is completed, the photoelectrochemical biosensor is obtained.

[0025] In an exemplary embodiment of the present disclosure, the preparing a probe solution and activating the probe solution comprises:

[0026] dissolving the aptamer in a buffer solution to prepare the probe solution;

[0027] Adding a mixed solution of EDC and NHS to the probe solution to activate the probe solution;

[0028] Wherein, the activation time is 0.5 to 1.5 hours, and the activation temperature is 4 to 25°C.

[0029] In an exemplary embodiment of the present disclosure, the aptamer used in the probe solution includes: a thiolated 5-HT aptamer or a 5′-terminal thiolated dopamine aptamer;

[0030] The nucleic acid sequence of the thiolated 5-HT aptamer is: 5′-HS-SH-CTC TCG GGA CGACTG GTAGGCAGA TAG GGG AAG CTG ATT CGA TGC GTG GGT CGT CCC-3;

[0031] The nucleic acid sequence of the 5′-terminal thiol-modified dopamine aptamer is: 5′-(SH)-(CH2)6-GGG AAU UCC GCGUGU GCG CCG CGG AAG AGG GAA UAU AGA GGC CAG CAC AUA GUG AGG CCC UCC UCC C-3′.

[0032] In an exemplary embodiment of the present disclosure, the incubation temperature is 28-39° C., the humidity is 40-80%, and the incubation time is 1-2 hours.

[0033] According to one aspect of the present disclosure, a photoelectric biosensor is provided. The photoelectric biosensor is prepared according to any one of the above-mentioned methods for preparing a photoelectric biosensor.

[0034] In the technical solution provided in the embodiment of the present disclosure, on the one hand, since the working electrode slurry is printed on the electrode substrate, the PBAC interface harmonizing adsorption layer carbon slurry is also printed on the surface of the working electrode slurry by screen printing technology. The PBAC interface harmonizing adsorption layer carbon slurry can provide a suitable embedded space structure for the colloidal gold particles, firmly fix the gold nanoparticles, and provide sites for the precise and firm binding of the thiol aptamer probe, which is more conducive to the fixation of the probe, improves the stability and reliability of the probe, and also improves the accuracy of the number of probes. On the other hand, the pore structure of the PBAC interface harmonizing adsorption layer carbon slurry provides a fast channel for ion transport in the electrolyte, which is more conducive to the separation of photogenerated carriers and holes. The connected pore structure effectively reduces photon scattering, and at the same time has sufficient active surface, which effectively enhances the carrier separation efficiency and improves the sensitivity of the photoelectrochemical biosensor. On the other hand, since the sensor can be prepared using screen printing technology, the prepared working electrode has good uniformity, which simplifies the preparation steps of the sensor, facilitates assembly, reduces detection costs, enhances operability, is easy to manufacture and has good repeatability, and can be mass-produced, while ensuring the excellent linear range and sensitivity of the sensor.

[0035] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings herein are incorporated into the specification and constitute a part of the specification, illustrate embodiments consistent with the present disclosure, and together with the specification are used to explain the principles of the present disclosure. Obviously, the accompanying drawings described below are only some embodiments of the present disclosure, and for ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without creative work.

[0037] Figure 1 The flowchart schematically shows a method for preparing a photoelectric biosensor in an embodiment of the present disclosure.

[0038] Figure 2 The preparation process and reaction principle diagram of the photoelectric biosensor are schematically shown.

[0039] Figure 3 This is the X-ray diffraction spectrum of the PBAC material in Example 1.

[0040] Figure 4 This is the N2 adsorption-desorption curve of the PBAC material in Example 1.

[0041] Figure 5 This is a high-resolution scanning electron microscope image of the PBAC material in Example 1.

[0042] Figure 6 This is the photocurrent response diagram of the sensor prepared in Example 1.

[0043] Figure 7 This is a graph showing the photocurrent response test results of the TiO2 / PBAC / Au / 5-HT aptamer photoelectrochemical biosensor corresponding to Example 1 for testing 5-HT at different concentrations.

[0044] Figure 8 This is a graph showing the photocurrent response test results of the TiO2 / PBAC / Au / dopamine aptamer photoelectrochemical biosensor corresponding to Example 2 for testing dopamine at different concentrations.

[0045] Fig. 9 This is the photocurrent response diagram corresponding to Comparative Example 1.

[0046] Fig.10 This is a graph showing the photocurrent response test results of the TiO2 / Au / apt photoelectrochemical biosensor in Comparative Example 1 for different concentrations of 5-HT. DETAILED DESCRIPTION

[0047] Example embodiments will now be described more fully with reference to the accompanying drawings. However, example embodiments can be implemented in a variety of forms and should not be construed as being limited to the examples set forth herein; on the contrary, these embodiments are provided so that the present disclosure will be more comprehensive and complete, and the concepts of the example embodiments are fully conveyed to those skilled in the art. The described features, structures, or characteristics may be combined in one or more embodiments in any suitable manner. In the following description, many specific details are provided to provide a full understanding of the embodiments of the present disclosure. However, those skilled in the art will appreciate that the technical solutions of the present disclosure may be practiced while omitting one or more of the specific details, or other methods, components, devices, steps, etc. may be adopted. In other cases, known technical solutions are not shown or described in detail to avoid obscuring various aspects of the present disclosure.

[0048] The terms "a", "an", "the" and "said" used in this specification are used to indicate the presence of one or more elements / components, etc.; the terms "including" and "having" are used to express an open-ended inclusion and mean that there may be additional elements / components, etc. in addition to the listed elements / components, etc.; the terms "first" and "second" etc. are used only as labels and are not intended to limit the quantity of their objects.

[0049] Neurotransmitter markers such as serum 5-hydroxytryptamine (5-HT), dopamine or norepinephrine are widely present in mammalian tissues, especially in the cerebral cortex and synapses. Changes in markers may lead to some symptoms and problems. Therefore, the detection of neurotransmitter markers is extremely important.

[0050] In the related technology, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), immunoassay and fluorescence spectrophotometry are generally used to detect neurotransmitter markers. In the above methods, there are usually high requirements for equipment cost, technology and experimental conditions, and there may be problems such as complex operation, large errors in measurement results and possible cross-reactions, and the universality is poor. Photoelectrochemical biosensor has the advantages of simple device, convenient operation, low cost and easy miniaturization. At the same time, combined with the photoelectric reaction of semiconductor materials, it also has the characteristics of low background signal and higher sensitivity, which can realize the rapid and accurate detection of 5-HT and dopamine.

