One-step biosensor

Abstract

The invention discloses a one-step biosensor, and aims to solve the problems that a traditional detection device is tedious in steps and needs multiple operations to complete the detection process. The device mainly comprises a substrate layer and a channel layer, liquid with same or different specific biomolecules flows through multiple groups of gold particles through multiple groups of specific pipelines, so that the specific biomolecules are hung on the gold particles, whole blood in the channel layer is separated into plasma and blood cells through electrodes on the substrate layer, and after the plasma and a buffer solution are fully mixed, the blood cells in the channel layer are separated. The method comprises the following steps: enabling the plasma to flow through gold particles decorated by specific biomolecules, and combining target molecules in the plasma with the specific biomolecules to realize red shift and color change of SPR peaks, so that the same or different target molecules are identified, and the content of the target molecules is judged.

Description

Technical Field

[0001] The present application relates to the field of biosensors, and in particular to a one-step biosensor. Background Art

[0002] Gold nanoparticles (AuNPs) are widely used in biosensors because their surface plasmon resonance (SPR) properties provide a highly sensitive and selective detection method for molecular diagnostics. Through bioconjugation technology, AuNPs can be bound to molecules such as DNA, RNA, or antibodies to specifically recognize their target. In the presence of the target, AuNPs aggregate, triggering a red shift in the SPR peak and a color change in the solution, forming a visible detection signal. This mechanism, based on changes in aggregation state, endows sensors with high sensitivity and selectivity, making them valuable for early disease diagnosis.

[0003] Traditional biosensors often have a low degree of integration and have significant defects in practical applications: First, during inspection, the blood cells and plasma of the whole blood must be separated by other instruments, and then the plasma must be injected into the traditional biosensor. This not only adds extra steps and leads to low work efficiency, but also requires the assistance of other instruments, which increases the detection cost; second, before detection, the detection area components need to be pre-processed outside the device and then assembled. The complex operation further reduces work efficiency; third, traditional biosensors usually have only one detection area and cannot detect multiple targets or cross-validate signals at the same time, resulting in low detection efficiency and reliability.

[0004] In summary, existing biosensors have obvious deficiencies in operation portability and result reliability. There is an urgent need for a new biosensor that is easy to operate, has high detection efficiency and reliable detection results.

[0005] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art known to those skilled in the art. Summary of the Invention

[0006] In view of at least one of the above technical problems, the present disclosure provides a one-step biosensor, which mainly solves the technical problems of low detection efficiency and high detection cost of traditional biosensors.

[0007] According to one aspect of the present disclosure, a one-step biosensor is provided, comprising a base layer, and a channel layer correspondingly disposed above the base layer; the channel layer having a groove on its bottom surface for contacting the base layer as a liquid channel; the channel layer having corresponding liquid inlet or outlet elbows at the inlet and outlet of the liquid channel, respectively; the liquid channel comprising a whole blood separation channel having a separation cavity, a buffer channel disposed in parallel with the whole blood separation channel, a sample mixing channel connected to the whole blood separation channel and the buffer channel downstream, a biomolecule channel connected in parallel to the sample mixing channel downstream, and a detection channel; the detection channel having a reaction chamber disposed therein; A separation electrode is provided at a position of the base layer corresponding to the whole blood separation area; and a gold nanoparticle layer for detecting the liquid in the reaction chamber is provided at a position of the base layer corresponding to the reaction chamber.

[0008] Furthermore, the base layer is made of ITO glass material, and the channel layer is made of transparent non-conductive material.

[0009] Furthermore, the whole blood separation channel includes a whole blood input passage, a separation chamber, and a blood cell output passage arranged in series; a plasma passage is provided between the separation chamber and the entrance of the sample mixing channel, and the plasma passage is composed of a series of tightly arranged and mutually parallel slender pipes vertically connected to the separation chamber and a trumpet-shaped cavity connected to the slender pipes, and the convergent end of the trumpet-shaped cavity is connected to the entrance of the sample mixing channel.

[0010] Furthermore, the buffer solution is phosphate buffered saline.

[0011] Furthermore, the sample mixing channel is arranged in a serpentine shape, including a straight section and a U-shaped curved section, and the inner wall of the straight section is a left-right staggered sawtooth structure.

