Biosynthesis chip and biosynthesis device

Abstract

Provided are a biosynthesis chip and a biosynthesis device. The biosynthesis chip comprises a substrate, a plurality of electrode units and a plurality of modification structures, wherein the plurality of electrode units are located on the substrate, and are configured to provide a voltage for monomer synthesis; and the plurality of modification structures are located on the substrate, and are configured to provide an active group for the monomer synthesis. Each electrode unit corresponds to at least two modification structures, and the at least two modification structures are arranged spaced apart from each other, such that a targeted and selective reaction with a biomonomer can be carried out, thereby improving the selectivity of biosynthesis and the accuracy of biodetection. Moreover, each modification structure facilitates the provision of sufficient reaction space and resources for biosynthesis, interference and competition between reaction regions are small, the plurality of modification structures which are arranged spaced apart from each other can respond quickly, and have high biosynthesis efficiency, which is beneficial to achieving a high-throughput biosynthesis effect.

Description

Biosynthetic chips and biosynthetic devices

[0001] This application claims priority to Chinese Patent Application No. 202410832123.5, filed on June 25, 2024, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] Embodiments of this disclosure relate to a biosynthetic chip and a biosynthetic apparatus. Background Technology

[0003] Synthetic biology is an emerging and interdisciplinary field that uses gene modules and engineering techniques to assemble and construct life systems that do not exist in nature. The essence of synthetic biology lies in design and creation, and its core foundation is DNA. Traditional technologies mainly rely on splicing and modifying existing genes within organisms; that is, they can only operate on existing "code" and cannot write new "code." Unlike traditional technologies, DNA monomer synthesis technology allows humans to directly write and synthesize entirely new DNA sequences—that is, write new "code." Therefore, compared to obtaining genes from organisms, direct gene synthesis offers greater flexibility. Summary of the Invention

[0004] At least one embodiment of this disclosure provides a biosynthetic chip including a substrate, a plurality of electrode units, and a plurality of modification structures. The plurality of electrode units are located on the substrate and are configured to provide a voltage for monomer synthesis. The plurality of modification structures are located on the substrate, wherein the modification structures are configured to provide active groups for monomer synthesis. Each electrode unit corresponds to at least two modification structures, and the at least two modification structures are spaced apart from each other.

[0005] For example, a biosynthetic chip provided according to at least one embodiment of the present disclosure, wherein the at least two modified structure arrays corresponding to each electrode unit are arranged.

[0006] For example, a biosynthetic chip provided according to at least one embodiment of the present disclosure, wherein the orthographic projection of the modified structure onto the substrate is circular, elliptical, annular, or polygonal.

[0007] For example, a biosynthetic chip provided according to at least one embodiment of the present disclosure, wherein each of the electrode units includes a first electrode and a second electrode, and the at least two modification structures corresponding to the electrode unit are located on the side of the first electrode of the electrode unit away from the substrate.

[0008] For example, in a biosynthetic chip provided according to at least one embodiment of the present disclosure, the electrode unit includes a first electrode and a second electrode, and the at least two modification structures corresponding to the electrode unit are located in the same layer as the first electrode of the electrode unit.

[0009] For example, in a biosynthetic chip provided according to at least one embodiment of the present disclosure, the material of the first electrode includes at least one of tantalum, molybdenum, titanium and their derivatives.

[0010] For example, a biosynthetic chip provided according to at least one embodiment of the present disclosure, wherein the modified structure is obtained by modifying a base structure, and the material of the base structure includes at least one of insulating materials, semiconductor materials, and conductive materials.

[0011] For example, a biosynthetic chip provided according to at least one embodiment of the present disclosure, wherein the insulating material comprises silicon dioxide, the semiconductor material comprises at least one of graphene, graphene oxide and carbon nanotubes; and the conductive material comprises at least one of polypyrrole, polyaniline, poly(3,4-ethylenedioxythiophene), poly(thiophene), a conductive composite of sodium polystyrene sulfonate, poly(p-styrene sulfonic acid), and poly(N-methylpyrrolidone).

[0012] For example, a biosynthetic chip provided according to at least one embodiment of the present disclosure, wherein the material of the base structure includes silicon dioxide, and the modification treatment includes treating the base structure with a sodium ethoxide solution.

[0013] For example, a biosynthetic chip provided according to at least one embodiment of the present disclosure, wherein the material of the basic structure includes graphene, and the modification treatment includes treating the basic structure with a mixed solution of 1-pyrenebutanol and methanol; or, the material of the basic structure includes graphene oxide, and the modification treatment includes catalytic reaction of the basic structure.

[0014] For example, according to at least one embodiment of the present disclosure, the biosynthetic chip further includes a sensing layer corresponding to the electrode unit, a first detection electrode, and a second detection electrode, wherein at least a portion of the sensing layer is located between the at least two modification structures corresponding to the electrode unit and the first electrode of the electrode unit, and the first detection electrode and the second detection electrode are respectively connected to different positions of the sensing layer.

[0015] For example, in a biosynthetic chip provided according to at least one embodiment of the present disclosure, the material of the sensing layer includes a semiconductor material or a conductive material, the second electrode of the electrode unit is located on the side of the sensing layer away from the substrate, and the second electrode is insulated from the sensing layer.

[0016] For example, in a biosynthetic chip provided according to at least one embodiment of the present disclosure, the material of the sensing layer includes a conductive material, the second electrode of the electrode unit is disposed at a distance from the sensing layer, and the first electrode, the second electrode, the first detection electrode, and the second detection electrode are all located in the same layer.

[0017] For example, in a biosynthetic chip provided according to at least one embodiment of the present disclosure, the modified structure is obtained by modifying a base structure, and the material of the sensing layer is the same as the material of the base structure corresponding to the modified structure.

[0018] For example, in a biosynthetic chip provided according to at least one embodiment of the present disclosure, the semiconductor material includes at least one of graphene, graphene oxide, and carbon nanotubes.

[0019] For example, in a biosynthetic chip provided according to at least one embodiment of the present disclosure, the conductive material includes at least one of polypyrrole, polyaniline, poly(3,4-ethylenedioxythiophene), poly(thiophene), a conductive composite of sodium polystyrene sulfonate, poly(p-styrene sulfonic acid), and poly(N-methylpyrrolidone).

[0020] For example, a biosynthetic chip provided according to at least one embodiment of the present disclosure is configured to perform biological target detection.

[0021] For example, a biosynthetic chip provided according to at least one embodiment of the present disclosure further includes: an inlet, an outlet, a microchannel, and a receiving chamber, wherein the receiving chamber is connected to the inlet and the outlet respectively through the microchannel, and the orthographic projection of the receiving chamber on the substrate at least partially overlaps with the orthographic projection of the modified structure on the substrate.

[0022] At least one embodiment of this disclosure also provides a biosynthesis apparatus, including the biosynthesis chip described in any of the preceding claims.

[0023] At least one embodiment of this disclosure also provides another biosynthetic chip, including a substrate, a plurality of electrode units, and a plurality of modification structures. The plurality of electrode units are located on the substrate and are configured to provide a voltage for monomer synthesis. The plurality of modification structures are located on the substrate, wherein the modification structures are configured to provide active groups for monomer synthesis. Each electrode unit corresponds to at least one of the modification structures. The modification structures are obtained by modifying a base structure. The material of the base structure includes at least one of insulating materials, semiconductor materials, and conductive materials.

[0024] For example, a biosynthetic chip provided according to at least one embodiment of the present disclosure, wherein the insulating material comprises silicon dioxide.

[0025] For example, a biosynthetic chip provided according to at least one embodiment of the present disclosure, wherein the semiconductor material includes graphene, graphene oxide or carbon nanotubes, and the conductive material includes at least one of polypyrrole, polyaniline, poly(3,4-ethylenedioxythiophene), poly(thiophene), poly(p-styrenesulfonic acid) and poly(N-methylpyrrolidone).

[0026] For example, in a biosynthetic chip provided according to at least one embodiment of the present disclosure, the electrode unit includes a first electrode and a second electrode, and the at least one modified structure corresponding to the electrode unit is located on the side of the first electrode of the electrode unit away from the substrate; or, the at least one modified structure corresponding to the electrode unit is located in the same layer as the first electrode of the electrode unit.

[0027] For example, according to at least one embodiment of the present disclosure, a biosynthetic chip is provided, wherein the electrode unit includes a first electrode and a second electrode, the at least one modification structure corresponding to the electrode unit is located on the side of the first electrode of the electrode unit away from the substrate, wherein the biosynthetic chip further includes a sensing layer, a first detection electrode and a second detection electrode corresponding to the electrode unit, at least a portion of the sensing layer is located between the at least one modification structure corresponding to the electrode unit and the first electrode of the electrode unit, and the first detection electrode and the second detection electrode are respectively connected to different positions on the sensing layer.

[0028] For example, in a biosynthetic chip provided according to at least one embodiment of the present disclosure, the material of the sensing layer includes the semiconductor material or the conductive material, the second electrode of the electrode unit is located on the side of the sensing layer away from the substrate, and the second electrode is insulated from the sensing layer.

[0029] For example, in a biosynthetic chip provided according to at least one embodiment of the present disclosure, the material of the sensing layer includes the conductive material, the second electrode of the electrode unit is disposed at a distance from the sensing layer, and the first electrode, the second electrode, the first detection electrode, and the second detection electrode are all located in the same layer.

[0030] For example, a biosynthetic chip provided according to at least one embodiment of the present disclosure, wherein the biosynthetic chip is configured to perform biological target detection.

[0031] At least one embodiment of this disclosure also provides another biosynthesis device, including the biosynthesis chip described in any of the preceding claims. Attached Figure Description

[0032] To more clearly illustrate the technical solutions of the embodiments of this disclosure, the accompanying drawings of the embodiments will be briefly described below. Obviously, the drawings described below only relate to some embodiments of this disclosure and are not intended to limit this disclosure.

[0033] Figure 1 is a schematic diagram of a biosynthetic chip provided in at least one embodiment of the present disclosure performing biosynthesis.

[0034] Figures 2 to 5 are top views of different biosynthetic chips provided in at least one embodiment of this disclosure.

[0035] Figure 6 is a schematic diagram of another biosynthetic chip provided in at least one embodiment of the present disclosure performing biosynthesis.

[0036] Figure 7 is a schematic diagram of a conductive pattern in a biosynthetic chip provided in at least one embodiment of the present disclosure.

[0037] Figure 8 is a schematic diagram of setting an insulating film layer on the conductive pattern shown in Figure 7.

[0038] Figure 9 is a schematic diagram of electrode units disposed on the insulating film layer shown in Figure 8.

[0039] Figure 10 is a schematic diagram of another biosynthetic chip provided in at least one embodiment of this disclosure.

[0040] Figure 11 is a connection diagram for testing the biosynthetic chip in Figure 10.

[0041] Figure 12 is a top view of the biosynthetic chip shown in Figure 10.

[0042] Figure 13 is a schematic diagram of the structure of another biosynthetic chip provided in at least one embodiment of the present disclosure.

[0043] Figure 14 is a top view of the biosynthetic chip in Figure 13.

[0044] Figure 15 is a connection diagram for testing the biosynthetic chip in Figure 13. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions of the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this disclosure. Based on the described embodiments of this disclosure, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this disclosure.

[0046] Unless otherwise defined, the technical or scientific terms used in this disclosure shall have the ordinary meaning understood by one of ordinary skill in the art to which this disclosure pertains. The terms “first,” “second,” and similar terms used in this disclosure do not indicate any order, quantity, or importance, but are merely used to distinguish different components. Terms such as “comprising” or “including” mean that an element or object preceding the word encompasses the elements or objects listed following the word and their equivalents, without excluding other elements or objects.