[0051] The precise fixation of photoelectrochemical biosensor probes has a crucial impact on the performance and application of the sensor. In some embodiments, commonly used fixation methods include: physical adsorption of two-dimensional materials, chemical cross-linking and covalent bonding, but they all have their own stability and reliability issues. For example, physical adsorption may be easily affected by environmental factors and fall off, chemical cross-linking may introduce new chemical reactions, and covalent bonding may change the active site of the probe.

[0052] In order to solve the above technical problems, the present disclosure provides a method for preparing a photoelectric biosensor, which is used to prepare a three-electrode integrated photoelectrochemical biosensor based on hierarchical porous carbon. Figure 1 As shown in , the preparation method of the photoelectric biosensor mainly includes the following steps:

[0053] In step S110, a biomass-derived hierarchical porous carbon PBAC material is prepared, a PBAC interface blending adsorption layer carbon slurry is prepared according to the biomass-derived hierarchical porous carbon PBAC material, and a working electrode slurry is prepared;

[0054] In step S120, the ITO conductive glass is chemically etched to prepare an electrode substrate, the working electrode slurry is printed on the electrode substrate using a screen printing technique, and the PBAC interface blending adsorption layer carbon slurry is printed on the electrode substrate to obtain a working electrode;

[0055] In step S130, a colloidal gold solution is dripped onto the working electrode, and after drying, a probe solution is dripped onto the surface of the working electrode again, and incubated in a constant temperature box. After the reaction is complete, a photoelectrochemical biosensor is obtained.

[0056] Next, the preparation method of the photoelectric biosensor in the embodiment of the present disclosure is described in detail.

[0057] In step S110, a biomass-derived hierarchical porous carbon PBAC material is prepared, a PBAC interface harmonizing adsorption layer carbon slurry is prepared based on the biomass-derived hierarchical porous carbon PBAC material, and a working electrode slurry is prepared.

[0058] In the embodiments of the present disclosure, the material for preparing the biomass-derived hierarchical porous carbon PBAC material can be a persimmon branch, and the persimmon branch can be a persimmon branch with a diameter greater than 0.5 cm. In some embodiments, the persimmon branch can be treated based on a two-step carbonization activation method to prepare the biomass-derived hierarchical porous carbon PBAC material.

[0059] Exemplarily, the preparation steps of the biomass-derived hierarchical porous carbon PBAC material include the following steps:

[0060] Step 1: grind and dry the persimmon branches in a high-speed grinder and then sieve them for pretreatment to obtain biomass precursor powder.

[0061] The rotation speed of high-speed pulverization is 30000-35000 r / min, and the pulverization can be performed in a high-speed pulverizer. The drying temperature is 100-150° C., and the drying time is 12-48 hours. The sieve for sieving pretreatment is 50-300 mesh. The particle size of the prepared biomass precursor powder is 50-270 μm.

[0062] Step 2: Carbonize the biomass precursor powder by heating in a protective atmosphere or vacuum environment to obtain one-step carbonized porous carbon.

[0063] The protective atmosphere includes at least one of argon, nitrogen, helium and neon. The temperature-raising carbonization conditions include: heating to 400-600°C at a heating rate of 1-10°C / min, and keeping at 400-600°C for 1-5h.

[0064] The specific surface area of ​​one-step carbonized porous carbon is 400~870m 2 / g, specific surface area refers to the total area per unit mass of material. The total volume of pores in one-step carbonized porous carbon is 0.8-1.2cm 3 / g, wherein the volume of the first size of micropores (<1nm) accounts for 1-8% of the total volume of the pores, the volume of the second size of mesopores (1-10nm) accounts for 34-57% of the total volume of the pores, and the volume of the third size of macropores (>10nm) accounts for 42-58% of the total volume of the pores. In addition, the one-step carbonized porous carbon also includes at least one impurity element of O, F, N, P, and S. Taking the total mass of the one-step carbonized porous carbon as 100%, the one-step carbonized porous carbon includes the following elemental components in mass percentage: C element 72-93%, O element 5-13%, F element 0-3%, N element 0-2%, P element 0-2.5%, S element 0-1.5%.

[0065] Step 3: Mix the one-step carbonized porous carbon with an activator, and activate it by heating in a protective atmosphere to obtain the two-step carbonized porous carbon to prepare a biomass-derived hierarchical porous carbon PBAC material.

[0066] The mass ratio of the one-step carbonized porous carbon to the activator is 1 to 1.5:1 to 4. The activator includes at least one of potassium hydroxide, sodium hydroxide, lithium hydroxide, sodium bicarbonate, potassium bicarbonate, lithium carbonate, sodium carbonate, potassium carbonate, ferrous chloride, ferric chloride, cupric chloride, cobalt chloride, zinc chloride, phosphoric acid, potassium dihydrogen phosphate, potassium hydrogen sulfate, sodium hydrogen sulfate, calcium hydroxide and zinc trifluoromethanesulfonate. The conditions for temperature activation in a protective atmosphere include: heating to 800 to 1000°C at a heating rate of 1 to 10°C / min, and keeping at 800 to 1000°C for 0.5 to 5h;

[0067] The specific surface area of ​​the two-step carbonized porous carbon is 1057~2483m 2 / g, the total volume of pores in the two-step carbonized porous carbon is 0.93~1.48cm 3 / g, wherein the volume of the first size of micropores (<1nm) accounts for 1-8% of the total volume of the pores, the volume of the second size of mesopores (1-10nm) accounts for 34-57% of the total volume of the pores, and the volume of the third size of macropores (>10nm) accounts for 42-58% of the total volume of the pores. The two-step carbonized porous carbon also includes at least one impurity element of O, F, N, P, and S. Taking the total mass of the two-step carbonized porous carbon as 100%, the two-step carbonized porous carbon includes the following elemental components in mass percentage: C element 78-95%, O element 5-13%, F element 0-3%, N element 0-2%, P element 0-2.5%, S element 0-1.5%.