[0012] Furthermore, the separation electrode includes a first electrode and a second electrode that are arranged opposite to each other, and the first electrode and the second electrode are respectively connected to an alternating current of 10V and 1MHz with opposite phases.

[0013] Furthermore, the first electrode is a strip electrode with a plurality of sharp teeth arranged in an evenly spaced array at the bottom at the end; the second electrode is arranged opposite to the first electrode and includes a plurality of grooves corresponding one-to-one to the sharp teeth of the first electrode, and an extension portion extending to the top of the groove is provided on one side of the groove, and the extension portion corresponds one-to-one to the sharp teeth.

[0014] Furthermore, a positioning mark for determining the position of the gold nanoparticle layer is provided on the substrate layer.

[0015] Furthermore, at least two groups of the biomolecule channels and the detection channels are provided in parallel downstream of the sample mixing channel.

[0016] One or more technical solutions provided in the embodiments of this application have at least any of the following technical effects or advantages: 1. Multifunctional integration improves work efficiency: By placing a whole blood separation area at the biosensor's sample inlet, blood cells and plasma are separated under the action of the electric field gradient of the separation electrode, simplifying the plasma extraction process. This area utilizes the differences in the dielectric properties of cells to separate blood cells and plasma through two electrodes with different voltages. This integrates the blood processing steps into the biosensor, reducing the number of operating steps and accelerating detection speed.

[0017] 2. An integrated design simplifies manual operations: Gold nanoparticles are printed onto the biosensor, a biomolecule liquid is passed through a specific channel, and specific biomolecules are attached to the gold nanoparticles. This reduces manual steps and avoids the influence of external impurities. This solution moves steps that are traditionally performed outside the device into the biosensor through specially designed tubing and fixed gold nanoparticles, eliminating the influence of improper operation or external environmental factors, significantly improving the reliability, efficiency, and automation level of the device.

[0018] 3. Simultaneous detection in multiple detection areas improves reliability: By setting up multiple detection areas, the same specific biomolecule is attached to multiple gold nanoparticles to detect samples, which improves the reliability of the test results. By attaching different specific biomolecules to multiple gold nanoparticles to detect samples, the detection efficiency is improved. By simultaneously completing multi-target detection or signal cross-validation in multiple monitoring areas, the detection efficiency and reliability of the test results are significantly improved.

[0019] 4. Leveraging the properties of gold particles to improve timeliness: The plasmon resonance properties of gold nanoparticles are used to diagnose molecules. Through bioconjugation technology, AuNPs are combined with molecules such as DNA, RNA, or antibodies to specifically identify the target. When the target is present, the aggregation of AuNPs triggers a red shift in the SPR peak and a change in the solution color, forming a visual detection signal. This mechanism, based on changes in aggregation state, is characterized by high timeliness, high sensitivity, and a low detection limit.

[0020] In summary, this application systematically solves the technical pain points of low installation efficiency, high maintenance cost, poor spatial compatibility and fragility of pump body of traditional joints through multi-dimensional innovation of rotational freedom, simplified sealing structure, tool adaptability and filtering function, and provides a connection solution with higher reliability and longer life cycle for the oil and gas recovery system of gas stations. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the overall structure of a one-step biosensor in one embodiment of the present application.

[0022] Figure 2 This is an overall explosion diagram of a one-step biosensor in one embodiment of the present application.

[0023] Figure 3 FIG. 1 is a schematic diagram of the substrate layer of a one-step biosensor according to an embodiment of the present application.

[0024] Figure 4 Schematic diagram of the channel layer of a one-step biosensor in one embodiment of the present application.

[0025] Figure 5 Schematic diagram of the microstructure of electrodes in the whole blood separation area of ​​a one-step biosensor in one embodiment of the present application.

[0026] Figure 6 Schematic diagram of the microstructure of the sample mixing channel of a one-step biosensor in one embodiment of the present application.