[0047] The terms “perpendicular,” “parallel,” and “identical” as used in this disclosure include the strictly defined meanings of “perpendicular,” “parallel,” and “identical,” as well as terms such as “approximately perpendicular,” “approximately parallel,” and “approximately identical” which contain a certain degree of error. Taking into account the measurement and the error associated with the measurement of a particular quantity (i.e., the limitations of the measurement system), they represent the acceptable deviation range for a particular value as determined by a person skilled in the art.

[0048] Synthetic biology is an emerging discipline based on multiple disciplines such as genetic engineering, systems biology, and computer engineering. Its applications in medicine, energy, information technology, and agriculture are growing rapidly, offering broad investment prospects and enormous development potential. Synthetic biology combines knowledge and technologies from biology, engineering, informatics, and chemistry, making it a new interdisciplinary research field. The core objective of synthetic biology is to design and construct new biological components (such as enzymes, genetic circuits, and cells), biological systems, and biological machines, or to redesign existing natural biological systems so that they can perform specific and useful functions.

[0049] Based on their technical principles, mainstream DNA monomer synthesis technologies can be divided into four generations. The first generation uses phosphoramidite-triester synthesis, generally referred to as column synthesis. The second generation is chip-based, including inkjet printing, photochemical methods, and electrochemical methods. The third generation is ultra-high-throughput synthesis, characterized by a combination of semiconductors and electrochemical methods, offering high throughput and low cost. The latest generation of DNA monomer synthesis technology is enzymatic synthesis, mainly including microarray methods, in vivo yeast DNA monomer synthesis, and ligation-mediated DNA synthesis. However, this technology is not yet mature and therefore has not been commercialized. Among these DNA monomer synthesis technologies, the second generation has entered the stage of domestic substitution, while the third generation of ultra-high-throughput synthesis has entered a period of rapid growth.

[0050] Synthesizing nucleic acid fragments on chips is an advanced technique in synthetic biology, typically involving the parallel synthesis of large numbers of short DNA fragments using microfluidic chips or solid-phase synthesis techniques. These techniques can significantly improve the efficiency of DNA synthesis, reduce costs, and accelerate the gene manufacturing and detection process.

[0051] Microfluidic chip technology is a key technology used to synthesize nucleic acid fragments on chips. By performing DNA synthesis on microfluidic chips, high-throughput production of DNA fragments can be achieved. Microfluidic chips can create tiny fluid channels and reaction chambers that can be used to synthesize DNA fragments, and the reaction conditions can be precisely controlled through an integrated fluid control system.

[0052] DNA in situ synthesis chips link nucleotide molecules sequentially at specific locations. Because a large number of oligonucleotide sequences can be synthesized on a small area, this technology allows for the mass production of chips with high-density nucleic acid sequences. For example, the chemical synthesis reactions of oligonucleotides in chip synthesis are carried out on a modified chip support. According to a predetermined base sequence, four corresponding phosphoramidite monomers (i.e., adenine A, thymine T, guanine G, and cytosine C) and other chemical reagents are sequentially added to a pre-modified solid support via a liquid system (e.g., a system including microfluidics) to complete the synthesis of the specified oligonucleotide sequence. During chip synthesis, the small volume of each oligonucleotide significantly reduces reagent consumption, achieving low-cost synthesis.

[0053] For example, DNA synthesis using electric current is a technique based on electrochemical principles. This method typically involves directly synthesizing DNA sequences on an electrode surface, adding nucleotides one by one by controlling a chemical reaction process using electric current. The key steps and principles of DNA synthesis based on electrochemical principles can include the following aspects:

[0054] (1) Electrode surface modification: First, a material suitable for DNA synthesis, such as self-assembled monolayers (SAMs) or polymers, needs to be modified on the electrode surface. These materials can provide a stable substrate to ensure the efficient synthesis and fixation of DNA strands.

[0055] (2) Electrochemical redox reactions: In the process of electrochemical DNA synthesis, redox reactions are driven by applying current. These reactions involve transferring electrons from the electrodes to nucleotide molecules, thereby activating and linking nucleotides.

[0056] (3) Adding nucleotides one by one: By controlling the current and voltage, specific nucleotides can be selectively activated and added. For example, by adjusting the voltage, one nucleotide (such as adenosine) on the electrode surface can be oxidized first, and then reduced with another nucleotide to form a phosphodiester bond, thereby linking the two nucleotides.

[0057] (4) Sequence control: By precisely controlling the current and voltage, precise control of the DNA sequence can be achieved. This method can be used to synthesize oligonucleotides or longer DNA fragments with specific sequences.

[0058] (5) Error correction: Errors in the electrochemical DNA synthesis process can be corrected by changing the electrochemical conditions. For example, if an incorrectly added nucleotide is detected, the reaction can be reversed by adjusting the voltage, the incorrect nucleotide can be removed, and then the correct nucleotide can be added.

[0059] (6) Termination and stabilization: After the DNA sequence is synthesized, the reaction needs to be terminated by electrochemical or other chemical methods, and the synthesized DNA chain needs to be stabilized to prevent further chemical reactions or degradation.

[0060] In summary, electrochemical DNA synthesis is a promising technology that allows for synthesis without enzymes, reducing reliance on bioactive substances, and enables highly customized DNA synthesis through precise electrochemical control. However, this method is currently still in the research and development stage and requires further optimization and large-scale application. For example, the potential problems and risks associated with this method may involve the following aspects:

[0061] (1) Stability of surface modification: The modified layer needs to remain stable during the synthesis process to ensure effective fixation and growth of the DNA strand. However, the modified layer may be damaged by chemical or physical factors, such as solvents, temperature changes or mechanical stress.

[0062] (2) Non-specific adsorption: Non-specific adsorption may occur on the electrode surface, that is, DNA or other molecules may be randomly adsorbed in non-target areas, which may lead to synthesis errors and background signals.

[0063] (3) Modification efficiency and uniformity: Forming a uniform modification layer on the electrode surface is a challenge, especially in large-scale synthesis or high-throughput applications. Inhomogeneous modification layers may lead to low synthesis efficiency and sequence deviation.

[0064] (4) Selectivity and specificity of the reaction: In electrochemical synthesis, it is necessary to ensure that the reaction has high selectivity and specificity in order to correctly add the target nucleotide. Side reactions and cross-reactions may introduce incorrect sequences.

[0065] (5) Selection of electrode materials: Different electrode materials (such as gold, platinum, silicon, etc.) have different electrochemical properties, which may affect the efficiency and stability of DNA synthesis. Selecting appropriate electrode materials is crucial for optimizing the synthesis process.

[0066] (6) Optimization of electrochemical conditions: Electrochemical synthesis requires precise control of parameters such as current, voltage, and reaction time. Inappropriate electrochemical conditions may lead to synthesis failure or the generation of incorrect sequences.

[0067] (7) Cleaning and regeneration of electrode surfaces: During the synthesis process, unreacted nucleotides, protecting groups, or other impurities may accumulate on the electrode surface. Therefore, it is necessary to clean and regenerate the electrode surface regularly to maintain synthesis efficiency.

[0068] (8) Integration and automation: Integrating electrochemical DNA synthesis into an automated system is a challenge that requires addressing issues such as fluid control, reaction monitoring and data processing.

[0069] (9) Cost and scalability: Although electrochemical DNA synthesis is theoretically cost-effective, achieving large-scale and high-throughput synthesis in practical applications still faces challenges in terms of cost and scalability.

[0070] (10) Error correction and verification: Electrochemically synthesized DNA sequences need to be error corrected and verified to ensure that the synthesized DNA sequences are accurate. This may require additional steps and techniques, such as electrochemical sequencing or external verification.

[0071] Solving these problems requires interdisciplinary research and innovation, involving expertise in materials science, chemistry, biology, and engineering. With technological advancements, these problems are expected to be solved, thereby driving the development and application of electrochemical DNA synthesis technology.

[0072] In their research, the inventors of this application discovered that in biosynthetic structures, such as DNA monomer synthesis structures, the electrode structure surface has modified structures for providing active groups for monomer synthesis, and multiple electrode structures correspond one-to-one with multiple modified structures. For example, the active group can be a hydroxyl group. By setting modified structures on the electrode structure surface, active sites can be introduced to interact with specific groups in the DNA monomer, thereby achieving efficient DNA monomer synthesis. However, the modified structures usually set on the electrode structure surface are typically monolithic structures, with the entire modified structure set on the electrode structure surface for DNA monomer synthesis. This setup makes it difficult to achieve precise control over the modified structure, making it difficult to selectively operate on the modified structure in specific regions, resulting in low synthesis efficiency. Furthermore, when using monolithic modified structures for DNA monomer synthesis, spatial competition and related interference can easily occur between different DNA strands, making it difficult to guarantee the accuracy of DNA monomer synthesis, thus requiring improvement in the efficiency of DNA monomer synthesis.

[0073] At least one embodiment of this disclosure provides a biosynthesis chip including a substrate, a plurality of electrode units, and a plurality of modification structures. The plurality of electrode units are located on the substrate and are configured to provide a voltage for monomer synthesis. The plurality of modification structures are located on the substrate and are configured to provide active groups for monomer synthesis. Each electrode unit corresponds to at least two modification structures, and the at least two modification structures are spaced apart from each other.

[0074] In the biosynthesis chip provided by at least one embodiment of this disclosure, at least two modification structures corresponding to each electrode unit are spaced apart from each other. Thus, each electrode unit corresponds to multiple sites for biosynthesis. The spaced modification structures can form multiple independent reaction regions and can react with base monomers in a targeted and selective manner, thereby improving the selectivity of biosynthesis and the accuracy of biological detection. At the same time, each modification structure is conducive to providing sufficient reaction space and resources for biosynthesis, and the interference and competition between each reaction region is small. Therefore, the spaced modification structures can respond quickly to the synthesis voltage, have high biosynthesis efficiency, and are conducive to achieving high-throughput biosynthesis.

[0075] At least one embodiment of this disclosure also provides a biosynthesis apparatus, which includes the biosynthesis chip provided in the above embodiments of this disclosure.

[0076] At least one embodiment of this disclosure also provides another biosynthetic chip, which includes a substrate, a plurality of electrode units, and a plurality of modification structures. The plurality of electrode units are located on the substrate and are configured to provide a voltage for monomer synthesis. The plurality of modification structures are located on the substrate and are configured to provide active groups for monomer synthesis. Each electrode unit corresponds to at least one modification structure. The modification structure is obtained by modifying the base structure. The material of the base structure includes at least one of silicon dioxide, semiconductor materials, and conductive materials.

[0077] Compared to using materials such as silicon dioxide and polysaccharides to form modified structures, using insulating materials, such as silicon dioxide, as the base structure of the modified structure facilitates a tighter bond between the base structure and its adjacent film layer (i.e., the film layer located between the modified structure and the substrate and in contact with the modified structure). This allows for the formation of a modified structure suitable for biosynthesis after modification treatment, and the base structure using this material exhibits excellent modification effects. Furthermore, using semiconductor or conductive materials as the base structure of the modified structure can effectively reduce the resistance between the first and second electrodes during biosynthesis, thereby reducing current loss caused by the modified structure and significantly improving the efficiency of biosynthesis.

[0078] At least one embodiment of this disclosure also provides another biosynthesis apparatus, which includes the biosynthesis chip described above provided in the embodiments of this disclosure.

[0079] The biosynthetic chip and biosynthetic device are described below with reference to the accompanying drawings and through some embodiments.

[0080] Figure 1 is a schematic diagram of biosynthesis performed by a biosynthetic chip provided in at least one embodiment of the present disclosure; Figures 2 to 5 are top views of different biosynthetic chips provided in at least one embodiment of the present disclosure. For example, Figures 2 to 5 can all be top views of an independent reaction unit in a biosynthetic chip.

[0081] As shown in Figure 1, at least one embodiment of the present disclosure provides a biosynthetic chip including a substrate 100, a plurality of electrode units 200, and a plurality of modified electrodes 300. For example, the material of the substrate 100 may be a substrate made of silicon, glass, or polymer. For example, the substrate 100 may be a silicon wafer, but the embodiments of the present disclosure are not limited thereto.