[0068] As a further improvement of the above technical solution, the preparation step of the biomass-derived hierarchical porous carbon PBAC material also includes: acid washing the two-step carbonized porous carbon obtained by the temperature activation. The acid washing process includes: mixing the two-step carbonized porous carbon with the acid solution, and then performing solid-liquid separation. The volume ratio of the acid solution to the two-step carbonized porous carbon is 150-300:1-10. Among them, the acid solution includes at least one of a hydrochloric acid solution, a sulfuric acid solution, a nitric acid solution, an acetic acid solution, a perchloric acid solution, a hypochlorous acid solution and a hydrofluoric acid solution with a concentration of 0.1-1M. The mixing method adopts stirring mixing, the stirring rate is 500-1200r / min, and the stirring time is 0.5-2h. Solid-liquid separation includes at least one of filtration and centrifugal separation. Among them, the filtration is carried out using a qualitative filter paper with a pore size of 20-60μm.

[0069] Further, after the two-step carbonized porous carbon is acid-washed, the acid-washed two-step carbonized porous carbon can be washed with water, and the washed two-step carbonized porous carbon can be dried to obtain a biomass-derived hierarchical porous carbon PBAC material. Optionally, the washed porous carbon material (two-step carbonized porous carbon) can be dried at 60-100° C. for 1-24 hours.

[0070] After obtaining the biomass-derived hierarchical porous carbon PBAC material, a PBAC interface harmonic adsorption layer carbon slurry can be prepared based on the biomass-derived hierarchical porous carbon PBAC material. Exemplarily, the PBAC porous carbon represented by the biomass-derived hierarchical porous carbon PBAC material can be mixed with an organic phase to prepare a PBAC interface harmonic adsorption layer carbon slurry, i.e., a carbon slurry. In some embodiments, the PBAC interface harmonic adsorption layer carbon slurry can be prepared by stirring and mixing the biomass-derived hierarchical porous carbon PBAC material, polyvinylidene fluoride adhesive, and N-methylpyrrolidone in a mass ratio of 80 to 90:5 to 10:85 to 110.

[0071] In addition, working electrode slurry can also be prepared. The working electrode slurry here can be TiO2 working electrode slurry or other types of working electrode slurry. The preparation process of TiO2 working electrode slurry includes: mixing TiO2 powder with an organic phase to prepare TiO2 working electrode slurry. First, diethylene glycol butyl ether acetate, diethylene glycol butyl ether, dehydrated sorbitan fatty acid ester, and ethyl cellulose are uniformly mixed at a mass ratio of 1-1.2:1.1-1.2:0.02-0.03:0.6-0.4 at 60-80°C to prepare a slurry liquid phase component, wherein the viscosity of ethyl cellulose is in the range of 50-220 mPa.s. TiO2 powder and slurry liquid phase components are mixed in a mass ratio of 0.9-1.2:2.4-2.8, and a self-rotating revolution stirring degassing machine is used to prepare a uniform TiO2 slurry. The revolution speed of the TiO2 slurry is 1200-2000 rpm, the rotation speed is 15-30% of the fixed revolution speed, the stirring time is 60-600 s, the vacuum degree is -30-60 kPa, the particle size distribution of the TiO2 powder is 0.2-5 μm. The viscosity of the prepared TiO2 slurry is 10000-30000 cps.

[0072] In step S120, the ITO conductive glass is chemically etched to prepare an electrode substrate, the working electrode slurry is printed on the electrode substrate using screen printing technology, and the PBAC interface blending adsorption layer carbon slurry is printed on the electrode substrate to obtain a working electrode.

[0073] In the embodiment of the present disclosure, the electrode substrate may be a three-electrode substrate or other suitable electrode substrate. The steps of printing the electrode by screen printing technology to obtain the working electrode include:

[0074] Step 1: After the etching paste is stirred evenly, the ITO conductive glass is partially etched by screen printing technology to make it non-conductive, retaining the conductive electrode shape, and then chemically etching is performed in a high temperature environment. Finally, the etching paste on the surface of the ITO glass is removed and cleaned to obtain an electrode substrate, for example, an integrated three-electrode substrate.

[0075] The conductive electrode shape can be, for example, a three-electrode shape. The etching paste for cleaning the ITO glass surface includes: using a 0.1-1M NaOH or KOH aqueous solution for cleaning. When partially etching to make it non-conductive, the surface four-probe test resistance is greater than 100MΩ.

[0076] Step 2: Print the TiO2 working electrode slurry on the working electrode position of the integrated three-electrode substrate through a screen printing process, and finally perform a calcination treatment to obtain the TiO2 working electrode.

[0077] The screen printing technology uses a screen with mesh size of 180 to 300. The calcination treatment conditions include: heating to 300 to 490°C at a heating rate of 1 to 10°C / min, keeping at 300 to 490°C for 0.5 to 2 hours, and cooling to 50°C at a cooling rate of 1 to 10°C / min.

[0078] Step three: print the PBAC interface harmonizing adsorption layer carbon slurry on the position of the TiO2 working electrode through a coating process, so as to print the PBAC interface harmonizing adsorption layer carbon slurry on the upper surface of the TiO2 working electrode, and complete the construction of the probe fixing layer on the working electrode surface after heat preservation treatment to obtain the working electrode.

[0079] The coating process includes drip coating, spin coating, spray coating, mask coating, and screen printing. The viscosity of the PBAC interface blending adsorption layer carbon slurry is 8000-15000cps, and the screen used in the screen printing technology is 180-300 mesh.

[0080] In some embodiments, the PBAC interface blending adsorption layer carbon slurry is loaded with 15 mg / cm on the working electrode surface. 2 ~230mg / cm 2 The thickness of the prepared PBAC interface harmonic adsorption layer is 200nm to 1300nm, that is, the thickness of the PBAC interface harmonic adsorption layer carbon slurry printed on the electrode substrate is 200nm to 1300nm. The temperature of the heat preservation treatment is 80 to 150°C, and the heat preservation time is 0.5 to 2h.

[0081] Next, continue to refer to Figure 1 As shown in FIG. 1 , in step S130 , a colloidal gold solution is dripped onto the working electrode, and after drying, a probe solution is dripped onto the surface of the working electrode again, and the mixture is incubated in a constant temperature box, and a photoelectrochemical biosensor is obtained after the reaction is complete.

[0082] In the embodiment of the present disclosure, the photoelectrochemical biosensor may be a TiO2 / PBAC / Au / apt photoelectrochemical biosensor. Colloidal gold is dripped onto the working electrode and dried, and then a probe solution is dripped again. After the reaction is complete, the preparation steps of the TiO2 / PBAC / Au / apt photoelectrochemical biosensor include the following:

[0083] Step 1: Add colloidal gold solution dropwise onto the surface of the working electrode, and embed the gold particles into the pore structure of the graded pore porous carbon with the corresponding pore diameter after heat preservation.