[0027] In the above figures, 1 is a base layer, 11 is a substrate, 12 is a first gold particle, 13 is a first positioning mark, 14 is a second gold particle, 15 is a second positioning mark, 16 is a first electrode, and 17 is a second electrode; 2 is a channel layer, 21 is a whole blood separation area, 211 is a whole blood input channel, 212 is a whole blood separation channel, 213 is a plasma channel, 214 is a blood cell output channel, 22 is a buffer channel, 23 is a sample mixing channel, 231 is a first diversion channel, 232 is a second diversion channel, 241 is a first biomolecule channel, 242 is a second biomolecule channel, 251 is a first reaction chamber, 252 is a second reaction chamber, 261 is a first detection channel, 262 is a second detection channel, and 27 is a channel plate; 3 is a channel elbow, 31 is a whole blood inlet elbow, 32 is a blood cell outlet elbow, 33 is a buffer inlet elbow, 34 is a first biomolecule inlet elbow, 35 is a first detection channel outlet elbow, 36 is a second biomolecule inlet elbow, and 37 is a second detection channel outlet elbow. DETAILED DESCRIPTION

[0028] In the description of this application, it should be understood that the terms "upper", "lower", "front", "back", "left", "right", "top", "bottom", "inside", "outside", "vertical", "horizontal", "clockwise", "counterclockwise", etc., indicating directions or positional relationships, are based on the directions or positional relationships shown in the accompanying drawings. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operated in a specific direction. Therefore, they cannot be understood as limitations on this application. The terms "first", "second", etc. mentioned in this application are used to distinguish the objects described and do not have any order or technical meaning. The terms "connection" and "connection" mentioned in this application, unless otherwise specified, include direct and indirect connections (connections).

[0029] Unless otherwise specified, the unit modules (components, structures, mechanisms) or sensors and other devices involved in the following embodiments are conventional commercially available products.

[0030] The present invention provides a one-step biosensor, solving the problem of cumbersome detection procedures in existing technologies, which require multiple operations to complete the test. This simplification of the process steps is achieved by attaching specific biomolecules to gold particles fixed to the device through channels integrated into the device.

[0031] The technical solution in the embodiments of the present application is to solve the above problems, and the overall idea is as follows: Specific biomolecules are attached to gold particles through biomolecule channels, and blood cells and plasma in whole blood are separated by electric field gradients, simplifying the operation steps of the biosensor; multi-target detection or signal cross-validation is achieved by setting up multiple gold nanoparticles.

[0032] In order to better understand the technical solution of the present application, the above technical solution will be described in detail below with reference to the accompanying drawings and specific implementation methods.

[0033] Example 1 Structure Description: Combine Figures 1 to 6 As shown, the one-step biosensor includes a base layer 1, a channel layer 2 mounted on the base layer 1, a groove-shaped channel formed at the bottom of the channel layer 2, and corresponding elbows 3 mounted at the entrance and exit of the channel layer 2. Their specific structures are as follows: 1. Base layer 1: The main body is the substrate 11, which is a square ITO glass with a thickness of 0.75 mm. It has a high transmittance and ensures the transmittance of photolithography exposure during electrode production and gold particle printing.

[0034] On the substrate 11, according to the designed positions corresponding to the whole blood separation area and the reaction chamber, the separation electrodes and the positioning marks are etched on the substrate by lithography technology; See Figure 5 , the separation electrodes include a first electrode 16 and a second electrode 17 to which alternating currents of 10V and 1MHz with opposite phases are applied. The first electrode 16 is a strip electrode, and a plurality of pointed teeth are provided at equal intervals below the strip electrode; the second electrode 17 is provided below the first electrode 16 and opposite to the first electrode 16. The second electrode 17 (including a plurality of grooves corresponding to the pointed teeth of the first electrode 16 respectively. One side wall of the groove extends to the upper part of the groove at the top of the groove to form an extension part, and each extension part is arranged opposite to the corresponding pointed tooth of the first electrode 16 respectively) is composed of a plurality of repeated "Ji" - shaped structures connected in sequence. The horizontal part of the "Ji" - shaped structure extends to the right for a part, and each "Ji" - shaped structure is connected by a right - angled turning line segment. This structure can increase the electrode surface area, help to increase the contact area between the electrode and the whole blood, and optimize the dielectrophoresis reaction of the whole blood. The triangular pointed teeth in the first electrode 16 are facing the part that the "Ji" - shaped structure in the second electrode 17 extends to the right. Among them, the length d3 of the upper part of the bottom edge of the "Ji" - shaped structure in the second electrode 17 is 120μm, the distance d4 from the bottom of the part that the "Ji" - shaped structure extends to the right to the upper part of the bottom edge of the "Ji" - shaped structure is 80μm, the distance d5 from the triangular pointed teeth in the first electrode 16 to the upper part of the part that the "Ji" - shaped structure in the second electrode 17 extends to the right is 45μm, the width d6 of the long strip in the first electrode 16 is 30μm, and the apex angle θ1 of the triangular pointed teeth in the first electrode 16 is 15°; The positioning marks are four cross - shaped convex platforms evenly distributed in two rows and two columns, aligned up and down and left and right, arranged in a regular and symmetric square array; the positioning marks are divided into two groups, namely the first positioning mark 13 and the second positioning mark 15, which respectively determine the positions of the first gold particle 12 and the second gold particle 14; There are two groups of gold particles with a diameter of 50nm each. They respectively determine their positions according to the centers of the square arrays of the two groups of positioning marks etched on the substrate. At the center of the square array of the positioning marks, the gold solution is irradiated with ultraviolet light to form the required gold pattern.

