Electro-biosensor and its preparation method

Abstract

The present disclosure provides an electro-biosensor and a preparation method thereof, relating to the field of biosensing technology. The electro-biosensor includes: a screen-printed electrode and a plurality of probes; the screen-printed electrode includes at least three working electrodes, a reference electrode and a counter electrode, the counter electrode is disposed within the region surrounded by the plurality of working electrodes, the reference electrode is located between the counter electrode and the working electrodes, and the reference electrode is disposed around each working electrode; each working electrode has a circular planar structure, and a carboxylated g-C3N4 thin film layer modified with ternary noble metals is formed on the surface of each working electrode, and the thin film layer includes a gradient composite structure formed by rhodium, platinum, and gold in sequence; the probes are arranged in one-to-one correspondence with the working electrodes, and at least some of the probes are adapted to different types of targets. The electro-biosensor has high biocompatibility and stability, can be used for multi-target detection, and improves the adaptability to complex analysis requirements.

Description

Technical Field

[0001] The present disclosure relates to the field of biosensing technology, and in particular, to a biosensor and a method for preparing the same. Background Art

[0002] An electrochemical biosensor (also known as an electro-biosensor) is an analytical device that combines a biorecognition element with an electrochemical transducer. It generates a detectable electrochemical signal through the specific interaction between the biorecognition element (such as enzymes, antibodies, DNA, cells, etc.) and the target analyte, thereby realizing quantitative or qualitative detection of the target analyte. Due to its high sensitivity, fast response speed and other characteristics, electro-biosensors have broad application prospects in the fields of medicine, environment, food, scientific research and industry.

[0003] Currently, due to the limitations of the materials and properties of the sensor materials of commonly used electro-biosensors, the stability of the sensor itself is poor, and the binding efficiency between the sensor and the biological probe is low, which in turn affects the detection sensitivity and detection results of the sensor, and the applicability of the sensor itself is poor, and it can only detect a single target analyte.

[0004] It should be noted that the information disclosed in the above background art section is only used to enhance the understanding of the background of the present disclosure, and therefore may include information that does not constitute the prior art known to those of ordinary skill in the art. Summary of the Invention

[0005] In view of this, an electro-biosensor and a method for preparing the same are provided. The electro-biosensor forms a ternary noble metal-modified carboxylated g-C3N4 thin film layer on the working electrode, so that each working electrode can be adapted to different types of probes. The electro-biosensor can be used for the detection of multiple target substances and has good stability and high detection sensitivity.

[0006] Other characteristics and advantages of the present disclosure will become apparent through the following detailed description, or will be partially learned through the practice of the present disclosure.

[0007] According to one aspect of the present disclosure, an electro-biosensor is provided, which includes:

[0008] A screen-printed electrode, the screen-printed electrode includes at least three working electrodes, a reference electrode and a counter electrode. The counter electrode is disposed in the region surrounded by the plurality of working electrodes. The reference electrode is located between the counter electrode and the working electrodes, and the reference electrode surrounds each of the working electrodes;

[0009] Among them, each of the working electrodes has a circular planar structure, and a carboxylated g-C3N4 thin film layer modified with ternary noble metals is formed on the surface of each of the working electrodes. The thin film layer includes a gradient composite structure formed by rhodium, platinum, and gold in sequence.

[0010] A plurality of probes, the probes are arranged in one-to-one correspondence with the working electrodes, the probes are used to detect target substances, and at least some of the probes are adapted to different types of target substances.

[0011] In an exemplary embodiment of the present disclosure, the thickness of the thin film layer is 100 nm to 500 nm, rhodium, platinum, and gold are formed in sequence from the direction of the working electrode pointing to the probe, and the mass ratios of rhodium, platinum, and gold doped in the thin film layer are 0.1% to 0.3%, 0.2% to 0.5%, and 0.3% to 0.6% in sequence.

[0012] In an exemplary embodiment of the present disclosure, the ratio of the distance between adjacent two working electrodes to the diameter of the working electrode is greater than or equal to three.

[0013] In an exemplary embodiment of the present disclosure, the part of the reference electrode surrounding each working electrode has a semi-wrapped concentric ring structure, the proportion of the reference electrode covering the circumference of the working electrode is 30% to 60%, and the area of the reference electrode is 15% to 20% of the total area of the working electrode.

[0014] In an exemplary embodiment of the present disclosure, the counter electrode adopts a continuously bent serpentine structure. The serpentine structure of the counter electrode includes at least 5 periodic bending units, the bending angle of each bending unit is 90° to 135°, and the width of the bending line of each bending unit is 0.5 mm to 1 mm.

[0015] According to another aspect of the present disclosure, a preparation method of an electro-biosensor is provided, and the preparation method includes:

[0016] Form an initial screen-printed electrode, the initial screen-printed electrode includes at least three initial working electrodes, a counter electrode, and a reference electrode. Among them, the reference electrode is located between the counter electrode and the initial working electrodes, and the part of the reference electrode surrounding each initial working electrode has a semi-wrapped concentric ring structure, and the counter electrode adopts a continuously bent serpentine structure;

[0017] Form a carboxylated g-C3N4 thin film layer modified with ternary noble metals on each of the initial working electrodes to form working electrodes, where the thin film layer includes a gradient composite structure formed by rhodium, platinum, and gold in sequence;

[0018] Probes are respectively formed on each of the working electrodes, and at least some of the probes are adapted to detect different types of targets.

[0019] In an exemplary embodiment of the present disclosure, forming a ternary noble metal-modified carboxylated g-C3N4 thin film layer on each of the initial working electrodes includes:

[0020] Preparing few-layer ternary noble metal-modified g-C3N4 powder;

[0021] Carboxylating the few-layer ternary noble metal-modified g-C3N4 powder to obtain a ternary noble metal-modified carboxylated g-C3N4 dispersion;

[0022] Spraying the ternary noble metal-modified carboxylated g-C3N4 dispersion on each of the initial working electrodes to form a ternary noble metal-modified carboxylated g-C3N4 thin film layer on each of the initial working electrodes.

[0023] In an exemplary embodiment of the present disclosure, the preparation of the few-layer ternary noble metal-modified g-C3N4 powder includes:

[0024] Preparing g-C3N4 precursor powder;

[0025] Placing the g-C3N4 precursor powder in a mixed solution and refluxing for a preset duration at a first preset temperature, wherein the mixed solution includes ethanol and glycerol with a volume ratio of not less than 3, the first preset temperature is 80°C to 90°C, and the preset duration is 4h to 8h;

[0026] During the reflux process, rhodium nitrate, chloroplatinic acid, and chloroauric acid are sequentially added to the mixed solution at intervals, and the addition amounts of rhodium nitrate, chloroplatinic acid, and chloroauric acid are respectively 0.1% to 0.3%, 0.2% to 0.5%, and 0.3% to 0.6% of the mass of the g-C3N4 precursor powder, and then suction filtration and drying are performed to obtain a ternary noble metal-modified g-C3N4 precursor powder;

[0027] Performing a first heat treatment on the ternary noble metal-modified g-C3N4 precursor powder to obtain few-layer ternary noble metal-modified g-C3N4 powder.