[0084] The preparation process of the colloidal gold solution includes: heating 100-300 ml of 0.5-2% chloroauric acid aqueous solution to a boil, adding 0.2-3 ml of 0.5-1.5% reducing agent, stirring and boiling for 15-30 minutes, removing the solution after it changes from purple-red to transparent orange-red, and storing it at 2-8°C for use. The reducing agent includes: at least one of ascorbic acid, vitamin E, tea polyphenols, reduced glutathione, trisodium citrate, uric acid, trisodium citrate, sodium sulfite, sodium nitrite, and potassium citrate. The amount of colloidal gold solution dripped onto each working electrode is 3-10 μL. The heat preservation treatment includes: keeping the working electrode dripped with the colloidal gold solution at 80-150°C for 0.2-0.5h to completely dry the surface of the working electrode dripped with the colloidal gold solution.

[0085] Step 2: After the probe solution is activated, it is added dropwise onto the surface of the working electrode and incubated in a constant temperature box. After the probe fixation and binding reaction is complete, the TiO2 / PBAC / Au / apt photoelectrochemical biosensor is obtained.

[0086] The aptamer can be dissolved in a buffer solution to prepare a probe solution. The aptamer can be a thiolated 5-HT aptamer or a 5′-terminal thiolated dopamine aptamer. The nucleic acid sequence of the thiolated 5-HT aptamer is: 5′-HS-SH-CTCTCG GGA CGA CTG GTA GGC AGA TAG GGG AAG CTG ATT CGA TGC GTG GGT CGT CCC-3. The nucleic acid sequence of the 5′-terminal thiolated dopamine aptamer is: 5′-(SH)-(CH2)6-GGG AAU UCC GCG UGU GCG CCG CGGAAG AGG GAA UAU AGA GGC CAG CAC AUA GUG AGG CCC UCC UCC C-3′. The type of aptamer is determined according to the type of substance to be detected by the sensor. Different types of substances require different aptamers.

[0087] Furthermore, the aptamer probe can be dissolved in a 0.01-0.1M PBS buffer solution to prepare a probe solution with a concentration of 5-50 μg / ml. The amount of the probe solution added can be 10 μL, as long as it can cover the working electrode.

[0088] On this basis, the thiol probe solution can be activated, and the activation treatment steps include: adding 30-150 μL of a mixed solution of 2-5 mmol / L EDC and 2-8 mmol / L NHS to the probe solution with thiol groups, and activating at 4-25° C. for 0.5-1.5 h.

[0089] After the probe solution is activated, the working electrode to which the probe solution is added can be placed in a constant temperature and humidity incubator for incubation. The incubation conditions include: temperature of 28-39° C., humidity of 40-80%, and incubation time of 1-2 hours. Based on this, a photoelectrochemical biosensor can be prepared.

[0090] After obtaining the photoelectrochemical biosensor, the photocurrent of the photoelectrochemical biosensor can be tested. For example, the test substance can be dropped on the surface of the TiO2 / PBAC / Au / apt electrode, incubated in a 37°C incubator for 15 to 20 minutes, and then taken out. After rinsing off the unbound test substance with ultrapure water, a 0.01M PBS buffer solution is used as the test liquid, and a purple light bead with a wavelength of 365nm, 3V, and 0.5W is used for back illumination to test the photocurrent to test the photoelectrochemical performance of the sensor.

[0091] In the embodiment of the present disclosure, by Figure 2The photoelectrochemical biosensor was prepared by the method in the paper. The hierarchical pore structure PBAC porous carbon in the TiO2 / PBAC / Au / apt photoelectrochemical biosensor has moderate conductivity. The reason why carbon compounds can conduct electricity is mainly attributed to the large pie bonds formed by many carbon atoms, which allow electrons to shuttle freely in them. The proportion of sp2 hybridized carbon atoms in PBAC is relatively small. This characteristic gives it suitable conductivity, which helps to quickly separate photogenerated carriers and holes, and avoids the problem of semiconductor interface conduction caused by excessive conductivity. Secondly, the hierarchical pore structure of PBAC includes micropores (<1nm), mesopores (1-10nm) and macropores (>10nm). The three-dimensional interconnected pore structure effectively reduces photon scattering and has sufficient active surface, which effectively enhances the carrier separation efficiency. At the same time, its three-dimensional pores can provide a suitable embedded space structure for colloidal gold particles, firmly fix gold nanoparticles, and provide sites for the precise and firm binding of thiol-modified aptamer probes. Finally, its good bioaffinity is more conducive to the fixation and modification of active probes and the full infiltration of electrolytes into electrodes. At the same time, the pore structure provides a fast channel for ion transport in the electrolyte, which is more conducive to the separation of photogenerated carriers and holes, and improves the sensitivity of the photoelectrochemical biosensor. The integrated photoelectric biosensor based on hierarchical pore porous carbon adopted in the present invention has the characteristics of good bioaffinity, accurate probe binding quantity, pore structure embedded gold nanoparticles, and strong gold-sulfur bonding, avoiding the problems in the related art such as weak physical adsorption, inaccurate probe quantity, changes in individual chemical bonds and interface bioaffinity.

[0092] In the disclosed embodiment of the three-electrode integrated photoelectric biosensor based on hierarchical porous carbon, each step of electrode preparation adopts screen printing technology, and the prepared printed electrodes have good uniformity between batches and do not require complicated processing, which simplifies the preparation steps, reduces the detection cost, and has strong operability while ensuring excellent linear range and sensitivity of detection.

[0093] Next, the preparation method of the photoelectric biosensor disclosed in the present invention is specifically described in conjunction with examples.

[0094] Example 1

[0095] The preparation of the optoelectronic biosensor based on the integrated electrode includes the following specific steps:

[0096] Step 1: prepare biomass-derived hierarchical porous carbon PBAC material, and prepare PBAC interface harmonizing adsorption layer carbon slurry, which is PBAC carbon slurry.

[0097] The persimmon branches were crushed in a high-speed crusher at 35000r / min, dried at 120°C for 12h, and then passed through a 200-mesh sieve to obtain a biomass precursor powder with a particle size of about 75μm; the biomass precursor powder was placed in a tubular furnace under a N2 environment, and the temperature was increased to 600°C at a heating rate of 5°C / min. After being kept at 600°C for 2h, the powder was cooled to room temperature along with the furnace to obtain a persimmon branch-derived carbon material.

