An ordered sensing interface based on DNA-enzyme complex nanostructure and construction method and application thereof

Abstract

The application relates to an ordered sensing interface based on a DNA-enzyme composite nanostructure and a construction method and application thereof. The ordered sensing interface comprises an electrode and a DNA-enzyme composite nanostructure connected to the electrode; the DNA-enzyme composite nanostructure comprises a DNA electronic transmission module and an enzyme catalysis module; the DNA electronic transmission module comprises a mediator-containing DNA sheet structure; the enzyme catalysis module comprises an enzyme substrate complex connected to the DNA sheet structure; and the enzyme substrate complex, the mediator-containing DNA sheet structure and the electrode are sequentially and orderly distributed in space in the order of enzyme substrate complex-mediator-containing DNA sheet structure-electrode. Through the design of the enzyme substrate complex and the DNA sheet structure, the enzyme is orderly fixed, the influence of spatial interference and non-specific interaction on electron transmission is reduced, and the embedded mediator can improve the electrode interface electron transmission rate.

Description

Technical Field

[0001] This application relates to biosensor electrode interface construction technology, and in particular to an ordered sensing interface based on a DNA-enzyme composite nanostructure, its construction method, and its application. Background Technology

[0002] Biosensors play a crucial role in fields such as medical diagnostics, environmental monitoring, and food safety. Their core lies in highly efficient charge-transfer interfaces, through which biomolecules such as enzymes can transfer electrons from biochemical reactions to electrodes, generating measurable electrical signals. In traditional enzyme-electrode interfaces, the transfer of electrons generated by substrate reactions from the enzyme to the electrode may be hampered by obstructed pathways or low transfer efficiency due to the random arrangement of enzyme molecules.

[0003] In recent years, DNA nanotechnology has shown great potential in constructing efficient charge transport interfaces due to its high programmability and precise spatial control capabilities. DNA nanostructures are nanostructures that guide the spatially specific localization of proteins.

[0004] HUH-tagged fusion proteins enable the reaction of POI / EOI (protein or enzyme of interest) with a specific DNA substrate sequence, thereby achieving spatially specific DNA-protein covalent directional linkage. This is also the basis for achieving spatially specific protein localization through DNA nanostructures.

[0005] This precise arrangement minimizes spatial interference and non-specific interactions between enzyme molecules, ensuring that each enzyme molecule is in an optimal reaction state, thereby improving catalytic efficiency and signal strength. However, because the phosphate backbone of the DNA nucleic acid chain carries a negative charge, the interface between the DNA sheet structure and the enzyme construction suffers from shielded electron transport channels, thus hindering the transfer of electrons generated by the enzyme molecules to the surface of the detection electrode. Summary of the Invention

[0006] This application provides one or more embodiments of an ordered sensing interface based on a DNA-enzyme composite nanostructure, its construction method, and its application, including the following technical solutions:

[0007] One or more embodiments of this application provide an ordered sensing interface based on a DNA-enzyme composite nanostructure, the ordered sensing interface comprising: an electrode, and a DNA-enzyme composite nanostructure connected to the electrode;

[0008] The DNA-enzyme composite nanostructure includes a DNA electron transport module and an enzyme catalysis module;

[0009] The DNA electron transport module includes a DNA sheet structure containing a mediator;

[0010] The enzyme catalytic module includes an enzyme substrate complex, which is attached to the DNA sheet structure.

[0011] The enzyme substrate complex, the mediator-containing DNA sheet structure, and the electrode are spatially ordered in the order of enzyme substrate complex - mediator-containing DNA sheet structure - electrode.

[0012] In some embodiments of this application, the mediator includes an electronic mediator;

[0013] The main body of the DNA electron transport module is a DNA sheet structure containing the electron mediator, which is used to directionally anchor the enzyme catalytic module on the electrode and realize directional electron transport.

[0014] In some embodiments of this application, the main body of the enzyme catalysis module is the enzyme substrate complex, which is used to catalyze the substrate to produce an electroactive substance, and the electrons produced by the catalytic reaction are directionally transferred to the electrode through the DNA electron transport module.

[0015] In some embodiments of this application, the mediator includes a cationic mediator.

[0016] In some embodiments of this application, the cationic electron mediator includes one or more of methylene blue, methyl chloride, ferrocene and its derivatives.

[0017] In some embodiments of this application, the enzyme substrate complex comprises a fusion enzyme and a substrate chain;

[0018] Optionally, the fusion enzyme includes a catalytic enzyme and a HUH-tagged enzyme;

[0019] Optionally, the catalytic enzyme includes, but is not limited to, sarcosine oxidase, uricase oxidase, and lactate dehydrogenase;

[0020] Optionally, the HUH tag enzyme includes TC1, Trwc, Tn608, TopoⅠ, DCV, Int-Tn, HI0217, TraI36, RayT AAV5-ReP, FBNYV-Rep, and SIRVI-Rep, etc.

[0021] Optionally, the substrate chain includes, but is not limited to, nucleic acids.

[0022] In some embodiments of this application, one end of the substrate chain is specifically recognized by the HUH tag enzyme and undergoes autocatalytic linkage, while the other end is complementary to the DNA electron transport module through base pairing.

[0023] In some embodiments of this application, the catalytic enzyme and the HUH-tagged enzyme are linked by a linker peptide to form a fusion enzyme; optionally, the linker peptide includes, but is not limited to, 4GS.

[0024] In the embodiments of this application, the synthesis of the DNA sheet structure is not limited to its sequence structure; the main purpose of the DNA sheet structure is to embody the function of the DNA electron transport module.

[0025] In some embodiments of this application, the DNA sheet structure includes an anchoring end scaffold chain binding portion and an enzyme substrate complex linker portion;

[0026] The anchoring end support chain joint includes a support chain joint where the thiol end is a free end.