[0082] As shown in Figure 1, multiple electrode units 200 are located on a substrate 100, and the electrode units 200 are configured to provide a voltage for monomer synthesis. As shown in Figure 2, each electrode unit 200 includes a first electrode 210 and a second electrode 220, with the first electrode 210 and the second electrode 220 having opposite polarities. For example, the first electrode 210 can be a positive electrode, and the second electrode 220 can be a negative electrode, but this is not limited to this. For example, during biosynthesis, a synthesis voltage exists between the first electrode 210 and the second electrode 220, which has an electrochemical deprotection function, and can be used to remove the protecting groups at the ends of the base monomers, thereby allowing the next synthesis reaction to proceed in an orderly manner. For example, the first electrode 210 and the second electrode 220 can be located in the same layer or in different layers, and the embodiments of this disclosure are not limited in this regard.

[0083] As shown in Figure 1, multiple modification structures 300 are located on the substrate 100. The modification structures 300 are configured to provide active groups 400 for monomer synthesis. For example, during DNA monomer synthesis, the modification structures 300 can be used to provide active groups such as hydroxyl groups, thereby introducing active sites to interact with specific groups in the DNA monomer, thus facilitating the orderly progress of DNA monomer synthesis.

[0084] As shown in Figure 2, each electrode unit 200 corresponds to at least two modification structures 300, and these at least two modification structures 300 are spaced apart from each other. As shown in Figures 1 and 2, the orthographic projection of the plurality of modification structures 300 corresponding to each electrode unit 200 onto the substrate 100 falls within the orthographic projection of the first electrode 210 of that electrode unit 200 onto the substrate 100. The sum of the orthographic projection areas of the plurality of modification structures 300 corresponding to each electrode unit 200 onto the substrate 100 is less than the orthographic projection area of ​​the first electrode 210 of that electrode unit 200 onto the substrate 100. Each electrode unit 200 can provide a synthesis voltage for its corresponding plurality of modification structures 300, and each modification structure 300 can independently interact with specific groups in the DNA monomer, thereby ensuring the orderly synthesis of the DNA monomer.

[0085] In the biosynthesis chip provided by at least one embodiment of this disclosure, at least two modification structures corresponding to each electrode unit are spaced apart from each other. Thus, each electrode unit corresponds to multiple sites for biosynthesis. The spaced modification structures can form multiple independent reaction regions, which can react with biological monomers in a targeted and selective manner, thereby improving the selectivity of biosynthesis and the accuracy of biological detection. At the same time, each modification structure is conducive to providing sufficient reaction space and resources for biosynthesis, and the interference and competition between each reaction region is small. Therefore, the spaced modification structures can respond quickly to the synthesis voltage, have high biosynthesis efficiency, and are conducive to achieving high-throughput biosynthesis.

[0086] For example, as shown in Figure 2, each electrode unit 200 corresponds to multiple modification structures 300, and the multiple modification structures 300 corresponding to each electrode unit 200 are arranged in an array. For example, the multiple modification structures 300 corresponding to each electrode unit 200 are arranged in an array in a multi-row, multi-column manner, and adjacent modification structures 300 are spaced apart from each other.

[0087] With this configuration, each electrode unit can provide a synthesis voltage for its corresponding multiple modified structures, and each modified structure can be biosynthesized independently. Furthermore, each modified structure can respond rapidly to the synthesis voltage, thus significantly improving biosynthesis efficiency and facilitating high-throughput synthesis.

[0088] For example, as shown in Figure 3, the distance between two adjacent modification structures 300 in each electrode unit 200 can be different. For instance, the multiple modification structures 300 corresponding to the electrode unit 200 include a first modification structure 310, a second modification structure 320, and a third modification structure 330. The minimum distance between the first modification structure 310 and the second modification structure 320 is a first distance. The distance between any two adjacent modification structures 300 in the first modification structure 310 is less than the first distance, and the distance between any two adjacent modification structures 300 in the second modification structure 320 is also less than the first distance. The minimum distance between the second modification structure 320 and the third modification structure 330 is a second distance. The distance between any two adjacent modification structures 300 in the second modification structure 320 is less than the second distance, and the distance between any two adjacent modification structures 300 in the third modification structure 330 is also less than the second distance.

[0089] This configuration allows the first modified structure 310, the second modified structure 320, and the third modified structure 330 to perform different biosynthetic operations, such as different DNA monomer synthesis. The modified structures 300 are clearly spaced apart, which reduces mutual interference during the biosynthesis process and facilitates the differentiation and identification of biosynthetic results. This enables high-throughput biosynthesis while maintaining selectivity in the biosynthesis process.

[0090] It should be noted that the embodiments of this disclosure do not limit the arrangement of the multiple modification structures corresponding to the electrode unit. Multiple modification structures with different numbers or intervals can be set in different regions of the electrode unit according to the needs of biosynthesis, thereby meeting the flexible needs of biosynthesis.

[0091] For example, as shown in Figures 2 to 5, the orthographic projection of the modification structure 300 onto the substrate 100 (see Figure 1) can be circular, elliptical, annular, or polygonal. For example, the shape and area of ​​the orthographic projection of multiple modification structures 300 overlapping the same electrode unit 200 onto the substrate 100 can be different, such as triangular, rectangular, elliptical, etc., and the embodiments of this disclosure do not limit this.

[0092] For example, as shown in Figure 4, the orthographic projections of multiple modification structures 300 overlapping with the same electrode unit 200 onto the substrate 100 (see Figure 1) are all rectangular, and the orthographic projection areas of these multiple modification structures 300 on the substrate 100 are not the same. For example, the modification structure 300 can be cuboid, but is not limited to this. For example, the heights of the multiple modification structures 300 relative to the substrate 100 can be unequal, thereby reducing the related interference of each modification structure 300 during the biosynthesis process, so as to ensure that the biosynthesized product has good quality.

[0093] For example, as shown in Figure 5, the orthographic projection of the modified structures 300 overlapping with the same electrode unit 200 onto the substrate 100 (see Figure 1) is annular or circular, and adjacent modified structures 300 are spaced apart, with the modified structure 300 closest to the second electrode 220 surrounding the other modified structures 300. In this configuration, the regions of each modified structure 300 have clear boundaries, which facilitates regional and selective biosynthesis. For example, by controlling different synthesis conditions (e.g., synthesis voltage, DNA monomers, etc.), different biosynthetic products can be synthesized on different modified structures 300, and these different biosynthetic products are spaced apart from each other, making them easy to distinguish and identify.

[0094] For example, as shown in Figure 1, at least two modification structures 300 corresponding to electrode unit 200 are located on the side of the first electrode 210 of electrode unit 200 away from the substrate 100. For example, the second electrode 220 of electrode unit 200 may be located on the same side as the first electrode 210 (not shown in the figure) and separated from the first electrode 210 by an insulating layer. The embodiments of this disclosure do not limit the arrangement of the second electrode 220, as long as a synthesis voltage can be applied between the first electrode 210 and the second electrode 220. For example, when there is a synthesis voltage between the first electrode 210 and the second electrode 220, the specific active groups provided by the modification structure 300, such as hydroxyl groups 400, can interact with specific groups (such as phosphate groups) in DNA monomer 500, thereby providing reaction sites for DNA monomer synthesis. It is understood that the synthesized DNA sequence 500 in Figure 1 is merely exemplary. In other embodiments, the four bases adenine A, thymine T, guanine G, and cytosine C can be linked in other orders. Specifically, the specific arrangements can be flexibly set according to the types and proportions of bases involved in the synthesis of DNA monomers and the synthesis conditions.

[0095] Figure 6 is a schematic diagram of another biosynthetic chip provided in at least one embodiment of the present disclosure performing biosynthesis.

[0096] For example, compared to the biosynthetic chip shown in FIG1, the arrangement of the first electrode 210 and the modification structure 300 in the biosynthetic chip shown in FIG6 is different, while other structures are the same. For example, as shown in FIG6, at least two modification structures 300 corresponding to electrode unit 200 are located on the same layer as the first electrode 210 of electrode unit 200. For example, at least a portion of the first electrode 210 is located between adjacent modification structures 300. In this scheme, referring to FIG2 and FIG4, the first electrode 210 in the same reaction unit is an integral structure, so that each part of the first electrode 210 can receive the same electrical signal, but the embodiments of this disclosure are not limited to this. For example, referring to FIG5, the first electrode 210 in the same reaction unit includes two spaced-apart parts, which can be connected by other connecting elements, so that they can receive the same electrical signal. For example, the connecting element for connecting different parts of the first electrode 210 can be located on the side of the first electrode 210 closer to the substrate 100, and the connecting element can be connected to different parts of the first electrode 210 through a via, which is not limited in the embodiments of this disclosure.

[0097] This configuration has two advantages. First, in the direction perpendicular to the substrate, it allows for a smaller distance between the monomers undergoing biosynthesis and the first electrode. This facilitates the application of the synthesis voltage between the first and second electrodes to each monomer used in the synthesis, for example, increasing the speed of deprotection of each monomer and thus accelerating the biosynthesis process. Second, it helps reduce the overall thickness of the biosynthesis chip, thereby enabling a thinner and lighter design.

[0098] Figure 7 is a schematic diagram of a conductive pattern in a biosynthetic chip provided in at least one embodiment of the present disclosure; Figure 8 is a schematic diagram of an insulating film layer disposed on the conductive pattern shown in Figure 7; Figure 9 is a schematic diagram of an electrode unit disposed on the insulating film layer shown in Figure 8.

[0099] For example, as shown in FIG7, the biosynthetic chip includes a conductive pattern, and the conductive pattern includes a plurality of conductive structures 110, the plurality of conductive structures 110 including a plurality of first conductive structures 1101 and a plurality of second conductive structures 1102. For example, the first conductive structure 1101 is configured to be connected to a first electrode 210 (see FIG9), and the second conductive structure 1102 is configured to be connected to a second electrode 220 (see FIG9), but the embodiments of this disclosure are not limited thereto. For example, the first conductive structure 1101 may also be connected to the second electrode 220, thereby connecting the second conductive structure 1102 to the first electrode 210.

[0100] For example, as shown in FIG8, the insulating film layer 120 covers at least a portion of the conductive pattern, and at least a portion of each conductive structure 110 is exposed by the insulating film layer (e.g., a portion 111 of the first conductive structure 110 and a portion 112 of the second conductive structure 120 shown in FIG8) to facilitate the application of electrical signals. The insulating film layer 120 includes a plurality of vias N such that the first electrode 210 (see FIG9) or the second electrode 220 (see FIG9) is connected to the corresponding conductive structure 110 (see FIG7) through the vias N.

[0101] For example, as shown in FIG9, the first electrode 210 and the second electrode 220 in the same electrode unit 200 are spaced apart, and the second electrode 220 surrounds the first electrode 210. The orthographic projection of the first electrode 210 on the substrate 100 is circular, and the orthographic projection of the second electrode 220 on the substrate 100 is annular, and the edge of the orthographic projection is square, but this is not limited in the embodiments of the present disclosure.

[0102] For example, as shown in Figures 7 to 9, the plurality of vias N in the insulating film layer 120 includes a first via N1 and a plurality of second vias N2. The first electrode 210 is connected to the first conductive structure 1101 through the first via N1, and the second electrode 220 is connected to the second conductive structure 1102 through the second via N2. Thus, electrical signals can be applied to the first conductive structure 110 and the second conductive structure 120 respectively, so that the first electrode 210 can receive the electrical signal from the first conductive structure 110, and the second electrode 220 can receive the electrical signal from the second conductive structure 120.