[0035] Channel layer 2: The main body is a channel plate 27 with a thickness of 4mm, and the material used is PDMS. The channel layer is used to fit the bottom surface of the base layer and is provided with grooves as liquid channels. All the channel inlets and outlets are arranged in a row and are all located at the lower part of the channel plate 27.

[0036] The lower left portion of the channel plate 27 is the whole blood separation area 21. The inlet of the whole blood input channel is connected to the whole blood input channel 211, and the horizontal separation chamber 212 is connected to the whole blood input channel 211. Above the separation chamber 212, multiple thin tubes are provided perpendicular to the separation chamber 212. These thin tubes together form the plasma channel 213 and converge at the end of the plasma channel 213. On the right side of the separation chamber 212, a blood cell output channel 214 is connected to the blood cell output port located to the right of the inlet of the whole blood input channel. Because the plasma channel 213 is composed of multiple thin tubes perpendicular to the plasma channel 213, and the blood cell output channel 214 is a wide tube immediately adjacent to the separation chamber 212, the flow rate of the plasma channel 213 is lower than that of the blood cell output channel 214. With the help of the Zweifach-Fung effect, blood cells tend to flow into the channel with higher flow rate due to inertia, while plasma enters the branch with lower flow rate, thereby improving the purity of the collected plasma. The provision of multiple channels can effectively prevent blood cells from clogging the plasma channel 213. A buffer channel 22 is located on the right side of the whole blood separation area 21. Its entrance is located to the right of the blood cell output port. The buffer channel 22 is connected in parallel with the plasma channel 213. The output ends of the buffer channel 22 and the plasma channel 213 merge into the sample mixing channel 23 located on the upper left of the channel plate 27. The sample mixing channel 23 includes straight segments and U-shaped bends, which are alternately combined to form a continuously rotating serpentine layout. The side walls of the straight segments of the sample mixing channel 23 are a sawtooth structure that alternates left and right. The sawtooth shape is a parallelogram with a side length d7 of 900μm, a height d8 of 550μm, and an internal angle θ2 of 30°. While increasing the actual length of the channel, the complexity of the inner wall of the pipeline is increased, which can ensure uniform mixing of plasma and buffer. The sample mixing channel 23 is divided into two groups of channels arranged on the upper right side of the channel plate 27, namely the first diversion channel 231 and the second diversion channel 232. Each group of channels is composed of a biomolecule channel and a detection channel. Intersecting with the first diversion channel 231 is the first biomolecule channel 241. The entrance of the first biomolecule channel 241 is located to the right of the inlet of the buffer channel 22. It leads to the upper portion of the channel plate 27 via a straight line, then turns through a bend to merge with the first diversion channel 231, aligning its flow direction with that of the first diversion channel 231. The merged channel forms a serpentine, sawtooth channel similar to the sample mixing channel 23, followed by the first detection channel 261. A vertically elliptical first reaction chamber 251 is located within the first detection channel 261. The outlet of the first detection channel 261 is located to the right of the entrance of the first biomolecule channel 241. Intersecting the second diversion channel 232 is the second biomolecule channel 242. The entrance of the second biomolecule channel 242 is located to the right of the exit of the first detection channel 261. It leads to the upper portion of the channel plate 27 via a straight line segment, then turns through a bend to merge into the second diversion channel 262, with its flow direction being consistent with that of the second diversion channel 262.