[0098] The persimmon branch derived carbon material and KOH were mixed evenly in a mass ratio of 1:4 and placed in a tube furnace under a N2 environment. The temperature was raised to 850°C at a heating rate of 5°C / min. After being kept at 850°C for 2 hours, the temperature was cooled to room temperature with the furnace. The activated material was repeatedly soaked in 1M HCl for 30 minutes, and then repeatedly filtered and washed with deionized water until the pH of the filtered supernatant was 7. The washed material was freeze-dried to obtain a biomass-derived hierarchical porous carbon PBAC material that can be used to prepare a slurry, namely, a persimmon branch derived hierarchical porous carbon PBAC material, whose specific surface area is 1898.8m 2 / g. The X-ray diffraction spectrum of the PBAC carbon material prepared according to Example 1 is as follows Figure 3 as shown in . Figure 4 This is the N2 adsorption-desorption curve of the PBAC material in Example 1 of the present invention. It can be seen that it has good adsorption capacity. Figure 5 This is a high-resolution scanning electron microscope image of the PBAC material in Example 1 of the present invention.

[0099] Further, a PBAC carbon slurry can be prepared. For example, 0.1 g of polyvinylidene fluoride and 0.87 g of persimmon branch-derived hierarchical porous carbon PBAC material are weighed and mixed evenly, and then 1.2 g of N-methylpyrrolidone is added, and the mixture is stirred in a homogenizer for 30 min to obtain a uniform PBAC interface harmonic adsorption layer carbon slurry, i.e., PBAC carbon slurry.

[0100] Step 2: Prepare integrated printed electrodes of PBAC material modified TiO2.

[0101] Using a 300-mesh three-electrode-shaped screen printing screen, the etching paste was screen-printed on the surface of the ITO conductive glass of 100 mm × 100 mm × 0.7 mm, and it was taken out after being kept at 150° C. for 30 minutes. The etching paste was used on the clean ITO glass surface cleaned with 0.1 M NaOH. The resistance of the ITO glass after etching was greater than 100 MΩ, and an electrode substrate was obtained. The electrode substrate can be an integrated three-electrode substrate.

[0102] Weigh 10.6g of diethylene glycol butyl ether acetate, 10.1g of diethylene glycol butyl ether, 0.52g of sorbitan fatty acid ester and 3.2g of ethyl cellulose, stir at 60°C to completely dissolve the ethyl cellulose, then add 1.3g of TiO2 powder with a particle size of 2-3μm after the solution is cooled, and use a rotation and revolution stirring degassing machine to prepare a slurry with an orbital speed of 1500rpm, rotation of 20% of the fixed orbital speed, stirring time of 600s, and vacuum degree of -50kPa to finally obtain TiO2 slurry.

[0103] Use a 250-mesh screen to screen-print TiO2 slurry once on the working electrode position of the three-electrode substrate, then keep it at 100°C for 30 minutes and take it out. Use a 200-mesh screen to screen-print PBAC interface blending adsorption layer carbon slurry once on the upper surface of the TiO2 working electrode, keep it at 100°C for 30 minutes and take it out. Then place the working electrode in a muffle furnace, heat it to 250°C at a heating rate of 5°C / min, keep it at 250°C for 50 minutes, and then cool it down to room temperature with the furnace, so that the PBAC carbon layer is firmly combined with the TiO2 electrode to obtain a PBAC-modified TiO2 working electrode. The PBAC loading on the working electrode surface is 26 mg / cm 2 .

[0104] Step 3: Add colloidal gold solution to the working electrode, assemble the probe after drying, and prepare the TiO2 / PBAC / Au / apt photoelectrochemical biosensor.

[0105] Exemplarily, 200 ml of 1% chloroauric acid aqueous solution is heated to boiling, 0.5 ml of 1% trisodium citrate is added, and the mixture is stirred and boiled for 15 minutes. After the solution changes from purple-red to transparent orange-red, the solution is removed and the prepared colloidal gold solution is stored at 4° C. for later use.

[0106] 5 μL of colloidal gold solution was dropped onto the surface of the TiO2 / PBAC electrode and kept at 100°C for 0.5 h to completely dry the electrode surface and completely embed the gold nanoparticles into the pore structure of PBAC to prepare a TiO2 / PBAC / Au electrode.

[0107] The method for preparing the probe solution may include: dissolving a 5-HT aptamer having a thiol-modified base sequence of HS-SH-5′-CTC TCGGGA CGA CTG GTA GGC AGA TAG GGG AAG CTG ATT CGA TGC GTG GGT CGT CCC-3′ in a 0.01 M PBS buffer solution to prepare a probe solution with a concentration of 10 μg / ml.

[0108] Then, 100 μL of a mixed solution of 5 mmol / L EDC and 5 mmol / L NHS in a volume ratio of 1:1 was added, and the probe solution was activated at 10°C for 1 hour. 5 μL of the activated probe solution was dripped onto the surface of the TiO2 / PBAC / Au electrode, and then the electrode was placed in a constant temperature and humidity incubator at a humidity of 75% and a temperature of 37°C for 1.5 hours. Finally, the TiO2 / PBAC / Au / serotonin aptamer photoelectrochemical biosensor for detecting 5-HT was prepared.

[0109] The photoelectric chemical biosensor prepared according to Example 1 was tested for photocurrent response, and the results were as follows: Figure 6 shown. Figure 6 The TiO2 photoelectrochemical biosensor, TiO2 / PBAC photoelectrochemical biosensor, TiO2 / PBAC / Au photoelectrochemical biosensor, and TiO2 / PBAC / Au / apt photoelectrochemical biosensor prepared according to Example 1 are shown.

[0110] The test used a 365nm wavelength, 3V, 0.5W purple lamp bead, and the lamp bead was 10mm away from the bottom of the integrated electrode. The electrolyte used to test the photocurrent response of the corresponding photoelectrochemical biosensor was 0.01M PBS buffer solution. The test results show that the photocurrent response of the TiO2 / PBAC sensing electrode is significantly higher than that of the TiO2 electrode, which indicates that the porous structure and conductivity of PBAC effectively enhance the interfacial electron transport. The photocurrent response of the TiO2 / PBAC / Au electrode prepared after colloidal gold composite is reduced, which indicates that the gold nanoparticles are embedded in the pore structure of porous carbon to hinder the interfacial reaction. The photocurrent response of the TiO2 / PBAC / Au / apt electrode after incubation with the aptamer is further reduced. Since the molecular size of the aptamer is about 1 to 5nm, the thiol aptamer is firmly combined with the nano-gold on the interface in the form of Au-S bonds, and then embedded in the hierarchical pore structure of the same scale, which cannot be oxidized and leads to a further reduction in the photocurrent.