[0103] For example, as shown in FIG9, the first electrode 210 and the second electrode 220 may be located on the same layer, but the embodiments of this disclosure are not limited thereto. In some embodiments, the second electrode 220 may also be located on the side of the first electrode 210 away from the substrate 100 (as shown in FIG1), and an insulating layer may also be provided between the first electrode 210 and the second electrode 220. The first electrode 210 and the second electrode 220 may be connected to the corresponding conductive structure 110 (see FIG7) through different vias, respectively. The embodiments of this disclosure do not limit the arrangement of the first electrode 210 and the second electrode 220.

[0104] Currently, most semiconductor electrochemical synthesis chips use precious metals such as gold and platinum as functional electrodes (e.g., the first electrode), mainly because these precious metals are chemically stable and have good acid and alkali resistance. However, the high cost of precious metals increases the cost of DNA monomer synthesis, and these functional electrodes are difficult and unstable to modify organically.

[0105] Based on this, in at least one embodiment of the present disclosure, the material of the first electrode 210 of the biosynthetic chip may include at least one of non-precious metals, such as tantalum, molybdenum, titanium and their derivatives, for example, titanium alloys, etc., thereby effectively reducing the cost of the biosynthetic chip. Since the process of forming modification structures on the surface of these non-precious metals is relatively mature, the modification efficiency can also be significantly improved.

[0106] For example, as shown in Figure 1, the modified structure 300 is obtained by modifying the base structure. The material of the base structure may include at least one of insulating materials, semiconductor materials, and conductive materials.

[0107] For example, as shown in Figure 1, when forming the modified structure 300, the base structure of the modified structure 300 can be patterned on the side of the first electrode 210 away from the substrate 100. For example, the material of the base structure may include, but is not limited to, silicon dioxide. For example, the silicon dioxide base structure can be treated with sodium ethoxide solution, thereby allowing the base structure to react with the sodium ethoxide solution to open the silicon-oxygen bonds of silicon dioxide, thereby generating hydroxyl groups, which can then be used for DNA monomer synthesis.

[0108] In at least one embodiment of this disclosure, compared to using materials such as silicon dioxide or polysaccharides to form the modified structure, by using silicon dioxide as the base structure of the modified structure, it is beneficial to make the base structure tightly bonded to its adjacent membrane layer (i.e., the membrane layer located between the modified structure and the substrate and in contact with the modified structure). Thus, after modification treatment, a modified structure for biosynthesis is formed. The base structure using this material has a good modification effect, thereby helping to ensure that the biosynthesis has a good effect.

[0109] In at least one embodiment of this disclosure, by making the base structure of the modified structure silicon dioxide and making the material of the first electrode include at least one of non-precious metals such as tantalum, molybdenum, and titanium alloys, it is beneficial to make the base structure tightly bonded to its adjacent film layer (i.e., the film layer located between the modified structure and the substrate and in contact with the modified structure), thereby achieving high modification efficiency and good modification effect while reducing costs.

[0110] For example, as shown in Figure 1, the base structure of the modified structure 300 may also include a semiconductor material, such as at least one of graphene, graphene oxide, and carbon nanotubes. For example, a linker molecule (e.g., a linker) for generating active groups can be introduced into the base material containing graphene or carbon nanotubes to achieve the biosynthetic process. For example, a mixed solution of 1-pyrene butanol and methanol can be used to react with the graphene-containing base structure to introduce hydroxyl groups. For example, the aforementioned semiconductor material may include graphene oxide (GO). Due to GO's excellent photothermal conversion effect and photocatalytic ability, oxygen-containing functional groups (such as hydroxyl groups, epoxy groups, etc.) on the GO surface can participate in the reaction during photothermal catalysis, generating or releasing hydroxyl groups for DNA monomer synthesis. The embodiments of this disclosure do not limit the modification treatment method corresponding to the base structure.

[0111] In at least one embodiment of this disclosure, compared to using insulating materials such as silicon dioxide and polysaccharides to form the modified structure, by using semiconductor materials such as graphene or carbon nanotubes as the base structure of the modified structure, the modification efficiency can be improved and the resistance between the first electrode and the second electrode can be effectively reduced. This facilitates an increase in the current used to generate acid (for example, the generated acid can effectively remove the protecting groups on the protected nucleotides, making them more likely to react with phosphorusamide-protected nucleotide monomers), thereby reducing the current loss caused by the modified structure and significantly improving the efficiency of biosynthesis.

[0112] For example, as shown in Figure 1, the basic structure of the modified structure 300 may also include a conductive material, such as a conductive complex of polypyrrole (PPy), polyaniline (PANI), poly(3,4-ethylenedioxythiophene), poly(thiophene), sodium polystyrene sulfonate (PSS), poly(styrene sulfonic acid), PSSA, and poly(N-methylpyrrole), PNMP. These conductive polymers not only provide a conductive substrate but also influence the efficiency of DNA fixation and electrochemical reactions through their chemical structure and properties.

[0113] For example, polypyrrole, polyaniline, poly(3,4-ethylenedioxythiophene), poly(thiophene), poly(p-styrenesulfonic acid), and poly(N-methylpyrrolidone) are all conductive polymers.

[0114] For example, polypyrrole can be electrochemically polymerized to form a thin film on the surface of the first electrode. Polypyrrole exhibits good biocompatibility and chemical stability, and its electrochemical properties and DNA immobilization ability can be tuned by introducing different dopants or functionalized groups.

[0115] For example, polyaniline possesses tunable electrochemical properties and good environmental stability. Polyaniline can be prepared by different synthetic methods (such as chemical polymerization or electrochemical polymerization), and different functional groups can be introduced through doping or copolymerization to enhance DNA immobilization and electrical signal generation capabilities.

[0116] For example, poly(3,4-ethylenedioxythiophene) exhibits high conductivity and good biocompatibility. A thin film of poly(3,4-ethylenedioxythiophene) can be formed on the surface of the first electrode via electrochemical polymerization, and DNA immobilization sites can be introduced through chemical modification.

[0117] For example, poly(thiophene) and its derivatives are a class of polymers with good electrical conductivity, which can be used to form thin films on the surface of the first electrode through methods such as electrochemical polymerization. The electrochemical properties and biocompatibility of these polymers can be tuned by introducing different substituents.

[0118] For example, sodium polystyrene sulfonate itself is non-conductive, but it can be combined with conductive polymers such as poly(3,4-ethylenedioxythiophene) to form conductive composite materials, namely, sulfonic acid group PEDOT (PEDOT:PSS), which can provide good DNA fixation ability.

[0119] For example, poly(p-styrene sulfonic acid) has sulfonic acid groups that can be used for DNA fixation and catalytic processes in electrochemical reactions.

[0120] For example, poly(N-methylpyrrolidone) is a derivative of polypyrrole, which has high conductivity and good biocompatibility. Poly(N-methylpyrrolidone) can be used in electrochemical DNA monomer synthesis processes, providing a stable electrochemical environment and a DNA immobilization platform.

[0121] For example, using the aforementioned materials, performance can be optimized through different synthetic strategies and chemical modifications to meet the requirements of electrochemical DNA monomer synthesis. For instance, monomers with specific functional groups can be introduced via copolymerization to enhance DNA immobilization and electrical signal generation capabilities. Furthermore, the surface morphology, pore structure, and electrochemical stability of these materials are crucial factors influencing their performance in electrochemical DNA synthesis. Precise control of these parameters can further improve the efficiency and accuracy of electrochemical DNA synthesis.

[0122] Therefore, the basic structure using the above-mentioned conductive material has good DNA immobilization ability and helps to reduce current loss caused by modified structure, thus significantly improving the efficiency of biosynthesis.

[0123] Figure 10 is a schematic diagram of another biosynthetic chip provided in at least one embodiment of the present disclosure; Figure 11 is a connection diagram for testing the biosynthetic chip in Figure 10.

[0124] For example, as shown in Figure 10, the biosynthetic chip includes a substrate 100 and a first electrode 210 located on the substrate 100. Furthermore, the biosynthetic chip also includes a sensing layer 260 corresponding to the electrode unit 200, a first detection electrode 410, and a second detection electrode 420. At least a portion of the sensing layer 260 is located between at least two modification structures 300 corresponding to the electrode unit 200 and the first electrode 210 of the electrode unit 200. The first detection electrode 410 and the second detection electrode 420 are respectively connected to different positions on the sensing layer 260. For example, to meet the requirements of the fabrication process, the size of the portion of the second detection electrode 420 (or the first detection electrode 410) overlapping with the sensing layer 260 is L1, and L1 is not less than 2 micrometers. The size of the portion of the second detection electrode 420 (or the first detection electrode 410) extending beyond the sensing layer 260 is L2, and L2 is not less than 2 micrometers.

[0125] For example, as shown in FIG10, the first electrode 210 is located between the sensing layer 260 and the substrate 100, and the first electrode 210 is covered by the sensing layer 260. For example, the modification structure 300 is located on the side of the sensing layer 260 away from the first electrode 210 and is in contact with the sensing layer 260. For example, the electrode unit 200 corresponds to a plurality of modification structures 300, and adjacent modification structures 300 are spaced apart from each other. FIG10 only schematically shows two modification structures 300, but is not limited thereto, and the embodiments of this disclosure do not limit the number of modification structures 300 corresponding to each electrode unit 200.

[0126] For example, as shown in Figure 10, the second electrode 220 of the electrode unit 200 is located on the side of the sensing layer 260 away from the substrate 100, and the second electrode 220 and the sensing layer 260 are insulated from each other. For example, an insulating layer 240 is provided between the second electrode 220 and the sensing layer 260, thereby making the second electrode 220 and the sensing layer 260 insulated from each other. For example, the biosynthetic chip also includes a cover plate 500, and the first electrode 210 and the second electrode 220 are both located in the cavity enclosed by the cover plate 500, thereby making the structure of the biosynthetic chip highly integrated, which is beneficial for miniaturization design.

[0127] For example, as shown in Figure 11, during biosynthesis (e.g., DNA monomer synthesis), the modified structure 300 can be used to provide active groups such as hydroxyl groups, thereby introducing active sites. A synthesis voltage V is provided between the first electrode 210 and the second electrode 220 to remove the protecting groups at the ends of the base monomers, thereby ensuring the orderly progress of the biosynthetic reaction.

[0128] For example, as shown in Figure 11, this biosynthetic chip can be configured for biological target detection. For instance, after biosynthesis, the synthesized DNA product can serve as a probe (e.g., multiple synthesized probes can serve as a probe modification layer). By bringing the sample to be tested into contact with and reacting with the synthesized probes, the probes can identify analytes (such as target molecules) in the sample. For instance, when the probes identify an analyte, the charge generated when the probes bind to the analyte can be transferred through the modification structure 300 to the sensing layer 260, thereby causing the first detection electrode 410 and the second detection electrode 420 connected to the sensing layer 260 to sense the charge change. For instance, a detection voltage Vsd can be applied between the first detection electrode 410 and the second detection electrode 420, and the electrical detection of the analyte can be achieved by observing the change in this detection voltage Vsd. For instance, various probes can be synthesized by controlling different synthesis conditions (e.g., synthesis voltage), thereby enabling the detection of various analytes. The embodiments of this disclosure do not limit the type of probes.

[0129] This configuration allows for several advantages. First, a synthesis voltage can be applied between the first and second electrodes to facilitate biosynthesis. The spaced modification structures also allow for better control over the types and effects of synthesized products (e.g., capture probes) in different regions, resulting in better uniformity of the synthesized products. Furthermore, different types of synthesized products can be controlled and synthesized within the same chamber (e.g., in different regions). Second, after biosynthesis, the first and second detection electrodes, together with the sensing layer, form a detection structure. This allows for the detection of analytes in the sample using the synthesized products. This structure exhibits high detection sensitivity, enabling the detection of biological targets while simultaneously synthesizing capture probes in situ.