[0027] In some embodiments of this application, SEQ ID NO.71 to SEQ ID NO.105 are selected as the partial scaffold chain binding portion for the synthesis of one of the DNA sheet structures;

[0028] Optionally, the combination of the partial scaffold chain binding portion and the enzyme substrate complex connection portion, such as SEQ ID NO.275 and SEQ ID NO.276, can constitute the binding portion sequence intervals SEQ ID NO.1 to SEQ ID NO.35 that bind to the proximal end of the enzyme catalytic module, and the binding portion sequence intervals SEQ ID NO.36 to SEQ ID NO.70 that bind to the distal end of the enzyme catalytic module;

[0029] Optionally, the distribution of the enzyme catalytic module on the DNA sheet structure can be achieved by controlling the quantity and / or order of SEQ ID NO.275 and / or SEQ ID NO.276 in combination with SEQ ID NO.71 to SEQ ID NO.105;

[0030] Optionally, the number of some support chain joints may be 0 to 35;

[0031] In some embodiments of this application, the stent chain joint further includes an anchoring end stent chain joint, the anchoring end stent chain joint having a thiol free end;

[0032] Optionally, the number of anchor end bracket chain joints is 3 to 9;

[0033] Optionally, the anchoring end support chain joint includes one or more of the support chain joints described in SEQ ID NO.270 to SEQ ID NO.274.

[0034] In some embodiments of this application, the stent chain connection includes one or more of the stent chain connections described in SEQ ID NO.106 to SEQ ID NO.268.

[0035] In some embodiments of this application, the sequence of the scaffold strands in the DNA nanosheet structure is shown in SEQ ID NO. 269.

[0036] In some embodiments of this application, the sequence of the substrate chain is shown in SEQ ID NO.277.

[0037] In some embodiments of this application, the electrode includes an electrode with gold at the interface; optionally, the metal electrode is a gold electrode, a gold-plated electrode, or a gold adsorption electrode.

[0038] In some embodiments of this application, the electrode includes one or more of a planar electrode and a printed electrode.

[0039] One or more embodiments of this application also provide a method for constructing the DNA-enzyme composite nanostructure, the method comprising the step of constructing an ordered sensing interface using the electrode, the DNA sheet structure, the mediator and the enzyme substrate complex.

[0040] In some embodiments of this application, the construction method includes the following steps:

[0041] (1) The electrodes are pretreated as follows: the electrodes are polished, cleaned, soaked and rinsed in piranha solution, and then dried.

[0042] (2) Prepare the DNA sheet structure and the fusion enzyme;

[0043] (3) First, modify the DNA sheet structure on the surface of the electrode, and then embed the mediator on the DNA sheet structure;

[0044] Alternatively, the mediator can be embedded into the DNA sheet structure first, and then the DNA sheet structure can be modified on the electrode;

[0045] (3) Seal the blank sites on the electrode interface; and,

[0046] (4) The substrate chain and the fusion enzyme are sequentially linked to the DNA sheet structure, or the enzyme-substrate complex is linked to the DNA sheet structure.

[0047] In some embodiments of this application, the construction method includes the following steps:

[0048] (1) The DNA sheet structure was prepared by a one-pot method using the scaffold chain and the scaffold chain junction;

[0049] (2) First, modify the DNA sheet structure on the surface of the electrode, and then embed the electron mediator on the DNA sheet structure;

[0050] Alternatively, the mediator can be embedded into the DNA sheet structure first, and then the DNA sheet structure can be modified on the metal electrode;

[0051] (3) Seal the blank sites on the electrode interface; and,

[0052] (4) The substrate chain and the fusion enzyme are sequentially linked to the DNA sheet structure, or the enzyme-substrate complex is linked to the DNA sheet structure.

[0053] In some embodiments of this application, the molar ratio of the stent chain joint to the stent chain is (7-13):1.

[0054] In some embodiments of this application, the modification conditions include: protection from light; using a solution containing the DNA sheet structure in an amount of 8 μL to 12 μL, wherein the concentration of the DNA sheet structure is 1 nM to 10 nM; a time of 3 hours to 20 hours; and / or a temperature of 3°C to 38°C.

[0055] In some embodiments of this application, the embedding conditions include: protection from light; using a solution containing methylene blue in an amount of 8 μL to 12 μL, wherein the concentration of methylene blue is 0.01 mM to 100 mM; a time of 1 hour to 12 hours; and / or a temperature of 3°C to 38°C.

[0056] In some embodiments of this application, the blocking conditions include: protection from light; the blocking reagent used includes one or more of SH-PEG-SH and 6-mercapto-1-hexanol (MCH); and / or a temperature of 3°C to 38°C.

[0057] Optionally, the amount of the blocking reagent containing SH-PEG-SH is 8 μL to 12 μL, wherein the concentration of SH-PEG-SH is 0.1 mM to 100 mM, and the time is 2 hours to 6 hours;

[0058] Optionally, the amount of the blocking agent containing MCH is 8 μL to 12 μL, wherein the concentration of MCH is 0.1 mM to 10 mM, and the time is 1 hour to 3 hours.

[0059] In some embodiments of this application, the conditions for sequentially connecting the substrate chain and the fusion enzyme include: the volume of the solution containing the substrate chain is 8 μL to 12 μL, wherein the concentration of the substrate chain is 1 μM to 1000 μM; the volume of the solution containing the fusion enzyme is 8 μL to 12 μL, wherein the concentration of the fusion enzyme is 0.5 mg / mL to 3 mg / mL; the time is 1 hour to 20 hours; and / or the temperature is 3°C to 38°C.

[0060] In some embodiments of this application, the conditions for linking the enzyme substrate complex include: the volume of the solution containing the enzyme substrate complex is 8 μL to 12 μL, wherein the concentration of the enzyme substrate complex is 0.5 mg / mL to 3 mg / mL; the time is 1 hour to 20 hours; and / or the temperature is 3°C to 38°C.

[0061] In some embodiments of this application, the resulting reaction products must be rinsed after the modification, embedding, blocking and linking reactions are completed;

[0062] Optionally, the eluent for each reaction independently includes water, 1 mM to 100 mM Tris, 0 mM to 1000 mM NaCl, and not less than 10 mM MgCl2, with a pH range of 6.5 to 8.

[0063] Optionally, the number of rinses may range from 2 to 6.