[0111] In addition, photoelectrochemical sensing tests can be performed on the test object by using test liquids of different concentrations.

[0112] First, add 0.5 mg of 5-HT powder into 0.5 ml of 0.01 M PBS buffer solution to completely dissolve the 5-HT powder and prepare a 5-HT stock solution with a concentration of 1 mg / ml. Then dilute it with ultrapure water at pH = 7.4 to obtain 5-HT standard test solutions with concentrations of 1 fg / ml, 100 fg / ml, 10 pg / ml, 1 ng / ml, and 100 ng / ml, respectively.

[0113] Different concentrations of 5-HT standard test solutions were added to the surface of the sensing electrode, incubated in a 37°C incubator for 15 minutes, and then taken out. After rinsing off the unbound test substances with ultrapure water, the photocurrent corresponding to different concentrations was tested by the integrated photoelectrochemical biosensor. The results are as follows: Figure 7 As shown, the concentrations are 100ng / ml, 1ng / ml, 10pg / ml, 100fg / ml, and 1fg / ml, respectively. The linear detection range of the TiO2 / PBAC / Au / apt aptamer sensor for the analyte 5-hydroxytryptamine 5-HT is 1fg / ml to 100ng / ml, and the detection limit is 0.3fg / ml. It can be seen that the TiO2 / PBAC / Au / apt can stably and firmly fix the aptamer and has an ultra-high detection sensitivity for the analyte 5-HT.

[0114] Example 2

[0115] The same method as in Example 1 was used to prepare an integrated electrode-based photoelectric biosensor for detecting dopamine. The specific steps included the following:

[0116] Step 1: Use the PBAC carbon slurry prepared in Example 1.

[0117] Step 2: Prepare integrated printed electrodes of PBAC material modified TiO2.

[0118] Using a 300-mesh three-electrode-shaped screen printing screen, the etching paste was screen-printed on the 100mm×100mm×0.7mm ITO conductive glass surface, which was kept at 150°C for 30 minutes and then taken out. The etching paste was applied to the clean ITO glass surface cleaned with 0.1M NaOH. The resistance of the etched ITO glass was greater than 100MΩ, and an integrated three-electrode substrate was obtained.

[0119] Weigh 9.6g of diethylene glycol butyl ether acetate, 9.1g of diethylene glycol butyl ether, 0.62g of sorbitan fatty acid ester and 3.6g of ethyl cellulose, stir at 70°C to completely dissolve the ethyl cellulose, then add 1.4g of TiO2 with a particle size of 2-3μm after the solution is cooled, and use a rotation and revolution stirring degassing machine to prepare a slurry, with an orbital speed of 1500rpm, a rotation speed of 20% of the fixed orbital speed, a stirring time of 600s, and a vacuum degree of -50kPa to finally obtain TiO2 slurry.

[0120] The TiO2 slurry was screen-printed once on the working electrode position of the three-electrode substrate using a 250-mesh screen plate, and then kept at 100°C for 30 min before being taken out. The PBAC carbon slurry was screen-printed three times on the upper surface of the TiO2 electrode using a 200-mesh screen plate, and then kept at 100°C for 30 min before being taken out. The electrode was then placed in a muffle furnace and heated to 250°C at a heating rate of 5°C / min. After being kept at 250°C for 50 min, the temperature was cooled to room temperature with the furnace, so that the PBAC carbon layer was firmly combined with the TiO2 electrode to obtain a PBAC-modified TiO2 integrated printed electrode with a PBAC loading of 56 mg / cm on the electrode surface. 2 .

[0121] Step 3: Add colloidal gold solution to the working electrode, and after drying, add probe solution to assemble the probe to obtain the TiO2 / PBAC / Au / apt photoelectrochemical biosensor.

[0122] A colloidal gold solution was prepared using the method of Example 1. 7 μL of the colloidal gold solution was dripped onto the surface of the TiO2 / PBAC electrode and kept at 100°C for 0.5 h to completely dry the electrode surface and completely embed the gold nanoparticles into the PBAC pore structure to prepare a TiO2 / PBAC / Au electrode.

[0123] The dopamine aptamer with a thiol-modified base sequence of 5′-(SH)-(CH2)6-GGG AAU UCC GCG UGU GCG CCG CGGAAG AGG GAA UAU AGA GGC CAG CAC AUA GUG AGG CCC UCC UCC C-3′ was dissolved in 0.01 M PBS buffer solution to prepare a probe solution with a concentration of 10 μg / ml.

[0124] Then, 100 μL of a mixed solution of 5 mmol / L EDC and 5 mmol / L NHS in a volume ratio of 1:1 was added, and the probe solution was activated at 10°C for 1 hour. 5 μL of the activated aptamer probe solution was dropped onto the surface of the TiO2 / PBAC / Au electrode, and then the electrode was placed in a constant temperature and humidity incubator at a humidity of 75% and a temperature of 37°C for 1.5 hours. Finally, the TiO2 / PBAC / Au / dopamine aptamer photoelectrochemical biosensor for detection was prepared.

[0125] The linear detection region and detection limit of the TiO2 / PBAC / Au / dopamine aptamer sensor prepared according to Example 2 were measured using the same method as in Example 1. The results are as follows: Figure 8 As shown, Figure 8 The concentrations in the assay were 1 μg / ml, 100 ng / ml, 1 ng / ml, 10 pg / ml, and 100 fg / ml, respectively.

[0126] The linear detection range of the TiO2 / PBAC / Au / dopamine aptamer sensor for the analyte dopamine is 100fg / ml to 1μg / ml, and the detection limit is 35.6fg / ml. It can be seen that PBAC and nano-Au particles can stabilize and firmly fix the aptamer probe, and have an ultra-high detection sensitivity for the analyte dopamine.

[0127] Comparative Example 1

[0128] The preparation of the optoelectronic biosensor based on the integrated electrode includes the following specific steps:

[0129] Step 1: Prepare integrated printed electrodes of TiO2.