[0130] For example, as shown in FIG10, the material of the sensing layer 260 may include a semiconductor material. For example, both the first detection electrode 410 and the second detection electrode 420 include conductive materials and overlap with the sensing layer 260. For example, one of the first detection electrode 410 and the second detection electrode 420 may serve as a source, the other of the first detection electrode 410 and the second detection electrode 420 may serve as a drain, and the second electrode 220 may serve as a gate, thereby the first detection electrode 410, the second detection electrode 420, and the second electrode 220 may constitute a thin-film transistor structure. For example, as shown in FIG11, when performing biological detection, a voltage Vg may be applied to the second electrode 220, thereby making both the first detection electrode 410 and the second detection electrode 420 conductive with the sensing layer 260, and then the electrical detection of the analyte may be achieved by detecting the change in the detection voltage Vsd between the first detection electrode 410 and the second detection electrode 420.

[0131] For example, as shown in FIG10, the material of the sensing layer 260 may include at least one of graphene and carbon nanotubes, but the embodiments of this disclosure are not limited thereto. For example, the sensing layer 260 using graphene may have sensitive sensing capabilities and be able to respond quickly to changes in electrical signals.

[0132] Compared to optical or magnetic detection and analysis methods that require molecular labeling, semiconductor-based label-free electronic biochemical sensing technology offers superior convenience and real-time performance. Graphene biochemical sensors enable precise monitoring of human biomarkers and environmental chemicals at extremely low concentrations, holding immense potential for early diagnosis and environmental assessment. Graphene field-effect biochemical sensors are ion-sensitive field-effect transistors. When ions or biochemical molecules bind to graphene, they alter the local electrical properties of graphene, such as charge distribution or potential, leading to changes in its conductivity. By measuring these changes in conductivity, highly sensitive detection of ions or biochemical molecules can be achieved. Due to graphene's extremely high carrier mobility and low electrical noise, graphene field-effect biochemical sensors have attracted widespread attention.

[0133] However, charge detection-based graphene field-effect sensors face several challenges in practical applications. Typically, chemical modification can be used to modify the target capture probe, but this process makes it difficult to control the uniformity of the target capture probe. Furthermore, when modifying multiple target capture probes, compartmentalization is required, placing high demands on the design and operation of the new pinnacle structure, thus affecting the device's uniformity, stability, and reproducibility. These challenges urgently need to be addressed.

[0134] By employing semiconductor materials such as graphene or carbon nanotubes in the sensing layer, the resistance between the first and second electrodes can be effectively reduced, thereby minimizing current loss caused by the modified structure and significantly improving the efficiency of biosynthesis. Simultaneously, with the first electrode located between the sensing layer and the substrate, controlling the synthesis voltage between the first and second electrodes allows for probe synthesis on the surface of the sensing layer away from the first electrode. This facilitates control over probe distribution, ensuring good probe uniformity and enabling the synthesis of multiple probes within the same chamber. Furthermore, during biodetection, by using the aforementioned semiconductor materials in the sensing layer, it can be electrically connected to both the first and second detection electrodes, allowing for the simultaneous in-situ synthesis of capture probes and detection of biological targets.

[0135] In some embodiments of this disclosure, as shown in FIG10, the material of the sensing layer 260 may also include a conductive material, thereby directly connecting the sensing layer 260 to the first detection electrode 410 and the second detection electrode 420. During biological detection, it is not necessary to apply a voltage to the second electrode 220; only a voltage needs to be applied between the first detection electrode 410 and the second detection electrode 420. Therefore, electrical detection of the analyte can be achieved by detecting the voltage change between the first detection electrode 410 and the second detection electrode 420, which helps to reduce the difficulty of the detection operation.

[0136] Figure 12 is a top view of the biosynthetic chip shown in Figure 10.

[0137] For example, the sensing layer 260 may correspond to multiple electrode units 200. Figure 12 schematically shows four electrode units 200. The second electrodes 220 of adjacent electrode units 200 are connected to each other. The orthographic projections of the second electrodes 220 of the four electrode units 200 onto the substrate 100 (see Figure 10) form a closed ring. The first electrodes 210 of adjacent electrode units 200 are spaced apart from each other, and each first electrode 210 is located on the side of the second electrode 220 closer to the substrate 100. For example, the first detection electrode 410 and the second detection electrode 420 are respectively disposed on opposite sides of the four electrode units 200 and are both connected to the sensing layer 260. For example, during biosynthesis, the first electrodes 210 of the four electrode units 200 can be connected to different conductive structures, and the second electrodes 220 can be connected to a single conductive structure. By applying electrical signals to the conductive structures to which the first electrodes 210 are connected, different synthesis voltages can be generated to perform different biosynthetic processes. For example, when performing biological detection, if the sensing layer 260 includes a semiconductor material, by applying a voltage to the second electrode 220, both the first detection electrode 410 and the second detection electrode 420 can be connected to the sensing layer 260, thereby allowing the detection of the analyte using any one of the four electrode units 200 corresponding to a biosynthetic product.

[0138] As shown in Figure 10, the preparation method of this biosynthetic chip may include the following steps S110 to S160.

[0139] Step S110: Fabricate the first electrode, namely: form a Mo-AlNd-Mo three-layer metal structure at 240°C by physical vapor deposition (PVD) or chemical vapor deposition (CVD), wherein the thicknesses of Mo, AlNd and Mo are 200 Å, 3000 Å and 800 Å respectively, and pattern it by photoreactive ion etching (Photo RIE) technology to achieve a first electrode with a specific shape.

[0140] Step S120: Graphene transfer is performed, namely: a uniform and dense monolayer of graphene is grown on a copper foil using methods such as CVD; PMMA (polymethyl methacrylate) is used to support the graphene film, forming a sandwich structure of PMMA + graphene + copper foil; then the graphene is etched in an ammonium persulfate or ferric chloride solution. Finally, it is transferred to a substrate, and the PMMA is removed to obtain the transferred graphene sample.

[0141] Step S130: Pattern the graphene sample, i.e., use a hard mask (e.g., SiO2) to protect the areas that do not need to be etched, and use plasma etching (Plasm) to pattern the graphene.

[0142] Step S140: Set an insulating layer, i.e.: apply photoresist, for example, submicron photoresist, and since each monomer in the submicron photoresist has 8 active base rings, it can be simply referred to as SU-8, and then form an insulating layer (such as insulating layer 240) by photolithography and development.

[0143] Step S150: Form a first detection electrode and a second detection electrode. A Mo-AlNd-Mo three-layer metal structure is formed at 240°C by physical vapor deposition (PVD) or chemical vapor deposition (CVD). The thicknesses of Mo, AlNd and Mo in this structure are 200 Å, 3000 Å and 800 Å, respectively. The structure is patterned by photoreactive ion etching (Photo RIE) to achieve a first detection electrode and a second detection electrode with specific shapes.

[0144] Step S160: Forming a modified structure, for example, introducing a linker molecule (e.g., a methanol solution of 1-pyrenebutanol) to generate active groups, and incubating for 3 hours to generate hydroxyl groups. It should be noted that a portion of the graphene sample is used to form the modified structure, while the remaining portion serves as the sensing layer.

[0145] For example, for the biosynthesis chip shown in Figure 10, DNA target probe synthesis reagents can be injected, a synthesis voltage can be applied between the first electrode 210 and the second electrode 220, and the probe sequence can be synthesized according to the design requirements.

[0146] For example, as shown in Figure 11, under set voltage conditions (Vg = 400mV, Vds = 100mV), the presence and concentration of target biomolecules (such as DNA targets) are detected by measuring the current change (ΔI%) of the first detection electrode 410 and the second detection electrode 420. For example, a probe station and electrical analysis software can be used to collect and analyze data, and the concentration of the target biomolecules can be quantified by plotting a standard curve. For example, during detection, the target biomarker can be prepared into solutions with different concentration gradients and then reacted with the synthesized probes respectively. For example, based on the test results and combined with a data analysis model, early screening diagnoses can be provided for clinical samples. Scoring and staging criteria based on patient age, gender, and target biomarkers are constructed to evaluate the test samples and output the scores and staging results. For example, this detection process can be applied to the screening and diagnosis of Alzheimer's disease (AD), but is not limited to this.

[0147] Figure 13 is a structural schematic diagram of another biosynthetic chip provided in at least one embodiment of the present disclosure; Figure 14 is a top view of the biosynthetic chip in Figure 13; Figure 15 is a connection schematic diagram for testing the biosynthetic chip in Figure 13.

[0148] For example, compared with the biosynthetic chip provided in FIG10, the biosynthetic chip shown in FIG13 is different in that the arrangement of the first detection electrode 410, the second detection electrode 420, and the second electrode 220 is different. Other structures can be referred to the same description in the above embodiments.

[0149] For example, as shown in Figure 13, the second electrode 220 of the electrode unit 200 is spaced apart from the sensing layer 260, and the first electrode 210, the second electrode 220, the first detection electrode 410, and the second detection electrode 420 are all located in the same layer. A portion of the first detection electrode 410 is covered by the sensing layer 260, and a portion of the second detection electrode 420 is covered by the sensing layer 260, so that both the first detection electrode 410 and the second detection electrode 420 can be connected to the sensing layer 260.

[0150] For example, as shown in Figures 13 and 14, the second electrode 220 surrounds the first detection electrode 410, the second detection electrode 420, and the sensing layer 260. The first detection electrode 410 is located between the sensing layer 260 and the second electrode 220, and the second detection electrode 420 is located between the sensing layer 260 and the second electrode 220. For example, the surfaces of the first detection electrode 410 and the second detection electrode 420 are provided with insulating structures 280, thereby insulating them from the second electrode 220, respectively. The first detection electrode 410, the second detection electrode 420, and the second electrode 220 are all located inside the cavity enclosed by the cover plate 500, thereby reducing the risk of external substances intruding.

[0151] The second electrode in this scheme is set up in a simpler way, which can reduce the correlation interference between the second electrode and other structures in the biosynthesis chip, thereby helping to ensure the accuracy of the electrical signal in the second electrode and making the biosynthesis and biodetection effects better.

[0152] For example, as shown in Figure 14, during biosynthesis (e.g., DNA monomer synthesis), the modified structure 300 can be used to provide active groups such as hydroxyl groups, thereby introducing active sites. A synthesis voltage V is provided between the first electrode 210 and the second electrode 220 to remove the protecting groups at the ends of the base monomers, thereby ensuring the orderly progress of the biosynthetic reaction.

[0153] For example, as shown in Figure 14, the material of the sensing layer 260 in this scheme may include a conductive material. After the biosynthesis process is completed, the synthesized DNA product can be used as a probe. By bringing the sample to be tested into contact with the synthesized probe and reacting with it, the probe can identify the analyte (such as a target molecule) in the sample. For example, when the probe identifies the analyte, the charge generated when the probe interacts with the analyte can be transferred to the sensing layer 260 through the modified structure 300, thereby causing the first detection electrode 410 and the second detection electrode 420 connected to the sensing layer 260 to sense the charge change, respectively. For example, a detection voltage Vsd can be applied between the first detection electrode 410 and the second detection electrode 420, and the electrical detection of the analyte can be achieved by detecting the change in the detection voltage Vsd.

[0154] By using a conductive material for the sensing layer, the resistance between the first and second electrodes can be effectively reduced, thereby minimizing current loss caused by the modified structure and significantly improving the efficiency of biosynthesis. During biosensoring, since the sensing layer can be directly connected to both the first and second detection electrodes, the detection of biological targets can be performed simultaneously with the in-situ synthesis of the capture probe.

[0155] For example, as shown in FIG14, the conductive material in the sensing layer 260 may also include at least one of polypyrrole, polyaniline, poly(3,4-ethylenedioxythiophene), poly(thiophene), a conductive composite of sodium polystyrene sulfonate, poly(p-styrene sulfonic acid), and poly(N-methylpyrrolidone). However, the embodiments of this disclosure are not limited thereto. For the relevant characteristics of these conductive materials, please refer to the relevant descriptions of the above embodiments, which will not be repeated here.