[0064] In some embodiments of this application, the preparation steps of the enzyme substrate complex include: mixing the fusion enzyme and the substrate chain in a divalent metal ion buffer solution to carry out a recognition reaction, thereby preparing the enzyme substrate complex; the divalent metal ions used include one or more of manganese ions and magnesium ions;

[0065] Optionally, the conditions for the recognition reaction include: protection from light; a reaction time of 1 hour to 16 hours; a reaction temperature of 3°C to 38°C; and / or, the initial reaction system includes a fusion enzyme solution and a substrate chain solution in a volume ratio of 1:(0.5 to 2), wherein the concentration of the fusion enzyme in the fusion enzyme solution is 0.5 mg / mL to 3 mg / mL, the concentration of the substrate chain in the substrate chain solution is 1 μM to 1000 μM, and the concentration of the divalent metal ions in the reaction system is 4 to 10 mM.

[0066] In some embodiments of this application, the metal electrode is modified after the following pretreatment steps: the metal electrode is polished, cleaned, then soaked and rinsed in a piranha solution, and then dried.

[0067] Optionally, the abrasive used for grinding and polishing includes alumina; further optionally, the alumina includes a first alumina with a particle size of 0.25μm to 0.35μm and a second alumina with a particle size of 0.03μm to 0.07μm; even more optionally, the grinding and polishing time using the first alumina is 4 minutes to 6 minutes; even more optionally, the grinding and polishing time using the second alumina is 4 minutes to 6 minutes.

[0068] Optionally, the cleaning solution used for cleaning includes one or more of water, ethanol, and acetone; further optionally, the water, ethanol, and acetone are used for cleaning; even further, the water, ethanol, and acetone are each used for ultrasonic cleaning for 10 to 20 minutes.

[0069] Optionally, the soaking time is 25 to 35 minutes;

[0070] Optionally, the rinsing fluid used includes water;

[0071] Alternatively, drying methods may include nitrogen drying.

[0072] One or more embodiments of this application provide a detection device, the detection device including the ordered sensing interface described above.

[0073] One or more embodiments of this application provide a method for detecting a target substrate in a sample, the detection method comprising the following steps:

[0074] The target substrate in the sample to be tested is detected using a three-electrode chemical detection system or the detection device that includes the ordered sensing interface.

[0075] The enzyme substrate complex specifically recognizes the target substrate and undergoes a catalytic reaction.

[0076] The mediator in the detection system may or may not include methylene blue.

[0077] In some embodiments of this application, the detection system meets the following conditions: the concentration of the mediator is 5 μM to 20 μM; the concentration of the target substrate is 0 μM to 10000 μM; and / or the medium includes water, 1 mM to 100 mM Tris, 0 mM to 1000 mM NaCl, and not less than 10 mM MgCl2, and the pH value ranges from 6.5 to 8.

[0078] In some embodiments of this application, the detection conditions include: the detection operating voltage is -0.4V to 0.6V; differential pulse voltammetry, cyclic voltammetry, AC impedance spectroscopy, or time-current curve method is used; and / or a platinum wire is used as the counter electrode and a silver chloride electrode is used as the reference electrode.

[0079] Details of one or more embodiments of this application are set forth in the following description, and other features, objects, and advantages of this application will become apparent from the specification and its claims. Attached Figure Description

[0080] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0081] Figure 1 These are schematic diagrams of the DNA sheet-like structure designs in Examples 1 and 2;

[0082] Figure 2 This is a schematic diagram of the proximal binding of the DNA sheet structure to the fusion enzyme substrate complex in Example 1;

[0083] Figure 3 This is a flowchart of the one-pot preparation of DNA sheet structures in Examples 1 and 2;

[0084] Figure 4 AFM characterization diagram of the DNA sheet structure in Example 1;

[0085] Figure 5 This is a schematic diagram of running nucleic acid gels on the DNA sheet-like structures in Examples 1 and 2;

[0086] Figure 6 This is a characterization diagram of the DNA sheet structure recognizing and binding to the substrate chain in Example 1;

[0087] Figure 7 This is a schematic diagram of the binding of the DNA sheet structure to the distal end of the fusion enzyme substrate complex in Example 2;

[0088] Figure 8 AFM characterization diagram of the DNA sheet structure in Example 2;

[0089] Figure 9 These are schematic diagrams illustrating the construction of charge-directed transport interfaces based on DNA sheet structures in Examples 3 and 4.

[0090] Figure 10 This is a characterization diagram of the activity of the HUH-tagged enzyme (TC1) in the enzyme substrate complex in Example 3;

[0091] Figure 11 The results show the changes in electron transfer rate detected by different biosensors in Example 3;

[0092] Figure 12 The cyclic voltammetry curve changes during the electrode interface construction process in Example 3;

[0093] Figure 13 This is an AFM characterization diagram of the DNA sheet structure containing 35 scaffold strand binding sites in Example 3 after binding with the enzyme substrate complex on a mica sheet;

[0094] Figure 14 The variation of the differential pulse voltammetry curve during the electrode interface construction process in Example 4;

[0095] Figure 15 This is an AFM characterization diagram of the DNA sheet structure containing 7 scaffold strand binding sites in Example 4 after binding to the enzyme substrate complex on a mica sheet. Detailed Implementation

[0096] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0097] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0098] the term

[0099] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0100] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0101] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0102] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0103] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0104] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0105] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0106] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0107] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0108] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0109] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0110] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0111] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0112] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0113] A first aspect of this application provides an ordered sensing interface based on a DNA-enzyme composite nanostructure, the ordered sensing interface comprising: an electrode, and a DNA-enzyme composite nanostructure connected to the electrode.

[0114] The DNA-enzyme composite nanostructure includes a DNA electron transport module and an enzyme catalysis module;

[0115] The DNA electron transport module includes a DNA sheet structure containing a mediator;

[0116] The enzyme catalytic module includes an enzyme substrate complex, which is attached to the DNA sheet structure.

[0117] The enzyme substrate complex, the mediator-containing DNA sheet structure, and the electrode are spatially ordered in the order of enzyme substrate complex - mediator-containing DNA sheet structure - electrode.

[0118] In some embodiments of this application, the mediator includes an electronic mediator;

[0119] The main body of the DNA electron transport module is a DNA sheet structure containing the electron mediator, which is used to directionally anchor the enzyme catalytic module on the electrode and realize directional electron transport.