[0130] Using a 300-mesh three-electrode-shaped screen printing screen, the etching paste was screen-printed on the 100mm×100mm×0.7mm ITO conductive glass surface, which was kept at 150°C for 30 minutes and then taken out. The etching paste was applied to the clean ITO glass surface cleaned with 0.1M NaOH. The resistance of the etched ITO glass was greater than 100MΩ, and an integrated three-electrode substrate was obtained.

[0131] Weigh 10.6 g of diethylene glycol butyl ether acetate, 10.1 g of diethylene glycol butyl ether, 0.52 g of sorbitan fatty acid ester and 3.2 g of ethyl cellulose, stir at 60°C to completely dissolve the ethyl cellulose, add 1.3 g of TiO2 with a particle size of 2 to 3 μm, cool and stir for 30 minutes to obtain TiO2 slurry.

[0132] Use a 250-mesh screen to screen-print TiO2 slurry on the working electrode position of the three-electrode substrate, then keep it at 100℃ for 30 minutes, take it out, heat it to 250℃ at a heating rate of 5℃ / min, keep it at 250℃ for 50 minutes, and then cool it to room temperature with the furnace to allow the organic solvent to fully evaporate and TiO2 to firmly combine with the ITO conductive glass to prepare an integrated printed electrode of TiO2.

[0133] Step 2: After drying the integrated electrode composite colloidal gold, assemble the probe to obtain the TiO2 / Au / apt photoelectrochemical biosensor.

[0134] Heat 200 ml of 1% chloroauric acid aqueous solution to boiling, add 0.5 ml of 1% trisodium citrate, continue stirring and boil for 15 minutes, remove the solution after it changes from purple-red to transparent orange-red, and store the prepared colloidal gold solution at 4°C for later use.

[0135] 5 μL of colloidal gold solution was dropped onto the surface of the TiO2 / PBAC electrode and kept at 100°C for 0.5 h to completely dry the electrode surface to prepare a TiO2 / Au electrode.

[0136] The 5-HT aptamer with the thiol-modified base sequence of 5-′HS-SH-CTC TCG GGA CGA CTG GTA GGC AGA TAGGGG AAG CTG ATT CGA TGC GTG GGT CGT CCC-3′ was dissolved in 0.01M PBS buffer solution to prepare a probe solution with a concentration of 10μg / ml. 5μL of the activated aptamer probe solution was dripped onto the surface of the TiO2 / Au electrode, and then the electrode was placed in a constant temperature and humidity incubator at a humidity of 75% and a temperature of 37°C for 1.5h. Finally, the TiO2 / Au / apt photoelectrochemical biosensor for detecting 5-HT was prepared.

[0137] The photoelectric chemical biosensor prepared according to Comparative Example 1 was tested for photocurrent response. The results are as follows: Fig. 9 shown. Fig. 9 The TiO2 photoelectrochemical biosensor, TiO2 / Au photoelectrochemical biosensor and TiO2 / Au / apt photoelectrochemical biosensor prepared according to Comparative Example 1 are shown.

[0138] When testing the photocurrent response of the photoelectrochemical biosensor, a 365nm wavelength, 3V, 0.5W purple lamp bead was used, and the lamp bead was 10mm away from the bottom of the integrated electrode. The electrolyte used to test the photocurrent response of the corresponding photoelectrochemical biosensor was 0.01M PBS buffer solution. The photoelectric response of the TiO2 / Au electrode prepared after the colloidal gold composite was reduced, indicating that the gold nanoparticles hindered the interface reaction. The photocurrent response of the TiO2 / Au / apt electrode after incubation with the aptamer was further reduced, indicating that it could not be oxidized and the photocurrent was further reduced.

[0139] In addition, photoelectrochemical sensing tests can be performed on the test object by using different concentrations of the test liquid. First, 0.5 mg of 5-HT powder is added to 0.5 ml of 0.01 M PBS buffer solution to completely dissolve the 5-HT powder and prepare a 5-HT mother solution with a concentration of 1 mg / ml. Then, it is diluted with ultrapure water at pH = 7.4 to obtain 5-HT standard test solutions with concentrations of 1 fg / ml, 100 fg / ml, 10 pg / ml, 1 ng / ml, and 100 ng / ml, respectively.

[0140] Furthermore, 5-HT buffer solutions of different concentrations were added to the surface of the sensing electrode, incubated in a 37°C incubator for 15 min, and then taken out. After the unbound analytes were rinsed off with ultrapure water, the photocurrents corresponding to different concentrations were tested using an integrated photoelectrochemical biosensor. The results are as follows: Fig.10As shown, the concentrations are 100 ng / ml, 1 ng / ml, 10 pg / ml, 100 fg / ml, and 1 fg / ml, respectively.

[0141] The TiO2 / Au / apt aptamer sensor is unstable in detecting the analyte 5-hydroxytryptamine 5-HT, which means that without the adsorption layer material structure firmly fixed on the surface of the TiO2 electrode, the stability of the aptamer binding cannot be guaranteed, and thus the accurate and sensitive detection of the analyte cannot be achieved.

[0142] In the disclosed embodiment, the porous PBAC interface harmonic adsorption layer has good bioaffinity for biological probes, and its hierarchical pore structure is conducive to the synergistic effect of fixing colloidal gold nanoparticles to firmly fix the probe. When excited by ultraviolet light, PBAC significantly promotes the rapid separation of electron-hole pairs in TiO2 photoelectric active materials due to its excellent bioaffinity, interface adsorption capacity and suitable conductivity. This performance optimization can significantly improve the detection sensitivity of the sensor and expand the linear range of detection. In the disclosed embodiment, it has the characteristics of high bioaffinity, high sensitivity, easy manufacturing and good repeatability, and can be widely used in the fields of photoelectric sensing and biochemical analysis.

[0143] In the embodiment of the present disclosure, a photoelectric biosensor prepared according to the method of step S110 to step S130 is also provided. The photoelectric biosensor can be used to detect neurotransmitter markers such as 5-HT and dopamine. The photoelectric biosensor has the characteristics of high bioaffinity, high sensitivity, easy manufacturing and good repeatability, and can be widely used in the field of photoelectric sensing and biochemical analysis.

[0144] In addition, the above-mentioned figures are only schematic illustrations of the processes included in the method according to the exemplary embodiments of the present disclosure, and are not intended to be limiting. It is easy to understand that the processes shown in the above-mentioned figures do not indicate or limit the time sequence of these processes. In addition, it is also easy to understand that these processes can be performed synchronously or asynchronously, for example, in multiple modules.