[0156] By using the aforementioned conductive materials, the sensing layer can possess excellent conductivity, which facilitates efficient electrical signal transmission and significantly enhances the detection capability for biological targets.

[0157] For example, as shown in Figures 10 and 13, in each biosynthetic chip, the modified structure 300 is obtained by modifying the base structure, and the material of the sensing layer 260 is the same as the material of the base structure corresponding to the modified structure 300. For example, the modified structure 300 and the sensing layer 260 can be two structures obtained by partially modifying the same original structure. The modified portion of the original structure forms the modified structure 300, and the unmodified portion serves as the sensing layer 260. However, the embodiments of this disclosure are not limited to this. In some embodiments of this disclosure, the material of the sensing layer 260 may also be different from the material of the base structure corresponding to the modified structure 300. The sensing layer 260 and the base structure corresponding to the modified structure 300 can be formed independently, and the embodiments of this disclosure do not limit this.

[0158] For example, as shown in Figure 10, both the base structure corresponding to the modified structure 300 and the sensing layer 260 can be made of semiconductor materials, such as graphene or carbon nanotubes, but are not limited thereto. For example, linker molecules (e.g., linkers) for generating active groups can be introduced into the base material containing graphene or carbon nanotubes to realize the biosynthesis process. For example, a mixed solution of 1-pyrenebutanol and methanol can be used to react with the base structure containing graphene to introduce hydroxyl groups. For example, the semiconductor material mentioned above can include graphene oxide (GO). Since GO has good photothermal conversion effect and photocatalytic ability, during the photothermal catalysis process, oxygen-containing functional groups (such as hydroxyl groups, epoxy groups, etc.) on the surface of GO can participate in the reaction to generate or release hydroxyl groups, which can then be used for DNA monomer synthesis. The embodiments of this disclosure do not limit the modification treatment method corresponding to the base structure.

[0159] For example, as shown in Figures 10 and 13, both the base structure corresponding to the modified structure 300 and the sensing layer 260 can be made of conductive materials, such as at least one of the conductive complexes of polypyrrole, polyaniline, poly(3,4-ethylenedioxythiophene), poly(thiophene), sodium polystyrene sulfonate, poly(p-styrene sulfonic acid), and poly(N-methylpyrrolidone). For example, performance can be optimized through different synthetic strategies and chemical modification methods to meet the needs of electrochemical DNA monomer synthesis. For example, monomers with specific functional groups can be introduced through copolymerization to enhance DNA immobilization ability and electrical signal generation ability. Thus, the base structure using the above-mentioned conductive materials has good DNA immobilization ability, helps reduce current loss caused by the modified structure, significantly improves the efficiency of biosynthesis, and enables the detection of biological targets while simultaneously achieving in-situ synthesis of capture probes.

[0160] For example, at least one embodiment of the biosynthetic chip provided in this disclosure further includes an inlet (see inlet 501 in Figure 10 or Figure 13), an outlet (see outlet 502 in Figure 10 or Figure 13), microchannels, and a receiving chamber. The receiving chamber is connected to the inlet and outlet via microchannels. For example, during bioassay, the test solution enters the receiving chamber from the inlet through the microchannel, enabling the synthesized probe to detect the biological target in the test solution. After detection, the solution flows through the microchannel to the outlet and is discharged. For example, the orthographic projection of the receiving chamber onto the substrate at least partially overlaps with the orthographic projection of the modified structure onto the substrate, allowing the probe to make sufficient contact with the test solution, thereby improving the detection efficiency of the biological target.

[0161] At least one embodiment of this disclosure also provides a biosynthesis apparatus, which includes the biosynthesis chip provided in any of the above embodiments. In addition, the apparatus may also include a signal unit and a microfluidic unit. The signal unit is configured to provide an electrical signal (e.g., the synthesis voltage mentioned above) to the biosynthesis chip and to detect the electrical signal. The microfluidic unit may include pipes, pumps, etc. for sample introduction and effluent, thereby controlling the sample introduction and effluent process of the biosynthesis chip. For details, please refer to the related art, which will not be described in detail here.

[0162] Referring to FIG1, at least one embodiment of this disclosure also provides another biosynthetic chip, which includes a substrate 100 and a plurality of electrode units 200 located on the substrate 100, each electrode unit 200 being configured to provide a voltage for monomer synthesis. For example, referring to FIG2, each electrode unit 200 includes a first electrode 210 and a second electrode 220, and the first electrode 210 and the second electrode 220 have opposite polarities. For example, the first electrode 210 can be a positive electrode and the second electrode 220 can be a negative electrode, but is not limited thereto. For example, during biosynthesis, a synthesis voltage exists between the first electrode 210 and the second electrode 220, which has an electrochemical deprotection function, which can be used to remove the protecting groups at the ends of the base monomers, thereby allowing the next synthesis reaction to proceed in an orderly manner. For example, the first electrode 210 and the second electrode 220 can be located in the same layer or in different layers, and the embodiments of this disclosure are not limited in this regard.

[0163] Referring to Figure 1, the biosynthesis chip also includes multiple modification structures 300 located on the substrate 100. The modification structures 300 are configured to provide active groups 400 for monomer synthesis. For example, during DNA monomer synthesis, the modification structures 300 can be used to provide active groups such as hydroxyl groups, thereby introducing active sites to interact with specific groups in the DNA monomer, thus ensuring the efficient synthesis of DNA monomers.

[0164] Referring to Figure 1, each electrode unit 200 corresponds to at least one modification structure 300, which is obtained by modifying the base structure. The base structure is made of at least one of insulating materials, semiconductor materials, and conductive materials. For example, each electrode unit 200 may correspond to one modification structure 300 or multiple modification structures 300, which is not limited here.

[0165] Referring to Figure 1, when forming the modified structure 300, the base structure of the modified structure 300 can be patterned on the side of the first electrode 210 away from the substrate 100. For example, the material of the base structure may include, but is not limited to, silicon dioxide. For example, the silicon dioxide base structure can be treated with sodium ethoxide solution, thereby allowing the base structure to react with the sodium ethoxide solution to open the silicon-oxygen bonds of silicon dioxide, thereby generating hydroxyl groups, which can then be used for DNA monomer synthesis.

[0166] Compared to using materials such as silicon dioxide and polysaccharides to form modified structures, using silicon dioxide as the base structure of the modified structure facilitates a tighter bond between the base structure and its adjacent membrane layer (i.e., the membrane layer located between the modified structure and the substrate and in contact with the modified structure). This allows for the formation of a modified structure for biosynthesis after modification treatment. The base structure using this material has a good modification effect, which helps to ensure good biosynthesis results.

[0167] Referring to Figure 1, the base structure of the modified structure 300 may also include a semiconductor material or a conductive material. For example, a linker molecule (e.g., a linker) for generating active groups can be introduced into the base structure using a semiconductor material or a conductive material to complete the modification process. For example, the base structure using a semiconductor material or a conductive material can also generate active groups (e.g., hydroxyl groups) under specific catalytic conditions. For example, in biosynthesis, semiconductor materials can be made conductive by external conditions (e.g., applying an electric field).

[0168] Compared to using insulating materials such as silicon dioxide and polysaccharides to form the modified structure, using semiconductor or conductive materials as the base structure of the modified structure can effectively reduce the resistance between the first and second electrodes during biosynthesis, thereby reducing the current loss caused by the modified structure and significantly improving the efficiency of biosynthesis.

[0169] For example, referring to Figure 1, the semiconductor material included in the base structure of modified structure 300 may include at least one of graphene and carbon nanotubes. For example, a linker molecule (e.g., a linker) for generating active groups can be introduced into the base material containing graphene or carbon nanotubes to achieve the biosynthetic process. For example, a mixed solution of 1-pyrene butanol and methanol can be used to react with the graphene-containing base structure to introduce hydroxyl groups. For example, the aforementioned semiconductor material may include graphene oxide (GO). Due to GO's excellent photothermal conversion effect and photocatalytic ability, oxygen-containing functional groups (such as hydroxyl groups, epoxy groups, etc.) on the GO surface can participate in the reaction during photothermal catalysis, generating or releasing hydroxyl groups for DNA monomer synthesis. The embodiments of this disclosure do not limit the modification treatment method corresponding to the base structure.

[0170] Compared to using insulating materials such as silicon dioxide and polysaccharides to form the modified structure, using semiconductor materials such as graphene or carbon nanotubes as the base structure of the modified structure can improve the modification efficiency and effectively reduce the resistance between the first and second electrodes, thereby reducing the current loss caused by the modified structure and significantly improving the efficiency of biosynthesis.

[0171] For example, referring to Figure 1, the conductive material included in the basic structure of the modified structure 300 may include at least one of the following: polypyrrole (PPy), polyaniline (PANI), poly(3,4-ethylenedioxythiophene) (PEDOT), poly(thiophene) (PTh), a conductive composite of sodium polystyrene sulfonate (PSS), poly(styrene sulfonic acid) (PSSA), and poly(N-methylpyrrole) (PNMP).

[0172] For example, polypyrrole, polyaniline, poly(3,4-ethylenedioxythiophene), poly(thiophene), poly(p-styrenesulfonic acid), and poly(N-methylpyrrolidone) are all conductive polymers.

[0173] For example, polypyrrole can be electrochemically polymerized to form a thin film on the surface of the first electrode. Polypyrrole exhibits good biocompatibility and chemical stability, and its electrochemical properties and DNA immobilization ability can be tuned by introducing different dopants or functionalized groups.

[0174] For example, polyaniline possesses tunable electrochemical properties and good environmental stability. Polyaniline can be prepared by different synthetic methods (such as chemical polymerization or electrochemical polymerization), and different functional groups can be introduced through doping or copolymerization to enhance DNA immobilization and electrical signal generation capabilities.

[0175] For example, poly(3,4-ethylenedioxythiophene) exhibits high conductivity and good biocompatibility. A thin film of poly(3,4-ethylenedioxythiophene) can be formed on the surface of the first electrode via electrochemical polymerization, and DNA immobilization sites can be introduced through chemical modification.

[0176] For example, poly(thiophene) and its derivatives are a class of polymers with good electrical conductivity, which can be used to form thin films on the surface of the first electrode through methods such as electrochemical polymerization. The electrochemical properties and biocompatibility of these polymers can be tuned by introducing different substituents.

[0177] For example, sodium polystyrene sulfonate itself is non-conductive, but it can be combined with conductive polymers such as poly(3,4-ethylenedioxythiophene) to form conductive composite materials, namely, sulfonic acid group PEDOT (PEDOT:PSS), which can provide good DNA fixation ability.

[0178] For example, poly(p-styrene sulfonic acid) has sulfonic acid groups that can be used for DNA fixation and catalytic processes in electrochemical reactions.

[0179] For example, poly(N-methylpyrrolidone) is a derivative of polypyrrole, which has high conductivity and good biocompatibility. Poly(N-methylpyrrolidone) can be used in electrochemical DNA monomer synthesis processes, providing a stable electrochemical environment and a DNA immobilization platform.

[0180] For example, using the aforementioned materials, performance can be optimized through different synthetic strategies and chemical modifications to meet the needs of electrochemical DNA monomer synthesis. For instance, monomers with specific functional groups can be introduced via copolymerization to enhance DNA immobilization and electrical signal generation. Thus, the basic structure using the aforementioned conductive materials exhibits excellent DNA immobilization capabilities and helps reduce current loss caused by modified structures, significantly improving the efficiency of biosynthesis.