[0028] In an exemplary embodiment of the present disclosure, the first heat treatment includes:

[0029] Heating the ternary noble metal-modified g-C3N4 precursor powder to a first temperature and maintaining for a first duration to obtain ternary noble metal-modified g-C3N4 powder, wherein the first temperature is 550°C to 650°C, the first duration is 1h to 2h, and the heating rate is 2°C / min to 5°C / min;

[0030] Keep the ternary noble metal modified g-C3N4 powder at the second temperature for the second duration to obtain the few-layer ternary noble metal modified g-C3N4 powder, where the second temperature is 500°C to 550°C and the second duration is 1 h to 2 h.

[0031] In an exemplary embodiment of the present disclosure, the precursor powder for preparing g-C3N4 includes:

[0032] Dissolve melamine and urea powders in deionized aqueous solution, and then add a preset amount of solid phosphorous acid to obtain a nitrogen-rich g-C3N4 precursor aqueous solution, where the mass ratio of melamine to urea is greater than or equal to 2;

[0033] After performing a second heat treatment on the nitrogen-rich g-C3N4 precursor aqueous solution, perform suction filtration and rinsing to obtain the precursor powder of g-C3N4. The heat preservation temperature of the second heat treatment is 160°C to 180°C, and the heat preservation time is 12 h to 16 h.

[0034] The electro-biosensor provided by the present disclosure forms a ternary noble metal modified carboxylated g-C3N4 thin film layer on at least three working electrodes, and the thin film layer includes a gradient composite structure formed by rhodium, platinum, and gold in sequence. Through the multi-scale synergistic effect of rhodium, platinum, and gold, the biocompatibility of each working electrode can be improved, the binding efficiency and stability between the working electrode and the probe can be improved, the test sensitivity can be increased, and thus the accuracy of the detection result can be improved; this electro-biosensor can perform repetitive detections; in addition, since multiple working electrodes of this electro-biosensor can be matched with different types of target substances, simultaneous detection of multiple targets can be achieved, the detection efficiency is increased, and it has adaptability to various complex analysis requirements.

[0035] The preparation method of the electro-biosensor provided by the present disclosure can form a ternary noble metal modified carboxylated g-C3N4 thin film layer on multiple working electrodes in the electro-biosensor, and thus form an electro-biosensor with good stability and high sensitivity, which can be matched with different target substances and is suitable for multi-target detection.

[0036] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings here are incorporated into the specification and form a part of this specification, showing embodiments consistent with the present disclosure, and are used together with the specification to explain the principles of the present disclosure. Obviously, the accompanying drawings in the following description are only some embodiments of the present disclosure. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0038] Figure 1 Schematic diagram of the structure of the electro-biosensor with six working electrodes in the exemplary embodiments of the present disclosure.

[0039] Figure 2 Schematic diagram of the structure of the electro-biosensor with five working electrodes in the exemplary embodiments of the present disclosure.

[0040] Figure 3 Schematic diagram of the structure of the electro-biosensor with four working electrodes in the exemplary embodiments of the present disclosure.

[0041] Figure 4 Schematic diagram of the structure of the electro-biosensor with three working electrodes in the exemplary embodiments of the present disclosure.

[0042] Figure 5 Curve relationship diagram of the thin film layer thickness, probe adsorption amount and peak current in the exemplary embodiments of the present disclosure.

[0043] Figure 6 Curve relationship diagram of the rhodium doping ratio, peak current and base current in the exemplary embodiments of the present disclosure.

[0044] Figure 7 Curve relationship diagram of the platinum doping ratio and the working electrode impedance in the exemplary embodiments of the present disclosure.

[0045] Figure 8 Curve relationship diagram of the gold doping ratio and the probe adsorption amount in the exemplary embodiments of the present disclosure.

[0046] Figure 9 XRD pattern of the ternary noble metal modified carboxylated g-C3N4 thin film layer in the exemplary embodiments of the present disclosure.

[0047] Figure 10 SEM image of the ternary noble metal modified carboxylated g-C3N4 thin film layer in the exemplary embodiments of the present disclosure.

[0048] Figure 11 TEM image of the ternary noble metal modified carboxylated g-C3N4 thin film layer in the exemplary embodiments of the present disclosure.

[0049] Figure 12 STEM element distribution map of the ternary noble metal modified carboxylated g-C3N4 thin film layer in the exemplary embodiments of the present disclosure.

[0050] Figure 13 In the exemplary embodiments of the present disclosure Figure 12 Corresponding STEM element distribution map of rhodium in the ternary noble metal modified carboxylated g-C3N4 thin film layer.

[0051] Figure 14 In the exemplary embodiments of the present disclosure Figure 12 STEM elemental distribution map of platinum in the corresponding ternary noble metal-modified carboxylated g-C3N4 thin film layer.

[0052] Figure 15 In the exemplary embodiments of the present disclosure Figure 12 STEM elemental distribution map of gold in the corresponding ternary noble metal-modified carboxylated g-C3N4 thin film layer.

[0053] Figure 16 Raman test diagram of the ternary noble metal-modified carboxylated g-C3N4 thin film layer in the exemplary embodiments of the present disclosure.

[0054] Figure 17 Fourier transform infrared spectrum diagram of the ternary noble metal-modified carboxylated g-C3N4 thin film layer in the exemplary embodiments of the present disclosure.

[0055] Figure 18 Flow chart of the preparation method of the electro-biosensor in the exemplary embodiments of the present disclosure.

[0056] Among them, the reference signs are explained as follows:

[0057] W, working electrode; S1, reference electrode; S2, counter electrode. Detailed implementation manners

[0058] Now, the exemplary embodiments will be described more comprehensively with reference to the accompanying drawings. However, the exemplary embodiments can be implemented in various forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the exemplary embodiments to those skilled in the art. The same reference signs in the drawings denote the same or similar structures, and thus their detailed descriptions will be omitted. In addition, the drawings are only schematic illustrations of the present disclosure and are not necessarily drawn to scale.

[0059] Although relative terms such as "upper" and "lower" are used in this specification to describe the relative relationship of one component of the icon to another component, these terms are used in this specification only for convenience, for example, according to the directions of the examples described in the accompanying drawings. It can be understood that if the device of the icon is turned upside down, the component described as "upper" will become the component "lower". When a structure is "on" another structure, it may mean that a structure is integrally formed on another structure, or that a structure is "directly" disposed on another structure, or that a structure is "indirectly" disposed on another structure through another structure.

[0060] The terms "a", "an", "the", "said" and "at least one" are used to indicate the presence of one or more elements / components / etc.; the terms "comprising" and "having" are used to mean an open inclusion and refer to the possibility of the existence of additional elements / components / etc. in addition to the listed elements / components / etc.; the terms "first", "second", "third", etc. are used only as labels and do not limit the quantity of their objects.