[0145] Those skilled in the art will readily appreciate other embodiments of the present disclosure after considering the specification and practicing the invention disclosed herein. This application is intended to cover any modification, use or adaptation of the present disclosure, which follows the general principles of the present disclosure and includes common knowledge or customary techniques in the art that are not disclosed in the present disclosure. The specification and examples are intended to be exemplary only, and the true scope and spirit of the present disclosure are indicated by the appended claims.

[0146] It should be understood that the present disclosure is not limited to the exact structures that have been described above and shown in the drawings, and that various modifications and changes may be made without departing from the scope thereof. The scope of the present disclosure is limited only by the appended claims.

Claims

1. A method for preparing a photoelectric biosensor, characterized in that: include: Prepare biomass-derived hierarchical porous carbon PBAC material, prepare PBAC interface blending adsorption layer carbon slurry according to the biomass-derived hierarchical porous carbon PBAC material, and prepare working electrode slurry; Chemically etching the ITO conductive glass to prepare an electrode substrate, printing the working electrode slurry on the electrode substrate using a screen printing technique, and printing the PBAC interface blending adsorption layer carbon slurry on the electrode substrate to obtain a working electrode; A colloidal gold solution is dripped onto the working electrode, and after drying, a probe solution is dripped onto the surface of the working electrode again, and the mixture is incubated in a constant temperature box. After the reaction is complete, a photoelectrochemical biosensor is obtained.

2. The method for preparing the photoelectric biosensor according to claim 1, characterized in that: The method for preparing the biomass-derived hierarchical porous carbon PBAC material comprises: The persimmon branches are crushed and dried by a high-speed crusher and then pre-screened to obtain a biomass precursor powder; Carbonizing the biomass precursor powder by heating in a protective atmosphere or a vacuum environment to obtain one-step carbonized porous carbon; The one-step carbonized porous carbon is mixed with an activator, and the mixture is activated by heating in the protective atmosphere to obtain the two-step carbonized porous carbon, so as to prepare the biomass-derived hierarchical pore porous carbon PBAC material.

3. The method for preparing the photoelectric biosensor according to claim 2, characterized in that: The mass ratio of the one-step carbonized porous carbon to the activator is 1-1.5:1-4; the specific surface area of ​​the one-step carbonized porous carbon is 400-870 m 2 / g, the total volume of the pores is 0.8 to 1.2 cm 3 / g; the specific surface area of ​​the two-step carbonized porous carbon is 1057-2483m 2 / g, the total volume of the pores is 0.93~1.48cm 3 / g.

4. The method for preparing the photoelectric biosensor according to claim 1, characterized in that: The ITO conductive glass is chemically etched to prepare an electrode substrate, the working electrode slurry is printed on the electrode substrate using a screen printing technique, and the PBAC interface blending adsorption layer carbon slurry is printed on the electrode substrate to obtain a working electrode, comprising: Based on the screen printing technology, the ITO conductive glass is partially etched with etching paste to retain the conductive electrode shape, and chemical etching is performed. After removal, the etching paste on the surface of the ITO glass is cleaned to obtain an electrode substrate; Printing the working electrode slurry on the working electrode position of the electrode substrate by screen printing, and calcining to obtain a working electrode; The PBAC interface harmonizing adsorption layer carbon slurry is printed on the working electrode position through a coating process to prepare the working electrode; the PBAC interface harmonizing adsorption layer carbon slurry is prepared by mixing PBAC porous carbon material with an organic phase.

5. The method for preparing the photoelectric biosensor according to claim 1, characterized in that: The process of preparing the working electrode slurry comprises: Mixing diethylene glycol butyl ether acetate, diethylene glycol butyl ether, sorbitan fatty acid ester, and ethyl cellulose to prepare a slurry liquid phase component; The TiO2 powder and the liquid phase component are mixed in accordance with the above, and the TiO2 slurry is prepared by using a rotation and revolution stirring degassing machine; Among them, the ratio of diethylene glycol butyl ether acetate, diethylene glycol butyl ether, anhydrous sorbitan fatty acid ester and ethyl cellulose is 1-1.2:1.1-1.2:0.02-0.03:0.6-0.4, and the viscosity range of the ethyl cellulose is 50-220 mPa.s; the mass ratio of TiO2 powder to the liquid phase component is 0.9-1.2:2.4-2.

8.

6. The method for preparing the photoelectric biosensor according to claim 1, characterized in that: The colloidal gold solution is dripped onto the working electrode, and after drying, a probe solution is dripped onto the surface of the working electrode again, and incubated in a constant temperature box, and a photoelectrochemical biosensor is prepared after the reaction is complete, comprising: The colloidal gold solution is dripped onto the surface of the working electrode and then heat-insulated. The dripping amount of the colloidal gold solution is 10-15 μL / cm 2 ; After preparing the probe solution and activating the probe solution, the activated probe solution is dripped onto the surface of the working electrode and incubated in a constant temperature box. After the probe fixation and binding reaction is completed, the photoelectrochemical biosensor is obtained.

7. The method for preparing the photoelectric biosensor according to claim 6, characterized in that: The step of preparing the probe solution and activating the probe solution comprises: dissolving the aptamer in a buffer solution to prepare the probe solution; Adding a mixed solution of EDC and NHS to the probe solution to activate the probe solution; Wherein, the activation time is 0.5 to 1.5 hours, and the activation temperature is 4 to 25°C.

8. The method for preparing the photoelectric biosensor according to claim 1, characterized in that: The aptamers used in the probe solution include: thiolated 5-HT aptamers or 5′-terminal thiolated dopamine aptamers; The nucleic acid sequence of the thiolated 5-HT aptamer is: 5′-HS-SH-CTC TCG GGA CGACTG GTA GGCAGA TAG GGG AAG CTG ATT CGA TGC GTG GGT CGT CCC-3; The nucleic acid sequence of the 5′-terminal thiol-modified dopamine aptamer is: 5′-(SH)-(CH2)6-GGG AAU UCC GCG UGUGCG CCG CGG AAG AGG GAA UAU AGA GGC CAG CAC AUA GUG AGG CCC UCC UCC C-3′.

9. The method for preparing the photoelectric biosensor according to claim 6, characterized in that: The incubation temperature is 28-39° C., the humidity is 40-80%, and the incubation time is 1-2 hours.

10. A photoelectric biosensor, characterized in that: The photoelectric biosensor is prepared according to the method for preparing a photoelectric biosensor according to any one of claims 1 to 9.

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