[0181] For example, referring to FIG1, at least one modification structure 300 corresponding to electrode unit 200 is located on the side of the first electrode 210 of electrode unit 200 away from the substrate 100. For example, the second electrode 220 of electrode unit 200 may be located on the same side as the first electrode 210 (not shown in the figure) and separated from the first electrode 210 by an insulating layer. The embodiments of this disclosure do not limit the arrangement of the second electrode 220, as long as a synthesis voltage can be applied between the first electrode 210 and the second electrode 220. For example, when there is a synthesis voltage between the first electrode 210 and the second electrode 220, the specific active group provided by the modification structure 300, such as hydroxyl group 400, can interact with specific groups (such as phosphate groups) in DNA monomer 500, thereby providing reaction sites for DNA monomer synthesis. It is understood that the synthesized DNA sequence 500 in Figure 1 is merely exemplary. In other embodiments, the four bases adenine A, thymine T, guanine G, and cytosine C can be linked in other orders. Specifically, the specific arrangements can be flexibly set according to the types and proportions of bases involved in the synthesis of DNA monomers and the synthesis conditions.

[0182] For example, referring to FIG6, at least one modification structure 300 corresponding to electrode unit 200 is located on the same layer as the first electrode 210 of electrode unit 200. For example, at least a portion of the first electrode 210 is located between adjacent modification structures 300. In this scheme, referring to FIGS. 2 and 4, the first electrode 210 in the same reaction unit is an integral structure, so that each part of the first electrode 210 can receive the same electrical signal, but the embodiments of this disclosure are not limited thereto. For example, referring to FIG5, the first electrode 210 in the same reaction unit includes two spaced-apart parts, which can be connected by other connecting elements so that they can receive the same electrical signal. For example, the connecting element for connecting different parts of the first electrode 210 can be located on the side of the first electrode 210 closer to the substrate 100, and the connecting element can be connected to different parts of the first electrode 210 through a via, which is not limited in the embodiments of this disclosure.

[0183] This configuration has two advantages. First, in the direction perpendicular to the substrate, it allows for a smaller distance between the monomers undergoing biosynthesis and the first electrode. This facilitates the application of the synthesis voltage between the first and second electrodes to each monomer used in the synthesis, for example, increasing the speed of deprotection of each monomer and thus accelerating the biosynthesis process. Second, it helps reduce the overall thickness of the biosynthesis chip, thereby enabling a thinner and lighter design.

[0184] For example, referring to FIG10, at least one modification structure 300 corresponding to electrode unit 200 is located on the side of the first electrode 210 of electrode unit 200 away from the substrate 100. The biosynthetic chip also includes a sensing layer 260 corresponding to electrode unit 200, a first detection electrode 410 and a second detection electrode 420. At least a portion of the sensing layer 260 is located between at least one modification structure 300 corresponding to electrode unit 200 and the first electrode 210 of electrode unit 200. The first detection electrode 410 and the second detection electrode 420 are respectively connected to different positions of the sensing layer 260. For example, an insulating layer 240 is provided between the second electrode 220 and the sensing layer 260, thereby insulating the second electrode 220 and the sensing layer 260 from each other.

[0185] For example, referring to FIG10, the first electrode 210 is located between the sensing layer 260 and the substrate 100, and the first electrode 210 is covered by the sensing layer 260. For example, the modification structure 300 is located on the side of the sensing layer 260 away from the first electrode 210 and is in contact with the sensing layer 260. FIG10 only schematically shows that the electrode unit 200 corresponds to two modification structures 300, but is not limited thereto. For example, the electrode unit 200 may also correspond to one modification structure 300, and the embodiments of this disclosure do not limit the number of modification structures 300 corresponding to each electrode unit 200. For example, the first electrode 210 may be made of a conductive material, such as platinum, gold, aluminum or molybdenum, thereby enabling the first electrode 210 to have good conductivity.

[0186] For example, referring to Figure 11, during biosynthesis (e.g., DNA monomer synthesis), the modified structure 300 can be used to provide active groups such as hydroxyl groups, thereby introducing active sites. A synthesis voltage V is provided between the first electrode 210 and the second electrode 220 to remove the protecting groups at the ends of the base monomers, thereby ensuring the orderly progress of the biosynthetic reaction.

[0187] For example, referring to Figure 11, after biosynthesis, the synthesized DNA product can be used as a probe. By bringing the sample to be tested into contact with the synthesized probe and allowing it to react, the probe can identify the analyte (such as a target molecule) in the sample. For example, when the probe identifies the analyte, the charge generated when the probe binds to the analyte can be transferred to the sensing layer 260 through the modified structure 300, thereby allowing the first detection electrode 410 and the second detection electrode 420, which are connected to the sensing layer 260, to sense the charge change. For example, a detection voltage Vsd can be applied between the first detection electrode 410 and the second detection electrode 420, thereby enabling the electrical detection of the analyte by observing the change in the detection voltage Vsd between the first detection electrode 410 and the second detection electrode 420.

[0188] For example, referring to FIG10, the material of the sensing layer 260 may include a semiconductor material. For example, both the first detection electrode 410 and the second detection electrode 420 include conductive materials and overlap with the sensing layer 260. For example, one of the first detection electrode 410 and the second detection electrode 420 may serve as a source, the other of the first detection electrode 410 and the second detection electrode 420 may serve as a drain, and the second electrode 220 may serve as a gate, thereby the first detection electrode 410, the second detection electrode 420, and the second electrode 220 may constitute a thin-film transistor structure. For example, referring to FIG11, when performing biological detection, a voltage Vg may be applied to the second electrode 220, thereby making both the first detection electrode 410 and the second detection electrode 420 conductive with the sensing layer 260, and then the electrical detection of the analyte can be achieved by detecting the change in the detection voltage Vsd between the first detection electrode 410 and the second detection electrode 420.

[0189] For example, referring to Figure 10, the material of the sensing layer 260 may include at least one of graphene and carbon nanotubes, but the embodiments of this disclosure are not limited thereto. For example, the sensing layer 260 using graphene can have sensitive sensing capabilities and can respond quickly to changes in electrical signals. By using semiconductor materials such as graphene or carbon nanotubes for the sensing layer, the resistance between the first electrode and the second electrode can be effectively reduced, thereby helping to reduce current loss caused by the modified structure and significantly improving the efficiency of biosynthesis. Furthermore, during the biodetection process, by using the aforementioned semiconductor material for the sensing layer, the sensing layer can be connected to both the first detection electrode and the second detection electrode, thereby enabling the detection of biological targets while simultaneously achieving in-situ synthesis of the capture probe.

[0190] In some embodiments of this disclosure, referring to FIG10, the material of the sensing layer 260 may also include a conductive material, thereby directly connecting the sensing layer 260 to the first detection electrode 410 and the second detection electrode 420. During biological detection, it is not necessary to apply a voltage to the second electrode 220; only a voltage needs to be applied between the first detection electrode 410 and the second detection electrode 420. Therefore, electrical detection of the analyte can be achieved by observing the voltage change between the first detection electrode 410 and the second detection electrode 420, which helps to reduce the difficulty of the detection operation.

[0191] For example, referring to FIG13, the second electrode 220 of the electrode unit 200 is spaced apart from the sensing layer 260, and the first electrode 210, the second electrode 220, the first detection electrode 410, and the second detection electrode 420 are all located in the same layer. A portion of the first detection electrode 410 is covered by the sensing layer 260, and a portion of the second detection electrode 420 is covered by the sensing layer 260, so that both the first detection electrode 410 and the second detection electrode 420 can be connected to the sensing layer 260.

[0192] For example, referring to Figures 13 and 14, the second electrode 220 surrounds the first detection electrode 410, the second detection electrode 420, and the sensing layer 260. The first detection electrode 410 is located between the sensing layer 260 and the second electrode 220, and the second detection electrode 420 is located between the sensing layer 260 and the second electrode 220. For example, the surfaces of the first detection electrode 410 and the second detection electrode 420 are provided with insulating structures 280, thereby insulating them from the second electrode 220, respectively.

[0193] The second electrode in this scheme is set up in a simpler way, which can reduce the correlation interference between the second electrode and other structures in the biosynthesis chip, thereby helping to ensure the accuracy of the electrical signal in the second electrode and making the biosynthesis and biodetection effects better.

[0194] For example, referring to Figure 14, during biosynthesis (e.g., DNA monomer synthesis), the modified structure 300 can be used to provide active groups such as hydroxyl groups, thereby introducing active sites. A synthesis voltage V is provided between the first electrode 210 and the second electrode 220 to remove the protecting groups at the ends of the base monomers, thereby ensuring the orderly progress of the biosynthetic reaction.

[0195] For example, referring to Figure 14, the material of the sensing layer 260 in this scheme may include a conductive material. For example, this biosynthetic chip can be configured for biological target detection. After the biosynthesis process is completed, the synthesized DNA product can serve as a probe. By bringing the sample to be tested into contact with the synthesized probe and allowing it to react, the probe can identify the analyte (such as a target molecule) in the sample. For example, when the probe identifies the analyte, the charge generated when the probe interacts with the analyte can be transferred to the sensing layer 260 through the modified structure 300, thereby causing the first detection electrode 410 and the second detection electrode 420 connected to the sensing layer 260 to sense the charge change. For example, a detection voltage Vsd can be applied between the first detection electrode 410 and the second detection electrode 420, and the electrical detection of the analyte can be achieved by detecting the change in this detection voltage Vsd.

[0196] By using a conductive material for the sensing layer, the resistance between the first and second electrodes can be effectively reduced, thereby minimizing current loss caused by the modified structure and significantly improving the efficiency of biosynthesis. During biosensoring, since the sensing layer can be directly connected to both the first and second detection electrodes, the detection of biological targets can be performed simultaneously with the in-situ synthesis of the capture probe.

[0197] For example, as shown in Figure 14, the conductive material in the sensing layer 260 may also include at least one of polypyrrole, polyaniline, poly(3,4-ethylenedioxythiophene), poly(thiophene), a conductive composite of sodium polystyrene sulfonate, poly(p-styrene sulfonic acid), and poly(N-methylpyrrolidone), but the embodiments of this disclosure are not limited thereto. For the relevant characteristics of these conductive materials, please refer to the descriptions in the above embodiments, which will not be repeated here. By employing the above conductive materials, the sensing layer can have good conductivity, which is beneficial for efficient electrical signal transmission and thus significantly enhances the detection capability of biological targets.

[0198] For example, microfluidic chips modified with PSSA and leaving hydroxyl groups can be used for electrochemical DNA synthesis, biosensor construction, or other molecular biology applications. The following describes the modification process of PSSA for electrochemical DNA synthesis on a microfluidic chip, using PSSA as an example:

[0199] (1) Cleaning of the surface of the basic chip structure: For example, the basic chip structure may include a substrate and a first electrode (such as a positive electrode) located on the substrate. The surface of the basic chip structure may be cleaned with deionized water, organic solvents (such as ethanol or acetone), and surfactants to remove organic residues and particulate impurities. Then, the surface of the basic chip structure is further cleaned and activated by ultraviolet ozone cleaning or plasma treatment to improve the surface reactivity of the basic chip structure.

[0200] (2) Surface activation of basic chip structure: The surface is modified by a silane coupling agent (e.g., 3-aminopropyltrimethoxysilane, APTMS), which can form a self-assembled monolayer (SAM) to introduce amino functional groups that can react with PSSA, in preparation for subsequent PSSA grafting.

[0201] (3) PSSA grafting: PSSA is dissolved in a suitable solvent (such as water or alcohol) and introduced into the microchannel through microfluidic technology. PSSA is grafted onto the surface of the activated base chip structure through physical adsorption or covalent bonding.

[0202] (4) Introduction of hydroxyl groups: During PSSA grafting, PSSA derivatives containing hydroxyl groups can be selectively introduced, or hydroxyl groups can be introduced through chemical modification after PSSA grafting. For example, hydroxyl-containing compounds (such as alcohols) can be reacted with sulfate groups on the PSSA layer through esterification, leaving hydroxyl groups. For example, the introduced hydroxyl groups may need to be temporarily protected to prevent unwanted reactions. This can be achieved by introducing protecting groups (such as silicon protecting groups). Before DNA synthesis, the protecting groups are removed through specific chemical treatments to restore the activity of the terminal hydroxyl groups.