[0061] In the related art, electrochemical biosensors can achieve high sensitivity and fast response, especially having unique advantages in the field of multi-target analysis. However, due to the limited electrochemical performance of the materials of existing sensors, the binding rate between the sensor and the biological probe reaches a bottleneck, making it difficult to achieve efficient detection of target analytes; in addition, existing sensors usually perform one-to-one detection with target analytes, resulting in low detection efficiency.

[0062] Based on this, embodiments of the present disclosure provide an electro-biosensor, as Figure 1 shown, combined with Figures 2 to 4 , the electro-biosensor includes: a screen-printed electrode and a plurality of probes.

[0063] Among them, the screen-printed electrode includes at least three working electrodes, a reference electrode and a counter electrode. The counter electrode is disposed within the area surrounded by the plurality of working electrodes. The reference electrode is located between the counter electrode and the working electrodes, and the reference electrode is disposed around each working electrode; among them, each working electrode has a circular planar structure, and a carboxylated g-C3N4 (Graphitic Carbon Nitride) thin film layer modified with ternary noble metals is formed on the surface of each working electrode. The thin film layer includes a gradient composite structure formed by rhodium, platinum, and gold in sequence; the probes are arranged in one-to-one correspondence with the working electrodes. The probes are used to detect targets, and at least some of the probes are adapted to different types of targets.

[0064] The electro-biosensor provided by the present disclosure, by providing at least three working electrodes, and a carboxylated g-C3N4 thin film layer modified with ternary noble metals is formed on each working electrode. The thin film layer includes a gradient composite structure formed by rhodium, platinum, and gold in sequence. Through the multi-scale synergistic effect of rhodium, platinum, and gold, the biocompatibility of each working electrode can be improved, the binding efficiency and stability between the working electrode and the probe can be improved, the test sensitivity can be increased, and thus the accuracy of the detection result can be improved; the electro-biosensor can perform repetitive detection; in addition, since the multiple working electrodes of the electro-biosensor can all match different types of targets, simultaneous detection of multiple targets can be achieved, the detection efficiency is improved, and it has adaptability to various complex analysis requirements.

[0065] The following will describe in detail each part of the electro-biosensor provided by the embodiments of the present disclosure with reference to the accompanying drawings:

[0066] In the embodiments provided by the present disclosure, the electro-biosensor includes a screen-printed electrode, and the screen-printed electrode includes at least three working electrodes, a reference electrode, and a counter electrode. In the following embodiments of the present disclosure, the electro-biosensor is simply referred to as a sensor. It should be understood that in addition to the working electrode, the counter electrode, and the reference electrode, the sensor provided by the present disclosure also includes other necessary structures for the normal operation of the sensor, such as electrode interfaces, etc. These necessary other structures are provided in the sensor of the present disclosure to provide a structural basis for the normal operation of the sensor. Details of these other necessary structures are not described herein.

[0067] The working electrode, the reference electrode, and the counter electrode can all be prepared by screen printing technology. The screen printing technology can improve the shape accuracy of each electrode preparation and the correctness of the relative position relationship, providing a structural basis for the subsequent preparation of the electro-biosensor.

[0068] Among them, the working electrode can be prepared with silver paste. Since silver paste has the characteristic of low resistance, it can improve the sensitivity of the sensor. The electro-biosensor also includes wires printed with silver paste for electrically connecting each working electrode to external devices. The working electrode has a circular planar structure, and this structure can solve the problem of uneven current density caused by the edge effect and improve the performance of each working electrode. Each working electrode has a circular planar structure. The diameters of multiple working electrodes can all be the same, can be partially the same, or can all be different. The sizes of multiple working electrodes can be selected and adaptively adjusted according to the actual design and usage requirements of the sensor. However, for the convenience of simplifying the manufacturing process and improving the versatility of the sensor, multiple working electrodes can adopt a structure with all the same diameter.

[0069] To meet the multi-target detection requirements of the sensor, the number of working electrodes is at least three. For example, the number of working electrodes can be three, four, five, six, or even more. The number of working electrodes can be determined according to the specific structural design requirements of the sensor and the number and types of target substances. The present disclosure does not make specific limitations. Multiple working electrodes can be arranged in an array to form an array-type electro-biosensor, which is more conducive to improving the reliability of multi-target detection.

[0070] Taking the structure with multiple working electrodes of the same diameter as an example, the ratio of the distance between two adjacent working electrodes to the diameter of the working electrode is greater than or equal to 3, which can effectively inhibit the overlap of the diffusion layers of adjacent electrodes and ensure the structural stability of the sensor. Specifically, the diameter of the working electrode can be 1 mm to 3 mm. For example, it can be 1 mm, 1.5 mm, 2 mm, 2.5 mm, or 3 mm, etc.; the distance between adjacent working electrodes can be 3 mm to 9 mm. For example, it can be 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, or 9 mm, etc. When the diameter of each working electrode and the distance between adjacent working electrodes are within the above ranges, it is necessary to ensure that the ratio of the distance between adjacent working electrodes to the diameter of the working electrode is greater than or equal to 3. For example, when the diameter of the working electrode is 2 mm, the distance between adjacent working electrodes can be 6 mm or more. It should be noted that the distance between two adjacent working electrodes in the present disclosure refers to the minimum distance between the two working electrodes.

[0071] A carboxylated g-C3N4 thin film layer modified with ternary noble metals is formed on the surface of each working electrode. The thin film layer includes a gradient composite structure formed by rhodium, platinum, and gold in sequence. Through the multi-scale cooperative mechanism among the ternary noble metals, the binding efficiency between the sensor and the probe is improved, and the detection sensitivity, stability, and reliability of the sensor are enhanced, thereby improving the overall performance of the sensor.

[0072] Among them, rhodium, platinum, and gold are formed in sequence from the working electrode towards the probe. During the formation of the thin film layer on the working electrode, first, a g-C3N4 layer is formed on the working electrode, and then rhodium is reductively deposited on the surface of the g-C3N4 layer to form a catalytically active core layer, which effectively promotes the proton reduction reaction and increases the sensitivity and selectivity of the sensor; then platinum is formed on the basis of rhodium to construct an electron transport channel, and carrier separation is achieved through the Schottky junction technology at the metal-semiconductor interface, thereby improving the speed and reliability of electron transport; finally, gold is formed on the basis of platinum. Gold forms a specific surface structure on the outermost layer, and its thiol group binding sites are conducive to the directional adsorption of probe molecules, so as to improve the binding efficiency between the working electrode and the probe, and further improve the detection sensitivity and stability of the sensor. In the present disclosure, the gradient composite structure formed by the ternary noble metals in the thin film layer can achieve cross-scale coupling through the exposure of the catalytic active sites of rhodium and the electron bridge effect of platinum. The ternary noble metals interact synergistically to improve the catalytic efficiency, electron transport efficiency, and probe binding efficiency. Compared with a single noble metal element, it is not a simple superposition of functions, but complements each other to jointly improve the functionality of the sensor.