[0120] In biology, "anchoring" refers to the process by which biological macromolecules (proteins, nucleic acids, etc.) or cells bind to other molecules, cellular structures, or the extracellular matrix through specific mechanisms, thereby performing their functions at a specific location. This can manifest in various ways, including but not limited to nucleic acid anchoring, where specific DNA interacts with proteins to achieve "anchoring." Anchoring can be direct or indirect.

[0121] In some embodiments of this application, the main body of the enzyme catalysis module is the enzyme substrate complex, which is used to catalyze the substrate to produce an electroactive substance, and the electrons produced by the catalytic reaction are directionally transferred to the electrode through the DNA electron transport module.

[0122] In some embodiments of this application, the mediator includes a cationic mediator.

[0123] In some embodiments of this application, the cationic electron mediator includes one or more of methylene blue, methyl chloride, ferrocene and its derivatives.

[0124] In some embodiments of this application, the enzyme substrate complex comprises a fusion enzyme and a substrate chain;

[0125] Optionally, the fusion enzyme includes a catalytic enzyme and a HUH-tagged enzyme;

[0126] Optionally, the catalytic enzyme includes, but is not limited to, sarcosine oxidase, uricase oxidase, and lactate dehydrogenase;

[0127] Optionally, the HUH tag enzyme includes TC1, Trwc, Tn608, TopoⅠ, DCV, Int-Tn, HI0217, TraI36, RayT AAV5-ReP, FBNYV-Rep, and SIRVI-Rep, etc.

[0128] Optionally, the substrate chain includes, but is not limited to, nucleic acids.

[0129] In some embodiments of this application, one end of the substrate chain is specifically recognized by the HUH tag enzyme and undergoes autocatalytic linkage, while the other end is complementary to the DNA electron transport module through base pairing.

[0130] In the embodiments of this application, the synthesis of the DNA sheet structure is not limited to its sequence structure; the main purpose of the DNA sheet structure is to embody the function of the DNA electron transport module.

[0131] In some embodiments of this application, the DNA sheet structure includes an anchoring end scaffold chain binding portion and an enzyme substrate complex linker portion;

[0132] The anchoring end support chain joint includes a support chain joint where the thiol end is a free end.

[0133] In some embodiments of this application, the scaffold chain binding portion in the DNA sheet structure includes an anchoring end scaffold chain binding portion, Handle A, and other scaffold chain binding portions;

[0134] The enzyme substrate complex includes a fusion enzyme and a substrate chain (i.e., Handle C in the examples). The fusion enzyme includes a catalytic enzyme and a HUH-tagged enzyme. One end of the substrate chain is specifically recognized by the HUH-tagged enzyme (in the example of TC1, i.e., Handle B), and the other end is complementary to the DNA electron transport module.

[0135] This application provides an ordered sensing interface based on a DNA-enzyme composite nanostructure, which is a novel ordered interface related to the sheet-like structure of DNA. By introducing an electron mediator (such as methylene blue molecules) into this sensing interface, the constructed electron transport channel can improve the electron transport rate at the enzyme electrode interface.

[0136] In some examples, the stent chain junction includes one or more stent chain junctions with sequences as shown in SEQ ID NO. 71 to SEQ ID NO. 105, or sequences having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) homology with the sequence of the stent chain junction and capable of binding with the selected stent chain. Optionally, the number of stent chain junctions is 0 to 35, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35.

[0137] In some examples, the anchoring end support chain junction has a free-terminal thiol group. This application does not particularly limit the anchoring end support chain junction; optionally, the number of anchoring end support chain junctions is 3 to 9, for example, 3, 4, 5, 6, 7, 8, or 9. This application does not particularly limit the sequence of the anchoring end support chain junctions; optionally, the anchoring end support chain junction includes one or more of the second staple chains described in SEQ ID NO. 270 to SEQ ID NO. 274, or has a sequence that shares at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) homology with the sequence of the second staple chain and can bind to the selected support chain.

[0138] In some examples, the stent chain junction also includes other stent chain junctions, including one or more of the other stent chain junctions described in SEQ ID NO. 106 to SEQ ID NO. 268, or sequences that have at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) homology with sequences of other stent chain junctions and can bind to the selected stent chain.

[0139] This application does not specifically limit the enzyme substrate complex linker. In some examples, the sequence of the enzyme substrate complex linker is as shown in SEQ ID NO.275 and SEQ ID NO.276 or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) homology with the sequence of the enzyme substrate complex linker.

[0140] The embodiments of this application do not specifically limit the scaffold strands in the DNA sheet structure. In some examples, the sequence of the scaffold strands in the DNA sheet structure is as shown in SEQ ID NO.269, or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) homology with SEQ ID NO.269.

[0141] This application does not specifically limit the type of HUH tagging enzyme. The selected HUH tagging enzyme can recognize DNA sequences. In some examples, the HUH tagging enzyme includes TC1 enzyme.

[0142] In some of these examples, the sequence of the substrate chain is as shown in SEQ ID NO.277 or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) homology with SEQ ID NO.277.

[0143] This application does not specifically limit the type of catalytic enzyme. Appropriate catalytic enzymes can be selected based on the application scenario of the biosensor and the target substance to be detected, including but not limited to sarcosine oxidase, lactate dehydrogenase, and uricase oxidase. Sarcosine oxidase is used to detect sarcosine. The purposes of sarcosine detection are twofold: first, to assess the sarcosine content in muscle tissue to understand muscle health; and second, to serve as a specific and reliable biomarker to improve the early diagnosis and prognosis of prostate cancer cases.

[0144] This application does not impose any particular limitation on the linking method between the catalytic enzyme and the HUH-tagged enzyme in the fusion enzyme, including but not limited to linking via linker peptides, such as linking via 4GS.

[0145] This application does not impose any particular limitation on the type of material of the electrode. In some examples, the electrode includes an electrode with gold (Au) at the interface, including but not limited to gold electrodes, gold-plated electrodes, or gold adsorption electrodes.

[0146] This application does not specifically limit the type of electrode, including but not limited to flat electrode and printed electrode.

[0147] A second aspect of this application provides a method for constructing an ordered sensing interface based on a DNA-enzyme composite nanostructure, the method comprising the step of constructing an ordered sensing interface using the electrode, the DNA sheet structure, the mediator, and the enzyme substrate complex.