[0037] The combined channel is a serpentine zigzag channel similar to the sample mixing channel 23, followed by a second detection channel 262. A vertical elliptical second reaction chamber 252 is provided on the second detection channel 262. The outlet of the second detection channel 262 is located to the right of the inlet of the second biomolecule channel 242. The combined channel is a serpentine zigzag channel similar to the sample mixing channel 23, followed by a vertical elliptical second reaction chamber, and then a straight channel leading to the outlet of the second detection channel, which is located to the right of the inlet of the second biomolecule channel. Elbows are installed at the entrances and exits of the above-mentioned channels, namely, a whole blood inlet elbow 31, a blood cell outlet elbow 32, a buffer inlet elbow 33, a first biomolecule inlet elbow 34, a first detection channel outlet elbow 35, a second biomolecule inlet elbow 36, and a second detection channel outlet elbow 37, to facilitate control of injection from the corresponding inlet and outflow from the corresponding outlet.

[0038] The connecting surfaces of the base layer 1 and the channel layer 2 have the same shape and size. After assembly, the horizontal portion of the electrode in the base layer 1 overlaps with the separation cavity 212 of the channel layer 2, and the gold particles are located in the center of the reaction chamber.

[0039] Workflow: First, a liquid containing the same biomolecule is injected into the first biomolecule channel 241 and the second biomolecule channel 242 through the first biomolecule inlet elbow 34 and the second biomolecule inlet elbow 36. Using the negative pressure applied to the pipeline by the pressure pumps installed on the first detection channel outlet elbow 35 and the second detection channel outlet elbow 37 and the pressure difference of the fluid, the liquid containing the biomolecule is sucked out through the first detection channel outlet elbow 35 and the second detection channel outlet elbow 37. During this process, the liquid containing the biomolecule passes through the gold particles in the reaction chamber, causing the specific biomolecules to be attached to the gold particles, thereby completing the modification of the gold particles.

[0040] Afterwards, the whole blood is injected into the whole blood separation area 21 through the whole blood inlet elbow 31, and the whole blood enters the separation chamber 212 through the whole blood input path 211. The first electrode 16 and the second electrode 17 have different voltages and are of special shapes, which allows the two electrodes to generate an electric field gradient in the separation chamber 212, and use the electric field to selectively manipulate charged or polarized particles to separate blood cells from plasma. The separated blood cells pass through the blood cell output path 214 and are discharged through the blood cell outlet elbow 32; the plasma enters the sample mixing area through the plasma path 213. At the same time, phosphate buffered saline is injected into the buffer channel 22 from the buffer inlet elbow 33 as a buffer, and enters the sample mixing channel 23 at the same time as the plasma.

[0041] The sample mixing channel 23 has a continuously rotating serpentine layout, with its straight sidewalls forming a staggered sawtooth structure. This layout and structure increases the actual length of the channel while also increasing the complexity of the inner wall, ensuring uniform mixing of plasma and buffer. Plasma and phosphate-buffered saline are thoroughly mixed in the sample mixing channel 23. The uniformly mixed plasma solution then splits into two branches, flowing into the first detection channel 261 and the second detection channel 262, and ultimately exits the device through the first detection channel outlet elbow 35 and the second detection channel outlet elbow 37. As the two plasma solutions flow through the first and second reaction chambers 251 and 252, the analytes present in them bind to the specific biomolecules on the modified first and second gold particles 12 and 14, respectively. Surface plasmon resonance (SPR) on the gold particles causes a red shift and color change in the SPR peak. Analyzing this red shift and color change in the SPR peak allows identification of the target molecule and determination of its content.

[0042] Example 2 Structure Description: The structure of this embodiment is the same as that of the first embodiment.

[0043] Workflow: Except for the following differences, the rest of the workflow is the same as that of Example 1: Liquids containing two different biomolecules are injected into the first biomolecule channel 241 and the second biomolecule channel 242 through the first biomolecule channel elbow 34 and the second biomolecule channel elbow 36 respectively.

[0044] When the two plasma solutions flow through the first reaction chamber 251 and the second reaction chamber 252, the detection substances therein respectively combine with the specific biological molecules on the modified first gold particles 12 and the second gold particles 14. Under the action of plasma resonance (SPR) on the surface of the gold particles, the SPR peak produces a red shift and a color change. By analyzing the red shift and color change of the SPR peak, the two different target molecules can be identified and the content of the two target molecules can be determined.