[0073] Specifically, the mass ratio of rhodium doped in the thin film layer can be 0.1% to 0.3%. For example, it can be ratios such as 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, etc.Figure 6 As shown Figure 6 The figure shows the change curve of the peak current of the working electrode DPV (Differential Pulse Voltammetry) of rhodium at different doping ratios. It can be seen from the figure that as the rhodium doping ratio increases, the DPV peak current gradually increases, but the background signal dark current also increases accordingly. When the doping ratio of rhodium is about 0.2%, the DPV peak current reaches the maximum value, that is, the doping ratio of rhodium is 0.2% as the optimal ratio. It should be noted that when the mass ratio of rhodium doping in the thin film layer is in the range of 0.1% - 0.3%, rhodium has a good performance improvement effect on the working electrode.

[0074] The mass ratio of platinum doping in the thin film layer can be 0.2% - 0.5%, for example, it can be ratios such as 0.2%, 0.3%, 0.4%, 0.5%, etc., as Figure 7 shown Figure 7 The figure shows the impedance change curves of platinum at different doping ratios respectively. It can be seen from the figure that as the platinum doping ratio increases, the impedance arc radius decreases accordingly, and the electron transport effect is better. However, due to cost factors and the limitation of the component composition ratio, the doping ratio of platinum is 0.4% as the optimal ratio. It should be noted that when the mass ratio of platinum doping in the thin film layer is in the range of 0.2% - 0.5%, platinum has a good performance improvement effect on the working electrode.

[0075] The mass ratio of gold doping in the thin film layer can be 0.3% - 0.6%, for example, it can be ratios such as 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, etc., as Figure 8 shown Figure 8 The figure shows the adsorption amount of the probe by gold at different doping ratios respectively. It can be seen from the figure that as the gold doping ratio increases, the probe adsorption amount increases accordingly, and the probe adsorption effect is better. When the doping ratio of gold is 0.5%, the peak value of the probe adsorption amount is reached. Therefore, the doping ratio of gold is 0.5% as the optimal ratio. It should be noted that when the mass ratio of gold doping in the thin film layer is in the range of 0.3% - 0.6%, gold has a good performance improvement effect on the working electrode.

[0076] In addition, due to the introduction of the noble metal gold, the adsorption amount of the probe by the working electrode is increased, that is, the binding efficiency between the working electrode and the probe is improved, and this working electrode is applicable to various types of probes. For example, the probe can be one or more of an aptamer, an enzyme, and an antibody, which improves the applicable range of the sensor and the adaptability to complex detection tasks.

[0077] The thin film layer further includes carboxylated g-C3N4. By introducing carboxyl functional groups into g-C3N4, the biocompatibility of the sensor is further improved, and the stability and detection sensitivity of the sensor are enhanced. The carboxylated g-C3N4 is combined with ternary noble metal modification to form a thin film layer, which can improve the overall functionality of the sensor. Among them, the thickness of the thin film layer can be 100nm - 500nm. For example, it can be 100nm, 200nm, 300nm, 400nm, 500nm, etc. Figure 5 The curve graph showing the relationship between the thickness of the thin film layer and the probe adsorption amount is presented. It can be seen from the graph that as the thickness of the thin film layer increases, the probe adsorption amount increases, but the peak current will decrease. That is, due to the semiconductor property of the device, as the thickness of the thin film layer increases, the peak current of the sensor will decrease, resulting in a decrease in the sensitivity of the sensor. Therefore, in order to increase the probe adsorption amount while ensuring the sensitivity of the sensor, the thickness of the thin film layer is in the range of 100nm - 500nm. Further, when the thickness of the thin film layer is 200nm, the probe adsorption amount and the peak current can reach the best balance point, that is, the optimal thickness of the thin film layer is 200nm.

[0078] Among them, the reference electrode can be prepared with silver chloride. Since the reference electrode needs to provide a stable potential reference point for the working electrode in the sensor, the silver chloride reference electrode has a small potential drift and a fast response speed, which can improve the accuracy and reliability of the potential measurement of the working electrode.

[0079] The reference electrode is located between the counter electrode and the working electrode, and the reference electrode is arranged around each working electrode. The part of the reference electrode around each working electrode is in a semi-wrapped concentric ring structure to improve the uniformity and stability of the potential around each working electrode. Among them, the proportion of the circumference of the reference electrode covering the working electrode is 30% - 60%. For example, it can be 30%, 35%, 40%, 45%, 50%, 55%, 60%, etc. Within the above range, both the uniformity and stability of the potential around the working electrode are ensured, and the response time delay can also be reduced. Among them, the area of the reference electrode is 15% - 20% of the total area of the working electrode. For example, it can be 15%, 16%, 17%, 18%, 19%, 20%, etc., further ensuring the stability of the reference potential. By adjusting the ratio of the circumference and area between the reference electrode and the working electrode, the stability of the reference electrode can be ensured, and the performance of the sensor can be further improved.

[0080] Among them, the counter electrode can be prepared with carbon paste. Since the counter electrode needs to ensure the circuit closure and the stability of current flow in the sensor, the carbon counter electrode has high conductivity and chemical stability, which can ensure the circuit closure of the sensor and improve the stability of current flow.

[0081] The counter electrode is disposed within the region surrounded by multiple working electrodes. The counter electrode adopts a continuously bent serpentine structure, which reduces electrode polarization while increasing or decreasing the reaction current, thereby improving the accuracy and stability of detection. Among them, the serpentine structure of the counter electrode can include at least 5 periodic bending units, the bending angle of each bending unit is 90° - 135°, and the width of the bending line of each bending unit is 0.5mm - 1mm, which improves the service life of the counter electrode while enhancing its functionality. In addition, the overall shape of the serpentine structure of the counter electrode needs to match the working electrode and the reference electrode. On the one hand, it can avoid structural interference between the electrodes, which may lead to a decline in the sensor function. On the other hand, it can also improve the compactness of the sensor structure.

[0082] In some specific embodiments, such as Figures 1 to 4 shown, the numbers of the working electrodes of the sensor are 3, 4, 5, and 6 respectively. Among them, as Figure 1 shown, when the number of working electrodes is 6, the 6 working electrodes are evenly and symmetrically distributed on both sides of the counter electrode, and each working electrode adopts a circular structure with the same diameter. The reference electrode is located between the counter electrode and the working electrodes. The counter electrode and the reference electrode are axially symmetric structures, and the reference electrode semi-wraps each working electrode respectively. The counter electrode is in a continuously bent first serpentine structure. The symmetric structure of the sensor can improve the stability and practicality of the device, reduce the detection error, improve the performance of the device, and facilitate the simplification of the manufacturing process.

[0083] Such as Figure 2 shown, when the number of working electrodes is 5, the counter electrode and the reference electrode with the same structure as the six working electrodes can be adopted. When setting the positions of the working electrodes, one working electrode can be reduced on the basis of the six-working-electrode structure. It should be noted that when the number of working electrodes is 5, the reference electrode and the counter electrode with different structures from the six working electrodes can also be adopted, and the structures of the reference electrode and the counter electrode can be adaptively adjusted and changed to adapt to the change in the number of working electrodes.