[0148] This application does not specifically limit the particular construction steps. In some examples, the construction method includes the following steps:

[0149] (1) The electrodes are pretreated as follows: the electrodes are polished, cleaned, soaked and rinsed in piranha solution, and then dried.

[0150] (2) Prepare the DNA sheet structure and the fusion enzyme;

[0151] (3) First, modify the DNA sheet structure on the surface of the electrode, and then embed the mediator on the DNA sheet structure;

[0152] Alternatively, the mediator can be embedded into the DNA sheet structure first, and then the DNA sheet structure can be modified on the electrode;

[0153] (3) Seal the blank sites on the electrode interface; and,

[0154] (4) The substrate chain and the fusion enzyme are sequentially linked to the DNA sheet structure, or the enzyme-substrate complex is linked to the DNA sheet structure.

[0155] Further, optionally, the construction method includes:

[0156] (1) The DNA sheet structure is prepared using the scaffold chain and the scaffold chain junction;

[0157] (2) First, modify the surface of the electrode with the DNA sheet structure, and then embed the methylene blue into the DNA sheet structure;

[0158] Alternatively, the methylene blue can be embedded into the DNA sheet structure first, and then the DNA sheet structure can be modified on the electrode.

[0159] (3) Seal off the blank sites on the electrode interface;

[0160] (4) The substrate chain and the fusion enzyme are sequentially linked to the DNA sheet structure, or the enzyme-substrate complex is linked to the DNA sheet structure.

[0161] In some of these examples, the molar ratio of the stent chain joint to the stent chain is (7-13):1, for example, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1.

[0162] The DNA sheet-like structure precursor (containing disulfide bonds) is reduced with a reducing agent to prepare a thiol-containing DNA sheet-like structure, including but not limited to the following method: A DNA sheet-like structure precursor solution (15 nM–25 nM, volume ratio) and a reducing agent TCEP solution are treated in the dark to break the disulfide bonds. Excess TCEP is then removed by ultrafiltration and centrifugation, and the DNA sheet-like structure is brought to a final volume. Optionally, the concentration of the DNA sheet-like structure precursor in the DNA sheet-like structure precursor solution is 15 nM–25 nM, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nM, optionally 20 nM. The concentration of TCEP in the TCEP solution is 10 μM to 200 μM, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 μM, optionally 100 μM. Optionally, the concentration of the fixed-volume DNA sheet structure is 4.5 nM to 5.5 nM, for example, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5 nM, optionally 5 nM. The light-protected treatment time is 20 minutes to 60 minutes (for example, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes), optionally 30 minutes.

[0163] In some examples, the modification conditions include: 1) protection from light; 2) the amount of solution containing the DNA sheet structure used is 8 μL to 12 μL (e.g., 8, 8.5, 9, 10, 10.5, 11, 11.5, 12 μL), wherein the concentration of the DNA sheet structure is 1 nM to 10 nM (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nM); 3) the time is 3 hours to 20 hours (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 hours); or / and 4) the temperature is 3°C to 38°C (e.g., 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 38°C). In this application, the modified conditions include one or more of the conditions shown in items 1) to 4).

[0164] In some examples, the embedding conditions include: (A) protection from light; (B) using a solution containing the methylene blue in an amount of 8 μL to 12 μL (e.g., 8, 8.5, 9, 10, 10.5, 11, 11.5, 12 μL), wherein the concentration of the methylene blue is 0.01 mM to 100 mM (e.g., 0.01, 0.1, 0.5, 1, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, ...). (A) 85, 90, 95, 100 mM); (C) time is 1 hour to 12 hours (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours); or / and, (D) temperature is 3℃ to 38℃ (e.g., 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 38℃). That is, in this application, the embedded conditions include one or more of the conditions shown in items (A) to (D).

[0165] In some examples, the blocking conditions include: A) protection from light; B) the use of blocking reagents including one or more of SH-PEG-SH and MCH; and / or C) a temperature of 3°C to 38°C (e.g., 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 38°C). That is, in this application, the blocking conditions satisfy one or more of the conditions shown in items A) to C). Optionally, the amount of the blocking reagent containing SH-PEG-SH is 8 μL to 12 μL (e.g., 8, 8.5, 9, 10, 10.5, 11, 11.5, 12 μL), wherein the concentration of SH-PEG-SH is 0.1 mM to 100 mM (e.g., 0.1, 0.5, 1, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mM); and the time is 2 hours to 6 hours (e.g., 2, 2.5, 3, 3.5, 4, 4.5, 6 hours). Optionally, the amount of the blocking reagent containing MCH is 8 μL to 12 μL (e.g., 8, 8.5, 9, 10, 10.5, 11, 11.5, 12 μL), wherein the concentration of MCH is 0.1 mM to 10 mM (e.g., 0.1, 0.5, 1, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mM), and the time is 1 hour to 3 hours (e.g., 1, 1.5, 2, 2.5, 3 hours).

[0166] In some examples, the conditions for sequentially linking the substrate chain and the fusion enzyme include: (I) the volume of the solution containing the substrate chain is 8 μL to 12 μL (e.g., 8, 8.5, 9, 10, 10.5, 11, 11.5, 12 μL), wherein the concentration of the substrate chain is 1 μM to 1000 μM (e.g., 1, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 μM); (II) the volume of the solution containing the fusion enzyme is 8 μL to 12 μL (e.g., 8 μL, 8.5, 9, 10, 10.5, 11, 11.5, 12 μL). (e.g., 8, 8.5, 9, 10, 10.5, 11, 11.5, 12 μL), wherein the concentration of the fusion enzyme is 0.5 mg / mL to 3 mg / mL (e.g., 0.5, 1, 1.5, 2, 2.5, 3 mg / mL); (III) time is 1 hour to 20 hours (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 hours); or / and, (IV) temperature is 3℃ to 38℃ (e.g., 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 38℃). In this application, the conditions for sequentially connecting the substrate chain and the fusion enzyme include one or more of the conditions shown in items (I) to (IV).