[0045] This device achieves significant technical effects through the following innovative designs: 1. Efficiently remove blood cells from whole blood: The whole blood separation area is set up, and the electric field gradient of the separation electrode separates blood cells from plasma, achieving a blood cell removal rate of over 97%. This reduces the number of operation steps and speeds up the detection process.

[0046] 2. The integrated design simplifies manual operation: Gold nanoparticles are printed onto the biosensor, and specific biomolecules are attached to the gold nanoparticles through specific channels. This allows the detection process to be completed in a single step on the biosensor, avoiding the impact of improper operation or external environmental factors on the test results, significantly improving the reliability and work efficiency of the device.

[0047] 3. Simultaneous detection in multiple detection zones improves reliability: By setting up multiple detection zones, the same specific biomolecule can be attached to multiple gold nanoparticles to detect samples, improving the reliability of the test results. By attaching different specific biomolecules to multiple gold nanoparticles to detect samples, the detection efficiency can be improved. Simultaneous detection of multiple targets or signal cross-validation across multiple monitoring zones significantly improves detection efficiency and the reliability of test results.

[0048] 4. Improving timeliness by using the properties of gold particles: Using the plasma resonance properties of the gold nanoparticle surface to diagnose molecules, it has high timeliness, high sensitivity and low detection limit.

[0049] Although some preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0050] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the inventive concept. Thus, if such changes and modifications fall within the scope of the claims of this application and their equivalents, this application is intended to include such changes and modifications.

Claims

1. A one-step biosensor comprising a base layer and a channel layer disposed above the base layer, wherein the channel layer is provided with a groove on its bottom surface for contact with the base layer as a liquid channel, and the channel layer is provided with corresponding liquid inlet or outlet elbows at the inlet and outlet of the liquid channel, characterized in that: The liquid channel includes a whole blood separation channel provided with a separation cavity, a buffer channel provided in parallel with the whole blood separation channel, a sample mixing channel connected to the whole blood separation channel and the buffer channel downstream, a biomolecule channel and a detection channel connected in parallel to the sample mixing channel downstream; a reaction chamber is provided in the detection channel; A separation electrode is provided at a position of the base layer corresponding to the whole blood separation area; and a gold nanoparticle layer for detecting the liquid in the reaction chamber is provided at a position of the base layer corresponding to the reaction chamber.

2. The one-step biosensor according to claim 1, wherein The base layer is made of ITO glass, and the channel layer is made of transparent non-conductive material.

3. The one-step biosensor according to claim 1, wherein The whole blood separation channel includes a whole blood input passage, a separation chamber, and a blood cell output passage arranged in series; a plasma passage is provided between the separation chamber and the entrance of the sample mixing channel, and the plasma passage is composed of a series of tightly arranged and mutually parallel slender pipes vertically connected to the separation chamber, and a trumpet-shaped cavity connected to the slender pipes, and the convergent end of the trumpet-shaped cavity is connected to the entrance of the sample mixing channel.

4. The one-step biosensor according to claim 1, wherein The buffer solution is phosphate buffered saline.

5. The one-step biosensor according to claim 1, wherein The sample mixing channel is arranged in a serpentine shape, including a straight section and a U-shaped curved section, and the side wall of the straight section is a sawtooth structure staggered left and right.

6. The one-step biosensor according to claim 1, wherein The separation electrode includes a first electrode and a second electrode that are arranged opposite to each other, and the first electrode and the second electrode are respectively connected to 10V, 1MHz alternating currents with opposite phases.

7. The one-step biosensor according to claim 6, characterized in that The first electrode is a strip electrode with a bottom end and a plurality of sharp teeth arranged in an evenly spaced array; the second electrode is arranged opposite to the first electrode and includes a plurality of grooves corresponding one-to-one to the sharp teeth of the first electrode, and an extension portion extending to the top of the groove is provided on one side of the groove, and the extension portion corresponds one-to-one to the sharp teeth.

8. The one-step biosensor according to claim 1, wherein Positioning marks for determining the position of the gold nanoparticle layer are provided on the substrate layer.

9. The one-step biosensor according to claim 1, wherein At least two groups of the biomolecule channels and the detection channels are provided in parallel downstream of the sample mixing channel.

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