[0203] (5) Stabilization of the modified layer: The PSSA-grafted base chip structure is subjected to heat treatment or chemical crosslinking to enhance the stability of the PSSA modified layer. For example, the PSSA modified layer can be stabilized by using a covalent crosslinking agent or a photoinitiated crosslinking method.

[0204] (6) Characterization and testing: Characterization techniques such as SEM and AFM can be used to examine the morphology and uniformity of the PSSA-modified layer. For example, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) can be used for electrochemical testing to evaluate the electrochemical performance of the PSSA-modified layer.

[0205] (7) DNA synthesis: Using the hydroxyl group introduced in step (4) above as a starting point, DNA chains are synthesized through chemical methods (such as the formation of phosphodiester bonds) or enzymatic methods (such as the use of DNA polymerase). For example, in electrochemical DNA synthesis, the location and sequence of DNA synthesis can be controlled by selectively activating specific microchannels.

[0206] (8) Post-processing and application: After DNA synthesis is completed, it may be necessary to wash and purify the DNA to remove unreacted monomers and byproducts.

[0207] Through the above steps, a PSSA layer can be successfully modified onto the surface of the basic microfluidic chip structure, providing hydroxyl groups and a stable platform for electrochemical DNA synthesis. This method not only improves the efficiency and accuracy of DNA synthesis but also facilitates large-scale DNA synthesis due to the high-throughput characteristics of microfluidic chips. Furthermore, the above process for electrochemical DNA synthesis offers the following advantages:

[0208] (1) Improve electrochemical performance: The conductivity of PSSA can enhance the electrochemical activity of electrodes on microfluidic chips, which is crucial for applications such as electrochemical DNA synthesis and biosensors.

[0209] (2) Enhanced DNA fixation: By introducing hydroxyl groups on the PSSA-modified layer, stable chemical bonding sites can be provided, thereby effectively fixing the DNA strand, which is very important for ensuring the accuracy and reliability of DNA synthesis.

[0210] (3) Achieving high-throughput synthesis: The use of microfluidic chips allows multiple synthetic reactions to be carried out in parallel in tiny channels, thereby improving the efficiency and throughput of DNA synthesis.

[0211] (4) Precise control of the synthesis process: Microfluidic technology provides the ability to precisely control the chemical reaction environment, including pH, temperature, reaction time, etc., which is crucial for optimizing DNA synthesis conditions and improving product quality.

[0212] (5) Reduce side reactions and improve selectivity: By precisely controlling the chemical reaction conditions in the microfluidic environment, unwanted side reactions can be reduced, and the selectivity and accuracy of DNA synthesis can be improved.

[0213] (6) Facilitates subsequent processing and application: DNA synthesized on microfluidic chips can be directly used in subsequent biological experiments, such as cloning, sequencing, or as part of a biosensor, without the need for complex purification steps.

[0214] (7) Reduce costs and improve scalability: The use of microfluidic chips can reduce the consumption of reagents and materials. At the same time, the automation and parallelization features make this platform highly scalable and suitable for large-scale production.

[0215] (8) Promote the development of integrated systems: By integrating conductive polymer modification and DNA synthesis functions on microfluidic chips, highly integrated biomanufacturing systems can be developed, which is of great significance for fields such as synthetic biology and personalized medicine.

[0216] In summary, the above process provides an efficient, controllable, and scalable platform for electrochemical DNA synthesis, which helps to promote the development of synthetic biology and biotechnology.

[0217] For example, at least one embodiment of the biosynthetic chip provided in this disclosure further includes an inlet (see inlet 501 in Figure 10 or Figure 13), an outlet (see outlet 502 in Figure 10 or Figure 13), microchannels, and a receiving chamber. The receiving chamber is connected to the inlet and outlet via microchannels. For example, during bioassay, the test solution enters the receiving chamber from the inlet through the microchannel, enabling the synthesized probe to detect the biological target in the test solution. After detection, the solution flows through the microchannel to the outlet and is discharged. For example, the orthographic projection of the receiving chamber onto the substrate at least partially overlaps with the orthographic projection of the modified structure onto the substrate, allowing the probe to make sufficient contact with the test solution, thereby improving the detection efficiency of the biological target.

[0218] At least one embodiment of this disclosure also provides another biosynthesis apparatus, which includes the biosynthesis chip provided in any of the above embodiments. In addition, the apparatus may also include a signal unit and a microfluidic unit. The signal unit is configured to provide an electrical signal (e.g., the synthesis voltage mentioned above) to the biosynthesis chip and to detect the electrical signal. The microfluidic unit may include pipes, pumps, etc. for sample introduction and effluent, thereby controlling the sample introduction and effluent process of the biosynthesis chip. For details, please refer to the related art, which will not be described in detail here.

[0219] The following points need to be explained:

[0220] (1) The accompanying drawings of the embodiments of this disclosure only involve the structures involved in the embodiments of this disclosure, and other structures can be referred to the general design.

[0221] (2) Where there is no conflict, features of the same embodiment and different embodiments of this disclosure may be combined with each other.

[0222] The above description is merely an exemplary embodiment of this disclosure and is not intended to limit the scope of protection of this disclosure, which is determined by the appended claims.

Claims

A biosynthetic chip, comprising: Base; Multiple electrode units are located on the substrate, and the electrode units are configured to provide a voltage for monomer synthesis; Multiple modified structures are located on the substrate, wherein the modified structures are configured to provide active groups for the synthesis of the monomer, each electrode unit corresponds to at least two of the modified structures, and the at least two modified structures are spaced apart from each other. According to claim 1, the biosynthetic chip, wherein, The at least two modified structure arrays corresponding to each electrode unit are arranged. The biosynthetic chip according to claim 1 or 2, wherein, The orthographic projection of the modified structure onto the substrate is circular, elliptical, annular, or polygonal. The biosynthetic chip according to any one of claims 1-3, wherein, Each of the electrode units includes a first electrode and a second electrode, and the at least two modification structures corresponding to the electrode unit are located on the side of the first electrode of the electrode unit away from the substrate. The biosynthetic chip according to any one of claims 1-3, wherein, The electrode unit includes a first electrode and a second electrode, and the at least two modification structures corresponding to the electrode unit are located in the same layer as the first electrode of the electrode unit. The biosynthetic chip according to any one of claims 1-5, wherein, The material of the first electrode includes at least one of tantalum, molybdenum, titanium and their derivatives. The biosynthetic chip according to any one of claims 1-6, wherein, The modified structure is obtained by modifying the base structure, and the material of the base structure includes at least one of insulating materials, semiconductor materials, and conductive materials. The biosynthetic chip according to claim 7, wherein, The insulating material includes silicon dioxide; the semiconductor material includes at least one of graphene, graphene oxide, and carbon nanotubes; and the conductive material includes at least one of polypyrrole, polyaniline, poly(3,4-ethylenedioxythiophene), poly(thiophene), poly(p-styrenesulfonic acid), and poly(N-methylpyrrolidone). The biosynthetic chip according to claim 7, wherein, The material of the base structure includes silicon dioxide, and the modification treatment includes treating the base structure with a sodium ethoxide solution. The biosynthetic chip according to claim 7, wherein, The material of the basic structure includes graphene, and the modification treatment includes treating the basic structure with a mixed solution of 1-pyrenebutanol and methanol; or, The material of the basic structure includes graphene oxide, and the modification treatment includes catalytic reaction of the basic structure. The biosynthetic chip according to claim 4 further includes a sensing layer, a first detection electrode, and a second detection electrode corresponding to the electrode unit. in, At least a portion of the sensing layer is located between the at least two modification structures corresponding to the electrode unit and the first electrode of the electrode unit, and the first detection electrode and the second detection electrode are respectively connected at different positions of the sensing layer. The biosynthetic chip according to claim 11, wherein, The material of the sensing layer includes a semiconductor material or a conductive material, and the second electrode of the electrode unit is located on the side of the sensing layer away from the substrate, and the second electrode is insulated from the sensing layer. The biosynthetic chip according to claim 11, wherein, The sensing layer is made of a conductive material. The second electrode of the electrode unit is spaced apart from the sensing layer, and the first electrode, the second electrode, the first detection electrode, and the second detection electrode are all located in the same layer. The biosynthetic chip according to claim 12 or 13, wherein, The modified structure is obtained by modifying the base structure, and the material of the sensing layer is the same as the material of the base structure corresponding to the modified structure. According to claim 12, the biosynthetic chip, wherein, The semiconductor material includes at least one of graphene, graphene oxide, and carbon nanotubes. The biosynthetic chip according to claim 12 or 13, wherein, The conductive material includes at least one of polypyrrole, polyaniline, poly(3,4-ethylenedioxythiophene), poly(thiophene), a conductive composite of sodium polystyrene sulfonate, poly(p-styrene sulfonic acid), and poly(N-methylpyrrolidone). The biosynthetic chip according to any one of claims 11-16, wherein, The biosynthetic chip is configured to perform biological target detection. The biosynthetic chip according to any one of claims 1-17 further comprises: The sample inlet, sample outlet, microchannel, and receiving chamber are provided. The receiving chamber is connected to the sample inlet and the sample outlet through the microchannel. The orthographic projection of the receiving chamber on the substrate at least partially overlaps with the orthographic projection of the modified structure on the substrate. A biosynthesis device comprising the biosynthesis chip according to any one of claims 1-18. A biosynthetic chip, comprising: Base; Multiple electrode units are located on the substrate, and the electrode units are configured to provide a voltage for monomer synthesis; Multiple modified structures are located on the substrate, wherein the modified structures are configured to provide active groups for the synthesis of the monomer, each electrode unit corresponds to at least one of the modified structures, the modified structures being obtained by modifying a base structure, the base structure being made of at least one of insulating materials, semiconductor materials, and conductive materials. The biosynthetic chip according to claim 20, wherein, The insulating material includes silicon dioxide. The biosynthetic chip according to claim 20, wherein, The semiconductor material includes graphene, graphene oxide, or carbon nanotubes, and the conductive material includes at least one of polypyrrole, polyaniline, poly(3,4-ethylenedioxythiophene), poly(thiophene), a conductive composite of sodium polystyrene sulfonate, poly(p-styrene sulfonic acid), and poly(N-methylpyrrolidone). The biosynthetic chip according to any one of claims 20-22, wherein, The electrode unit includes a first electrode and a second electrode. The at least one modified structure corresponding to the electrode unit is located on the side of the first electrode of the electrode unit away from the substrate; or, the at least one modified structure corresponding to the electrode unit is located in the same layer as the first electrode of the electrode unit. The biosynthetic chip according to claim 22, wherein, The electrode unit includes a first electrode and a second electrode, and the at least one modification structure corresponding to the electrode unit is located on the side of the first electrode of the electrode unit away from the substrate. The biosynthetic chip further includes a sensing layer corresponding to the electrode unit, a first detection electrode, and a second detection electrode. At least a portion of the sensing layer is located between the at least one modified structure corresponding to the electrode unit and the first electrode of the electrode unit. The first detection electrode and the second detection electrode are respectively connected to different positions on the sensing layer. The biosynthetic chip according to claim 24, wherein, The material of the sensing layer includes the semiconductor material or the conductive material, the second electrode of the electrode unit is located on the side of the sensing layer away from the substrate, and the second electrode is insulated from the sensing layer. The biosynthetic chip according to claim 24, wherein, The material of the sensing layer includes the conductive material, the second electrode of the electrode unit is spaced apart from the sensing layer, and the first electrode, the second electrode, the first detection electrode and the second detection electrode are all located in the same layer. The biosynthetic chip according to any one of claims 24-26, wherein, The biosynthetic chip is configured to perform biological target detection. A biosynthesis device comprising the biosynthesis chip according to any one of claims 20-27.

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