[0084] Such as Figure 3 shown, when the number of working electrodes is 4, the 4 working electrodes are evenly and symmetrically distributed on both sides of the counter electrode, and each working electrode adopts a circular structure with the same diameter. The reference electrode is located between the counter electrode and the working electrodes. The counter electrode and the reference electrode are axially symmetric structures, and the reference electrode semi-wraps each working electrode respectively. The counter electrode is in a continuously bent second serpentine structure. Among them, the second serpentine structure is different from the first serpentine structure, and the number of bending units of the second serpentine structure is less than that of the first serpentine structure.

[0085] Such as Figure 4As shown, when the number of working electrodes is three, a counter electrode and a reference electrode with the same structure as the four working electrodes can be used. When setting the positions of the working electrodes, one working electrode can be reduced on the basis of the structure of the four working electrodes. It should be noted that when the number of working electrodes is three, a reference electrode and a counter electrode with different structures from the four working electrodes can also be used, and the structures of the reference electrode and the counter electrode can be adaptively adjusted and changed to adapt to the change in the number of working electrodes.

[0086] In the embodiments provided by the present disclosure, the electro-biosensor includes a plurality of probes, and the probes are arranged in one-to-one correspondence with the working electrodes. The probes are used to detect target substances, and at least some of the probes are adapted to different types of target substances. Among them, the probes can be one or more of aptamers, antibodies, and enzymes. The probes are arranged on the working electrodes. Since there are multiple working electrodes in the sensor, the probes can be arranged on all the working electrodes, or on some of the working electrodes. However, in order to improve the utilization rate of the sensor, usually probes are arranged on each working electrode. Among them, at least some of the probes are adapted to different types of target substances to enable the sensor to detect different types of target substances and improve the adaptability of the sensor to complex detection requirements and complex analysis requirements.

[0087] The electro-biosensor provided by the present disclosure, by setting at least three working electrodes, and a ternary noble metal-modified carboxylated g-C3N4 thin film layer is formed on each working electrode. The thin film layer includes a gradient composite structure formed by rhodium, platinum, and gold in sequence. Through the multi-scale synergistic effect of rhodium, platinum, and gold, the biocompatibility of each working electrode can be improved, the binding efficiency and stability between the working electrode and the probe can be improved, the test sensitivity can be increased, and thus the accuracy of the detection result can be improved; the electro-biosensor can perform repetitive detection; in addition, since multiple working electrodes of the electro-biosensor can all match different types of target substances, simultaneous detection of multiple targets can be achieved, the detection efficiency is improved, and it has adaptability to various complex analysis requirements and complex detection requirements.

[0088] The embodiments of the present disclosure provide a preparation method of an electro-biosensor, as Figure 18 shown, the preparation method of the electro-biosensor includes: step S10 to step S30.

[0089] Among them, step S10: forming an initial screen-printed electrode, the initial screen-printed electrode includes at least three initial working electrodes, a counter electrode, and a reference electrode. Among them, the reference electrode is located between the counter electrode and the initial working electrodes, and the part of the reference electrode surrounding each initial working electrode has a semi-wrapped concentric ring structure, and the counter electrode adopts a continuously bent serpentine structure;

[0090] Step S20: Form a ternary noble metal-modified carboxylated g-C3N4 thin film layer on each initial working electrode to form a working electrode, wherein the thin film layer includes a gradient composite structure formed by rhodium, platinum, and gold in sequence;

[0091] Step S30: Form probes on each working electrode, and at least some of the probes are adapted to different types of detection targets.

[0092] The preparation method of the electro-biosensor provided by the present disclosure can form a ternary noble metal-modified carboxylated g-C3N4 thin film layer on multiple working electrodes in the electro-biosensor, and then form an electro-biosensor with good stability and high sensitivity. It can match various types of target substances, be suitable for multi-target detection, improve the adaptability of the sensor to complex detection requirements and complex analysis requirements, and thus improve the performance of the sensor.

[0093] It should be noted that the preparation method provided by the present disclosure can be used to prepare any electro-biosensor provided in the above embodiments.

[0094] The following details each step of the preparation method of the electro-biosensor provided by the embodiments of the present disclosure:

[0095] In the embodiment provided by the present disclosure, in step S10, an initial screen-printed electrode is formed. The initial screen-printed electrode includes at least three initial working electrodes, a counter electrode, and a reference electrode.

[0096] Among them, the initial screen-printed electrode is prepared by a screen-printing process. Each initial working electrode is prepared with silver paste, the reference electrode is prepared with silver chloride, and the counter electrode is prepared with carbon paste. In addition, it also includes silver paste wires prepared by a screen-printing process for realizing the electrical connection between the working electrode and external devices.

[0097] Each initial working electrode has a circular planar structure. The reference electrode is formed between the counter electrode and the initial working electrode, and the reference electrode surrounds each initial working electrode. The part of the reference electrode surrounding each initial working electrode has a semi-wrapped concentric ring structure. The counter electrode is arranged in the area surrounded by multiple initial working electrodes, and the counter electrode adopts a continuously bent serpentine structure. The specific shapes and structural parameters of the counter electrode and the reference electrode are the same as the data provided in the above embodiments, and will not be repeated here.

[0098] In the embodiment provided by the present disclosure, in step S20, a ternary noble metal-modified carboxylated g-C3N4 thin film layer is formed on each initial working electrode to form a working electrode.

[0099] Forming a ternary noble metal-modified carboxylated g-C3N4 thin film layer on each initial working electrode includes steps S21 to S23.

[0100] Among them, in step S21, prepare few-layer ternary noble metal-modified g-C3N4 powder; in step S22, carboxylate the few-layer ternary noble metal-modified g-C3N4 powder to obtain a ternary noble metal-modified carboxylated g-C3N4 dispersion; in step S23, spray the ternary noble metal-modified carboxylated g-C3N4 dispersion on each initial working electrode to form a ternary noble metal-modified carboxylated g-C3N4 thin film layer on each initial working electrode.

[0101] In step S21, the preparation of the few-layer ternary noble metal-modified g-C3N4 powder includes steps S211 to S214.

[0102] Step S211, prepare g-C3N4 precursor powder; step S212, place the g-C3N4 precursor powder in a mixed solution and reflux for a preset time at a first preset temperature; step S213, during the reflux process, sequentially and intermittently add rhodium nitrate, chloroplatinic acid, and chloroauric acid to the mixed solution, and the addition amounts of rhodium nitrate, chloroplatinic acid, and chloroauric acid are 0.1% - 0.3%, 0.2% - 0.5%, and 0.3% - 0.6% of the mass of the g-C3N4 precursor powder respectively, and then perform suction filtration and drying to obtain ternary noble metal-modified g-C3N4 precursor powder; step S214, perform a first heat treatment on the ternary noble metal-modified g-C3N4 precursor powder to obtain few-layer ternary noble metal-modified g-C3N4 powder.