[0167] In some examples, the conditions for linking the enzyme substrate complex include: I) the volume of the solution containing the enzyme substrate complex is 8 μL to 12 μL (e.g., 8, 8.5, 9, 10, 10.5, 11, 11.5, 12 μL), wherein the concentration of the enzyme substrate complex is 0.5 mg / mL to 3 mg / mL (e.g., 0.5, 1, 1.5, 2, 2.5, 3 mg / mL); II) The time is 1 hour to 20 hours (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 hours); or / and, the temperature is 3°C to 38°C (e.g., 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 38°C). That is, in this application, the conditions for linking the enzyme substrate complex include one or more of the conditions shown in (I) to (III).

[0168] In some examples, the reaction products are rinsed after the modification, embedding, blocking, and linking reactions are completed. Optionally, the rinsing solution for each reaction independently includes water and 1 mM to 100 mM (e.g., 1, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mM) Tris, and 0 mM to 1000 mM (e.g., 0, 5, 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 65) Tris. The rinsing solution contains 0, 700, 750, 800, 850, 900, 950, 1000 mM NaCl and not less than 10 mM (e.g., 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 mM) MgCl2, with a pH range of 6.5 to 8 (e.g., 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8); optionally, the rinsing may be performed 2 to 6 times (e.g., 2, 3, 4, 5, 6 times).

[0169] In some examples, the preparation steps of the enzyme substrate complex include: mixing the fusion enzyme and the substrate chain to perform a recognition reaction to prepare the enzyme substrate complex;

[0170] Optionally, the conditions for identifying the reaction include: (i) protection from light; (ii) a reaction time of 1 to 16 hours (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 hours); (iii) a reaction temperature of 3°C to 38°C (e.g., 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 38°C); or / and (iv) an initial reaction system comprising a volume ratio of 1:(0.5 to 2) (e.g., 1:0.5, 1:1:1:1). 5. A fusion enzyme solution and a substrate chain solution in a 1:2 ratio, wherein the concentration of the fusion enzyme in the fusion enzyme solution is 0.5 mg / mL to 3 mg / mL (e.g., 0.5, 1, 1.5, 2, 2.5, 3 mg / mL), and the concentration of the substrate chain in the substrate chain solution is 1 μM to 1000 μM (e.g., 1, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 μM). That is, the recognition reaction in this application satisfies one or more of the conditions shown in (i) to (iv).

[0171] In some examples, the metal electrode is pretreated before modification by the following steps: polishing, cleaning, immersing in a piranha solution, rinsing, and drying; optionally, the abrasive used for polishing includes alumina; further optionally, the alumina includes first alumina with a particle size of 0.25 μm to 0.35 μm (e.g., 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35 μm) and second alumina with a particle size of 0.03 μm to 0.07 μm (e.g., 0.03, 0.04, 0.05, 0.06, 0.07 μm); even more optionally, the polishing time using the first alumina is 4 to 6 minutes (e.g., 4, 4.5, ...). 5, 5.5, 6 minutes); More optionally, the polishing time using the second alumina is 4 to 6 minutes (e.g., 4, 4.5, 5, 5.5, 6 minutes); Optionally, the cleaning solution used for cleaning includes one or more of water, ethanol, and acetone; More optionally, the cleaning is performed using the aforementioned water, ethanol, and acetone; More preferably, the cleaning is performed using the aforementioned water, ethanol, and acetone for 10 to 20 minutes each (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 minutes); Optionally, the soaking time is 25 to 35 minutes (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 minutes); Optionally, the rinsing solution used for rinsing includes water; Optionally, the drying method includes nitrogen drying.

[0172] A third aspect of this application provides a detection device, the detection device including the ordered sensing interface described above.

[0173] A fourth aspect of this application provides a method for detecting a target substrate in a sample, the method comprising the following steps: using a three-electrode chemical detection system including the ordered sensing interface or the detection device to detect the target substrate in the sample to be tested; the enzyme substrate complex specifically recognizes the target substrate and undergoes a catalytic reaction.

[0174] The electron mediator in the detection system may or may not include methylene blue.

[0175] In some examples, the detection system satisfies the following conditions: i) the concentration of the electron mediator is 5 μM to 20 μM (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 μM); ii) the concentration of the target substrate is 0 μM to 10000 μM (e.g., 0, 5, 10, 50, 100, 150, 200, 250, ... 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000 mM); or / and, iii) media including water and 1 mM to 100 mM (e.g., 1, 10, 15, 2... 0, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100mM)) Tris, 0mM~1000mM (e.g. 1, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 250, 300, The detection system of this application satisfies one or more of the conditions shown in i) to iii). The concentrations of NaCl are 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, and 1000 mM, and MgCl2 are not less than 10 mM (e.g., 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40 mM). The pH range is 6.5 to 8.

[0176] In some examples, the detection conditions include: (a) the operating voltage of the detection is -0.4V to 0.6V (e.g., -0.4, -0.2, 0, 0.2, 0.4, 0.6V); (b) differential pulse voltammetry, cyclic voltammetry, AC impedance spectroscopy, or time-current curve method is used; and / or (c) a platinum wire is used as the counter electrode and a silver chloride electrode is used as the reference electrode. That is, the detection conditions in this application include one or more of the conditions shown in (a) to (c).

[0177] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0178] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0179] Example 1

[0180] This embodiment provides a method for synthesizing a proximal-bound DNA sheet structure. Figure 1 and Figure 2 ), including the following:

[0181] 1. Synthesis of DNA sheet structures

[0182] In this embodiment, 35 proximal scaffold chain binding portions were selected for synthesis. The specific combination includes: scaffold chain binding portions shown in SEQ ID NO.1 to SEQ ID NO.35, as shown in Table 1, which are used to bind other nucleic acids proximally.