[0103] Among them, in step S211, the preparation of the g-C3N4 precursor powder includes steps S2111 to S2112. Among them, in step S2111, dissolve melamine and urea powders in deionized aqueous solution, and then add a preset amount of solid phosphorous acid to obtain a nitrogen-rich g-C3N4 precursor aqueous solution, where the mass ratio of melamine to urea is greater than or equal to 2; in step S2112, after performing a second heat treatment on the nitrogen-rich g-C3N4 precursor aqueous solution, perform suction filtration and rinsing to obtain the precursor powder of g-C3N4. The heat preservation temperature of the second heat treatment is 160°C - 180°C, and the heat preservation time is 12h - 16h.

[0104] In step S2111, after dissolving melamine and urea powders in deionized aqueous solution and then adding a preset amount of solid phosphorous acid, the resulting mixed solution needs to be stirred at a temperature of 80°C, and the stirring time can be 30min - 60min to ensure the uniformity of the mixed solution. Among them, the mass of the preset amount of solid phosphorous acid can be 25% - 50% of the total mass of melamine and urea, which can promote the polycondensation reaction and also avoid affecting the subsequent loading of ternary metals due to excessive residual phosphorous acid.

[0105] In step S2112, the second heat treatment can specifically be heating an aqueous solution of a nitrogen-rich g-C3N4 precursor in a hydrothermal reaction kettle to 160°C to 180°C and holding for 12 h to 16 h. The product after the second heat treatment can be subjected to suction filtration and repeatedly rinsed with ultrapure water to obtain a g-C3N4 precursor powder.

[0106] Among them, in step S212, the mixed solution includes ethanol and glycerol with a volume ratio of not less than 3. Due to the characteristics of ethanol having a low boiling point and glycerol having high viscosity, the mixed solution can form a gradient heat transfer system, which is beneficial to improving the dispersion uniformity of the g-C3N4 precursor powder during the reflux process; in addition, the strong coordination ability of the glycerol hydroxyl group can effectively anchor metal ions, inhibit nanoparticle aggregation, improve the diffusion uniformity of subsequent metal ions, and thus improve the formation quality of the subsequent thin film layer. The first preset temperature is the reflux temperature, which can be 80°C to 90°C, and the preset duration is the reflux time, which can be 4 h to 8 h. The reflux temperature and reflux time can be adaptively adjusted according to the actual process design.

[0107] In step S213, during the reflux process, rhodium nitrate, chloroplatinic acid, and chloroauric acid are added to the mixed solution at intervals in sequence. Specifically, during the reflux process, after the temperature rises to the first preset temperature, rhodium nitrate with a mass ratio of 0.1% to 0.3%, chloroplatinic acid with a mass ratio of 0.2% to 0.5%, and chloroauric acid with a mass ratio of 0.3% to 0.6% are added to the mixed solution at intervals of every 5 min to 10 min. The obtained product is subjected to suction filtration and drying treatment to obtain a ternary noble metal-modified g-C3N4 precursor powder.

[0108] In step S214, the ternary noble metal-modified g-C3N4 precursor powder is subjected to a first heat treatment to obtain few-layer ternary noble metal-modified g-C3N4 powder. Among them, the first heat treatment includes: heating the ternary noble metal-modified g-C3N4 precursor powder to a first temperature and holding for a first duration to obtain ternary noble metal-modified g-C3N4 powder, where the first temperature is 550°C to 650°C, the first duration is 1 h to 2 h, and the heating rate is 2°C / min to 5°C / min; holding the ternary noble metal-modified g-C3N4 powder at a second temperature for a second duration to obtain few-layer ternary noble metal-modified g-C3N4 powder, where the second temperature is 500°C to 550°C and the second duration is 1 h to 2 h.

[0109] In step S22, the few-layer ternary noble metal-modified g-C3N4 powder is carboxylated to obtain a ternary noble metal-modified carboxylated g-C3N4 dispersion.

[0110] Among them, the carboxylation process includes: placing the obtained few-layer ternary noble metal-modified g-C3N4 powder into a citric acid solution with a mass fraction of 1% - 5%, where the mass ratio of the few-layer ternary noble metal-modified g-C3N4 powder to the citric acid solution ≤ 100, to obtain a mixed solution of the two; then placing the mixed solution into a reaction kettle for hydrothermal reaction, with the reaction conditions being a temperature of 120°C - 160°C and a time of 6h - 8h; filtering and rinsing the reaction product, and drying it at 60°C for 8h to obtain ternary noble metal-modified carboxylated g-C3N4 powder; dispersing the ternary noble metal-modified carboxylated g-C3N4 powder in ultrapure water with a powder mass fraction of 1% - 2%, and performing ultrasonic dispersion for 30min - 60min to obtain a ternary noble metal-modified carboxylated g-C3N4 dispersion.

[0111] The obtained ternary noble metal-modified carboxylated g-C3N4 thin film layer is as Figures 9 to 17 shown, as Figure 9 shown, it can be seen that hydrothermal carboxylation does not affect the crystal structure of g-C3N4; as Figure 10 and Figure 11 shown, they are respectively the microscopic morphology diagrams of ternary noble metal-modified carboxylated g-C3N4, and it can be seen that a better carboxyl group is formed in the thin film layer; Figure 12 , Figure 13 , Figure 14 and Figure 15 are the distributions of ternary noble metals under TEM, and it can be seen that the concentrations of the three noble metals are distributed from low to high in the order of rhodium, platinum, and gold; as Figures 16 to 17 shown, it can be seen that carboxyl groups are formed on the ternary noble metal-modified g-C3N4 powder after hydrothermal treatment.

[0112] In step S23, the ternary noble metal-modified carboxylated g-C3N4 dispersion is sprayed on each initial working electrode to form a ternary noble metal-modified carboxylated g-C3N4 thin film layer on each initial working electrode. Specifically, 1 ml - 2 ml of the ternary noble metal-modified carboxylated g-C3N4 dispersion is aspirated and sprayed on the initial working electrode one by one to form a ternary noble metal-modified carboxylated g-C3N4 thin film layer on each initial working electrode.

[0113] In the embodiments provided by the present disclosure, in step S30, probes are respectively formed on each working electrode, and at least some of the probes are adapted to detect different types of targets. Specifically, a probe solution is obtained, and the probe solution can be one or more of aptamers, antibodies, enzymes, etc.; the probes corresponding to the target are dispersed in a phosphate buffer solution, and then dropped onto the working electrode with a ternary noble metal-modified carboxylated g-C3N4 thin film layer by a drop-casting process. After incubation at 37°C for 2h, a final electro-biosensor is obtained. Among them, a protein-free blocking solution can be used to block the non-specific binding sites of the probes.

[0114] The preparation method of the electro-biosensor provided by the present disclosure can form a ternary noble metal-modified carboxylated g-C3N4 thin film layer on multiple working electrodes in the electro-biosensor, thereby forming an electro-biosensor with good stability and high sensitivity. It can match various types of target substances, is suitable for multi-target detection, improves the adaptability of the sensor to complex detection requirements and complex analysis requirements, and thus improves the performance of the sensor.

[0115] It should be noted that although the steps of the preparation method of the electro-biosensor in the present disclosure are described in a specific order in the drawings, this does not require or imply that these steps must be executed in this specific order, or that all the steps shown must be executed to achieve the desired result. Additionally or alternatively, certain steps can be omitted, multiple steps can be combined into one step for execution, and / or one step can be decomposed into multiple steps for execution, etc.

[0116] The following uses specific examples to illustrate the electro-biosensor provided by the present disclosure, its preparation method, and applications:

[0117] Example 1

[0118] Step 1: Place a PET film cut into 3 cm × 6 cm in ultrapure water and ultrasonicate for 30 min. Use silver paste to print the wire and six initial working electrodes, a reference electrode printed with silver chloride, and a counter electrode printed with carbon paste.

[0119] Step 2: Prepare ternary noble metal-modified carboxylated g-C3N4 powder.

[0120] Dissolve 1.5 g of melamine and 0.5 g of urea powder in 100 ml of deionized water to form a solution, then add 1.0 g of solid phosphorous acid and stir at 80 °C for 30 min until the solution is clear to obtain an aqueous solution of nitrogen-rich g-C3N4 precursor;

[0121] Place the aqueous solution of g-C3N4 precursor in a hydrothermal reaction kettle and heat it to 180 °C, and keep it warm for 16 h;

[0122] Filter the product obtained by hydrothermal treatment by suction filtration and repeatedly rinse it with ultrapure water to obtain g-C3N4 precursor powder;

[0123] Weigh 1 g of the g-C3N4 precursor powder and place it in a mixed solution of 100 ml of ethanol and glycerol. Reflux at 90 °C for 8 h, where the volume ratio of ethanol to glycerol is 4. During the reflux process, after heating to the set temperature, add rhodium nitrate, chloroplatinic acid, and chloroauric acid successively every 10 min. The amounts of the three metals added are 0.3%, 0.5%, and 0.6% of the mass of the g-C3N4 precursor powder respectively. Filter the obtained product by suction and dry it to obtain the g-C3N4 precursor powder modified with ternary noble metals;

[0124] Place the g-C3N4 precursor powder modified with ternary noble metals in a muffle furnace, heat it to 600 °C and hold for 2 h, with a heating rate of 5 °C / min, to obtain the g-C3N4 modified with ternary noble metals;

[0125] Place the g-C3N4 modified with ternary noble metals in a muffle furnace at 550 °C and hold for 1 h to obtain the few-layer g-C3N4 powder modified with ternary noble metals;

[0126] Take 0.1 g of the few-layer g-C3N4 powder modified with ternary noble metals and place it in 150 ml of a 5% citric acid solution by mass. Then place the mixed solution in a reaction kettle and hydrothermally react at 120 °C for 8 h. Filter the obtained product by suction, wash it, and dry it at 60 °C for 8 h to obtain the carboxylated g-C3N4 powder modified with ternary noble metals.

[0127] Step 3: Disperse the carboxylated g-C3N4 powder modified with ternary noble metals in ultrapure water, with the powder mass fraction being 2%, and ultrasonically disperse for 60 min to obtain the carboxylated g-C3N4 dispersion modified with ternary noble metals.

[0128] Step 4: Pipette 2 ml of the carboxylated g-C3N4 dispersion modified with ternary noble metals and spray it one by one onto the initial working electrode to form a working electrode with a carboxylated g-C3N4 thin film layer modified with ternary noble metals.

[0129] Step 5: Modify the aptamer probes corresponding to the six tumor markers on the six working electrodes respectively. After incubating at 37 °C for 2 h, an electro-biosensor for detecting the six tumor markers can be obtained.

[0130] Among them, the aptamers corresponding to the six tumor markers are shown in Table 1.

[0131] Table 1

[0132]

[0133] In Table 1, AFP is alpha-fetoprotein, CEA is carcinoembryonic antigen, CA125 is carbohydrate antigen 125, CA19-9 is cancer antigen 19-9, CA15-3 is carbohydrate antigen 15-3, and PSA is prostate specific antigen.

[0134] Among them, the detection results of six tumor markers obtained using the electro-biosensor provided by the present disclosure are shown in the figure, and the specific detection data are shown in Table 2.

[0135] Table 2

[0136]

[0137] Combining the data provided in Table 1 and Table 2, it can be seen that the electro-biosensor provided by the present disclosure can be applied to the detection of different tumor markers, that is, multi-target detection can be achieved, which is suitable for complex analysis requirements and complex detection requirements. The device has high sensitivity and high reliability of detection results.

[0138] Example 2

[0139] Step 1: Place a PET film cut into 3 cm × 6 cm in ultrapure water and ultrasonicate for 30 min. Use silver paste to print wires and five initial working electrodes, a reference electrode printed with silver chloride, and a counter electrode printed with carbon paste.

[0140] Step 2: Prepare carboxylated g-C3N4 powder modified with ternary noble metals.

[0141] Dissolve 2.0 g of melamine and 1.0 g of urea powder in 100 ml of deionized water to form a solution, and then add 0.8 g of solid phosphorous acid. Stir at 80 °C for 60 min until the solution becomes clear to obtain an aqueous solution of nitrogen-rich g-C3N4 precursor;

[0142] Place the aqueous solution of g-C3N4 precursor in a hydrothermal reaction kettle and heat it to 160 °C for 12 h;

[0143] Filter the product obtained by hydrothermal treatment and wash it repeatedly with ultrapure water to obtain g-C3N4 precursor powder;

[0144] Place 1 g of the g-C3N4 precursor powder weighed in a mixed solution of 100 ml of ethanol and glycerol, and reflux at 80-90 °C for 4 h, where the volume ratio of ethanol to glycerol is 3. During the reflux process, after heating to the set temperature, add rhodium nitrate, chloroplatinic acid, and chloroauric acid every 5 min in turn. The amounts of the three metals added are 0.1%, 0.2%, and 0.3% of the mass of the g-C3N4 precursor powder. Filter and dry the obtained product to obtain ternary noble metal-modified g-C3N4 precursor powder;

[0145] Heat the ternary noble metal-modified g-C3N4 precursor powder in a muffle furnace to 550 °C and keep it warm for 2 h, with a heating rate of 2 °C / min to obtain ternary noble metal-modified g-C3N4;

[0146] The ternary noble metal-modified g-C3N4 was placed in a muffle furnace at 500 °C and kept warm for 1 h to obtain few-layer ternary noble metal-modified g-C3N4 powder;

[0147] 0.1 g of the few-layer ternary noble metal-modified g-C3N4 powder was placed into 100 ml of a 1% mass fraction citric acid solution, and then the mixed solution was placed in a reaction kettle for hydrothermal treatment at 160 °C for 6 h. The obtained product was filtered by suction and rinsed, and dried at 60 °C for 8 h to obtain ternary noble metal-modified carboxylated g-C3N4 powder.

[0148] Step 3: The ternary noble metal-modified carboxylated g-C3N4 was dispersed in ultrapure water with a powder mass fraction of 1%, and ultrasonically dispersed for 30 min to obtain a ternary noble metal-modified carboxylated g-C3N4 dispersion.