[0183] In this embodiment, five scaffold chain binding portions containing thiol groups were selected as the anchoring ends of the synthetic electrode interface, and the sequences are shown in SEQ ID NO.270 to SEQ ID NO.274, as shown in Table 6 for the sDNA of the thiol free ends;

[0184] In this embodiment, other sequences involved are shown as SEQ ID NO.106 to SEQ ID NO.268, as shown in Table 4;

[0185] In this embodiment, the sequence of the proximal binding enzyme substrate complex linker is shown in SEQ ID NO.275;

[0186] In this embodiment, the sequence of the scaffold strand in the DNA sheet structure is shown in SEQ ID NO.269;

[0187] In this embodiment, the sequence of the substrate chain is shown in SEQ ID NO.277 (Handle C);

[0188] This embodiment uses a one-pot method to prepare DNA sheet-like structures, and the operation steps are as follows: Figure 3 As shown;

[0189] The one-pot preparation method involves mixing multiple scaffold chain linkers and then directly incubating them with a scaffold chain of long single-stranded viral genomic DNA (p7560, SEQ ID NO. 269) at a molar ratio of 10:1. The DNA sheet structure is prepared by reducing the DNA sheet structure precursor with a reducing agent to expose the free ends of thiol groups. Specifically, 20 nM of the DNA sheet structure precursor is mixed with 100 μM of TCEP (thiol reducing agent) at a volume ratio of 20 μL:20 μL in the dark for 30 minutes. Then, it is centrifuged 10 times using a 30 KD ultrafiltration tube. Finally, the volume of the DNA sheet structure is adjusted to 40 μL (the concentration of the DNA sheet structure is 10 nM) and stored at 4°C in the dark for later use.

[0190] 2. Characterization of DNA sheet structure

[0191] In this embodiment, the synthesized DNA sheet structure was diluted to 5 nM, then dropped onto the surface of a mica sheet, and its morphology was characterized using atomic force microscopy (AFM). The characterization results are as follows: Figure 4 As shown, its size is similar to Figure 1 The dimensions of the designs (84nm in length and 75nm in width) are basically the same.

[0192] In this embodiment, the mass and size of the DNA sheet-like structure were characterized using a nucleic acid gel electrophoresis method at 70V for 100min. The results are as follows. Figure 5 As shown, the proximal-bound DNA sheet structure (EE-5p-35) is similar in size to p7560, and the experimental results are accurate.

[0193] In this embodiment, the characterization of the binding ability of the DNA sheet structure to the substrate chain (Handle C) is as follows: Figure 6 As shown, the molecular weight of the DNA sheet structure (EE-5p-35) increases after binding to the substrate chain, indicating that the DNA sheet structure can recognize and bind to the substrate chain, laying the foundation for the construction of an ordered sensing interface.

[0194] Example 2

[0195] This embodiment provides a method for synthesizing a distally bound DNA sheet structure. Figure 7 ), including the following:

[0196] 1. Synthesis of DNA sheet structures

[0197] In this embodiment, five distal scaffold chain binding sites were selected for synthesis. The specific combination includes: the scaffold chain binding sites shown in SEQ ID NO.50 to SEQ ID NO.56, and Handle A used to bind other nucleic acids distally in Table 2;

[0198] In this embodiment, five scaffold chain binding portions containing thiol groups were selected as the anchoring ends for the synthetic electrode interface, with sequences shown in SEQ ID NO.270 to SEQ ID NO.274, as shown in Table 6 for the sDNA of the thiol free ends;

[0199] In this embodiment, other sequences involved are shown as SEQ ID NO.106 to SEQ ID NO.268;

[0200] In this embodiment, the sequence of the distal binding enzyme substrate complex linker is shown in SEQ ID NO.276;

[0201] In this embodiment, the sequence of the scaffold strand in the DNA sheet structure is shown in SEQ ID NO.269;

[0202] In this embodiment, the sequence of the substrate chain is shown in SEQ ID NO.277 (Handle C);

[0203] This embodiment uses a one-pot method to prepare DNA sheet-like structures, and the operation steps are as follows: Figure 3 As shown; after mixing different DNA sequences, incubate at 75°C for 5 min, then perform PCR annealing program, maintain at 65°C for 15 min, then decrease the temperature by 1°C for 15 min each time, until it reaches 4°C and maintains for 20 min, and finally maintain the system at 12°C.

[0204] At the end of the preparation process, ultrafiltration was used to purify and concentrate the DNA sheet structure.

[0205] The one-pot preparation method involves mixing multiple scaffold chain linkers and then directly incubating them with a scaffold chain of long single-stranded viral genomic DNA (p7560, SEQ ID NO. 269) at a molar ratio of 10:1. The DNA sheet structure is prepared by reducing the DNA sheet structure precursor with a reducing agent to expose the free ends of thiol groups. Specifically, 20 nM of the DNA sheet structure precursor is mixed with 100 μM of TCEP (thiol reducing agent) at a volume ratio of 20 μL:20 μL in the dark for 30 minutes. Then, it is centrifuged 10 times using a 30 KD ultrafiltration tube. Finally, the volume of the DNA sheet structure is adjusted to 40 μL (the concentration of the DNA sheet structure is 10 nM) and stored at 4°C in the dark for later use.

[0206] 2. Characterization of DNA sheet structure

[0207] In this embodiment, the synthesized DNA sheet structure was diluted to 5 nM, then dropped onto the surface of a mica sheet, and its morphology was characterized using atomic force microscopy (AFM). The characterization results are as follows: Figure 8 As shown, its size is similar to Figure 1 The dimensions of the designs are basically the same.

[0208] In this embodiment, the mass and size of the DNA sheet-like structure were characterized using a nucleic acid gel electrophoresis method at 70V for 100min. The results are as follows. Figure 5 As shown, the proximal-bound DNA sheet structure (EE-3p-7) is similar in size to p7560, and the experimental results are accurate.

[0209] Example 3

[0210] This embodiment provides a method for constructing an ordered sensing interface based on a DNA-enzyme composite nanostructure (e.g., Figure 9 (As shown) and applications, including the following:

[0211] 1. Preparation of enzyme substrate complex

[0212] 1.1 Preparation of fusion enzyme

[0213] The target enzyme gene, linker peptide (4GS), and TC1 gene were sequentially cloned into the pET32a vector using a one-step cloning kit (Vazyme) to obtain the pET32a-target enzyme-TC1 plasmid. This plasmid was transformed into *E. coli* BL21(DE3) to express the fusion enzyme. In this fusion enzyme, the target enzyme expressed by the target enzyme gene is sarcosine oxidase, and the sequence of the TC1 enzyme is shown in SEQ ID NO. 280. The TC1 enzyme naturally recognizes the substrate Handle B, the sequence of which is shown in SEQ ID NO. 278.