[0149] Step 4: 1 ml of the ternary noble metal-modified carboxylated g-C3N4 dispersion was aspirated and sprayed onto the initial working electrode one by one to form a working electrode with a ternary noble metal-modified carboxylated g-C3N4 thin film layer.

[0150] Step 5: Antibody probes corresponding to five antibiotics were respectively modified onto five working electrodes. After incubation at 37 °C for 2 h, an electro-biosensor for detecting five antibiotics was obtained.

[0151] Among them, the antibodies corresponding to the five antibiotics were tetracycline monoclonal antibody, gentamicin monoclonal antibody, cyclosporine monoclonal antibody, streptomycin monoclonal antibody, and erythromycin monoclonal antibody respectively. The detection results of the five antibiotics obtained by using the electro-biosensor provided by the present disclosure are shown in Table 3.

[0152] Table 3

[0153]

[0154] As can be seen from Table 3, the electro-biosensor provided by the present disclosure can be applied to the detection of different antibiotics, that is, multi-target detection can be realized, which is suitable for complex analysis requirements and complex detection requirements. The device has high sensitivity and high reliability of detection results.

[0155] Those skilled in the art will readily conceive of other embodiments of the present disclosure after considering the specification and practicing the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the present disclosure, which follow the general principles of the present disclosure and include common general knowledge or conventional technical means in the technical field not disclosed in the present disclosure. The specification and examples are only regarded as exemplary, and the true scope and spirit of the present disclosure are pointed out by the appended claims.

Claims

1. A method for preparing an electrical biosensor, characterized in that: include: Forming initial screen-printed electrodes, the initial screen-printed electrodes comprising at least three initial working electrodes, a counter electrode and a reference electrode, wherein the reference electrode is located between the counter electrode and the initial working electrode, and the portion of the reference electrode surrounding each of the initial working electrodes is in a semi-wrapped concentric ring structure, and the counter electrode is in a continuously bent serpentine structure; Forming a ternary noble metal-modified carboxylated g-C3N4 thin film layer on each of the initial working electrodes to form a working electrode, wherein the thin film layer includes a gradient composite structure formed by rhodium, platinum, and gold in sequence; Forming a ternary noble metal-modified carboxylated g-C3N4 thin film layer comprises: preparing a g-C3N4 precursor powder; Placing the g-C3N4 precursor powder in a mixed solution, and refluxing at a first preset temperature for a preset time, wherein the mixed solution comprises ethanol and glycerol in a volume ratio of not less than 3, the first preset temperature is 80°C to 90°C, and the preset time is 4h to 8h; During the reflux process, rhodium nitrate, chloroplatinic acid and chloroauric acid are sequentially added to the mixed solution at intervals, and the addition amounts of the rhodium nitrate, the chloroplatinic acid and the chloroauric acid are 0.1% to 0.3%, 0.2% to 0.5% and 0.3% to 0.6% of the mass of the g-C3N4 precursor powder, respectively, and the mixture is filtered and dried to obtain a ternary noble metal-modified g-C3N4 precursor powder; Performing a first heat treatment on the ternary noble metal modified g-C3N4 precursor powder to obtain a few-layer ternary noble metal modified g-C3N4 powder; Probes are formed on each of the working electrodes, and at least some of the probes are adapted to detect targets of different types.

2. The method for preparing the electrical biosensor according to claim 1, characterized in that: The step of forming a ternary noble metal-modified carboxylated g-C3N4 thin film layer on each of the initial working electrodes comprises: Carboxylating the few-layer ternary noble metal-modified g-C3N4 powder to obtain a ternary noble metal-modified carboxylated g-C3N4 dispersion; The ternary noble metal-modified carboxylated g-C3N4 dispersion is sprayed on each of the initial working electrodes to form a ternary noble metal-modified carboxylated g-C3N4 thin film layer on each of the initial working electrodes.

3. The method for preparing the electrical biosensor according to claim 2, characterized in that: The first heat treatment comprises: The ternary noble metal modified g-C3N4 precursor powder is heated to a first temperature and kept warm for a first time to obtain ternary noble metal modified g-C3N4 powder, wherein the first temperature is 550°C to 650°C, the first time is 1h to 2h, and the heating rate is 2°C / min to 5°C / min; The ternary noble metal modified g-C3N4 powder is kept at a second temperature for a second time to obtain the few-layer ternary noble metal modified g-C3N4 powder, wherein the second temperature is 500°C to 550°C, and the second time is 1h to 2h.

4. The method for preparing the electrical biosensor according to claim 2 or 3, characterized in that: The method for preparing the precursor powder of g-C3N4 comprises: Dissolving melamine and urea powder in a deionized water solution, and then adding a preset amount of solid phosphorous acid to obtain a nitrogen-rich g-C3N4 precursor aqueous solution, wherein the mass ratio of melamine to urea is greater than or equal to 2; After the nitrogen-rich g-C3N4 precursor aqueous solution is subjected to a second heat treatment, it is filtered and rinsed to obtain the g-C3N4 precursor powder. The insulation temperature of the second heat treatment is 160°C~180°C, and the insulation time is 12h~16h.

5. An electrical biosensor, prepared by the preparation method according to any one of claims 1 to 4, characterized in that: include: Screen-printed electrodes, the screen-printed electrodes comprising at least three working electrodes, a reference electrode and a counter electrode, the counter electrode being arranged in an area surrounded by the plurality of working electrodes, the reference electrode being located between the counter electrode and the working electrode, and the reference electrode being arranged around each of the working electrodes; Wherein, each of the working electrodes has a circular planar structure, and a carboxylated g-C3N4 thin film layer modified with a ternary noble metal is formed on the surface of each of the working electrodes, and the thin film layer includes a gradient composite structure formed by rhodium, platinum, and gold in sequence; the thickness of the thin film layer is 100nm~500nm, and rhodium, platinum, and gold are formed in sequence from the direction of the working electrode pointing to the probe, and the mass ratios of rhodium, platinum, and gold doped in the thin film layer are 0.1%~0.3%, 0.2%~0.5%, and 0.3%~0.6% respectively; A plurality of probes are provided corresponding to the working electrodes in a one-to-one manner, the probes are used to detect targets, and at least some of the probes are adapted to different types of targets.

6. The electrical biosensor according to claim 5, characterized in that The ratio of the distance between two adjacent working electrodes to the diameter of the working electrodes is greater than or equal to three.

7. The electrical biosensor according to claim 5, characterized in that The reference electrode surrounds each of the working electrodes in a semi-wrapped concentric ring structure, the reference electrode covers 30% to 60% of the circumference of the working electrode, and the area of ​​the reference electrode is 15% to 20% of the total area of ​​the working electrode.

8. The electrical biosensor according to claim 5, characterized in that The counter electrode adopts a continuously bent serpentine structure, and the serpentine structure of the counter electrode includes at least 5 periodic bending units, the bending angle of each bending unit is 90°~135°, and the bending line width of each bending unit is 0.5mm~1mm.

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