[0214] 1.2. Design Handle C

[0215] A Handle C was designed based on Handles A and B to connect the DNA sheet structure and the fusion enzyme, enabling the fusion enzyme to perform directional self-assembly on one side of the DNA sheet structure. The sequence of Handle C is shown in SEQ ID NO. 277. One end has the sequence of Handle B (semester shown in SEQ ID NO. 278), which can be specifically recognized by the TC1 enzyme. The other end has the sequence shown in SEQ ID NO. 279, which can be complementary to the fragment shown in SEQ ID NO. 275 in Handle A (proximal binding). Figure 2 This is a schematic diagram of the proximal binding of Handle A and the enzyme substrate complex as shown in Table 1.

[0216] 1.3 Preparation of enzyme substrate complex

[0217] The 1.5 mg / mL fusion enzyme was co-incubated with 100 μM Handle C at a volume ratio of 100 μL:100 μL at 37 °C in the dark for 3 hours. Then, the excess Handle C was removed by centrifugation 10 times using a 30 KD ultrafiltration tube. The concentration of the enzyme-substrate complex after co-incubation was adjusted to 1.5 mg / mL and stored at 4 °C for later use.

[0218] 2. Pretreatment electrode

[0219] The gold electrode surface was polished with 0.3 μm and 0.05 μm Al2O3 for 5 minutes each, then ultrasonically cleaned with water, ethanol and acetone for 15 minutes each, and dried with nitrogen. Next, the electrode was immersed in a fresh piranha solution with a volume ratio of 30% hydrogen peroxide and concentrated sulfuric acid of 3:7 for 30 minutes, then rinsed with distilled water, dried with nitrogen, and placed in a nitrogen atmosphere for later use.

[0220] 3. Construction of an ordered sensing interface

[0221] 3.1 Preparation of an ordered sensing interface for methylene blue-embedded DNA sheet structure 1

[0222] (1) Mix 10 μL of the DNA sheet structure from Example 1 with 10 μL of 10 mM methylene blue solution and incubate at 37°C in the dark for 2 hours.

[0223] (2) Next, take 10 μL of the DNA sheet structure and methylene blue mixture from step (1) and place it on the electrode surface. Incubate at 4°C in the dark for 12 hours. Then rinse three times with Tris-MgCl2 buffer solution and blow dry.

[0224] (3) Next, 10 μL of SH-PEG-SH was placed on the electrode surface and incubated at 4°C in the dark for 10 hours. The concentration of SH-PEG-SH was 10 mM. Then, it was rinsed three times with Tris-MgCl2 buffer solution and dried.

[0225] (4) Next, take 10 μL of enzyme substrate complex, incubate at 37°C for 3 hours, then rinse 3 times with buffer solution, blow dry with nitrogen and place in a nitrogen atmosphere for later use.

[0226] The Tris-MgCl2 buffer solution contains water, 10 mM Tris, and 10 mM MgCl2, with a pH of 7.5.

[0227] 3.2 Preparation of an ordered sensing interface for methylene blue-free DNA sheet structures 2

[0228] (1) Take 10 μL of the DNA sheet structure solution from Example 1 and mix it with 10 μL of Tris-MgCl2 buffer solution and place it on the electrode surface. Incubate at 4°C in the dark for 12 hours, then rinse three times with Tris-MgCl2 buffer solution and blow dry.

[0229] (2) Next, 10 μL of SH-PEG-SH was placed on the electrode surface and incubated at 4°C in the dark for 10 hours. The concentration of SH-PEG-SH was 10 mM. Then, it was rinsed three times with Tris-MgCl2 buffer solution and dried.

[0230] (3) Next, take 10 μL of enzyme substrate complex, incubate at 37°C for 3 hours, then rinse 3 times with buffer solution, blow dry with nitrogen and place in a nitrogen atmosphere for later use.

[0231] The Tris-MgCl2 buffer solution contains water, 10 mM Tris, and 10 mM MgCl2, with a pH of 7.5.

[0232] 4. Study on the activity of HUH-tagged enzyme in enzyme substrate complex

[0233] The enzyme substrate complex (SoxTC1F + Handles) and fusion enzyme prepared in section 1 were subjected to a protein gel electrophoresis test. The concentrations of the enzyme substrate complex and fusion enzyme (SoxTC1F) were 0.2 mg / mL, and the Handle chain was 10 μM. The voltage settings for the protein gel electrophoresis were 80V for 30 min and 120V for 50 min, with a dosage of 10 μL. The results are as follows. Figure 10 As shown, the molecular weight of the enzyme substrate complex is increased compared to the fusion enzyme, indicating that TC1 has the ability to recognize and bind substrate chains, laying the foundation for the construction of ordered sensing interfaces.

[0234] 5. Study on electron transfer rate

[0235] A three-electrode electrochemical testing system was constructed using the ordered sensing interface prepared in section 3. CV curves at different scan rates were obtained in a 100 μM sarcosine buffer solution system (Tris-MgCl2 buffer solution containing water, 10 mM Tris, and 10 mM MgCl2, pH = 7.5). Methylene blue was added to the buffer solution as an electron mediator in the solution system at a working concentration of 10 μM. The detection method was CV, with a working voltage range of -0.4 V to 0.6 V, a scan rate of 0.01 to 0.1 V / s, and a scan rate increment of 0.01 V / s.

[0236] The CV comparison results are as follows Figure 11 As shown, Figure 11 Figure a shows the result of the ordered sensing interface 1 modified with methylene blue. Figure 11Figure b shows the results of the unmodified methylene blue ordered sensing interface 2. Under the same test conditions, the methylene blue modified biosensor exhibits a larger current response at the methylene blue redox peak position, indicating that the electron transfer rate is improved, which also indirectly illustrates the process of charge-directed transfer.

[0237] 6. Ordered sensor interface test

[0238] A three-electrode electrochemical testing system was constructed using the ordered sensing interface prepared in step 3. Testing was conducted in a sarcosine buffer solution system (Tris-NaCl-MgCl2 buffer solution containing water, 10 mM Tris, 400 mM NaCl, and 10 mM MgCl2, pH = 7.5) with a concentration gradient from 0 μM to 100 μM. Methylene blue was added to the buffer solution as an electron mediator, with a working concentration of 10 μM. The detection method was CV, and the working voltage range was -0.4 V to 0.6 V. The results of the interface response current changes during the modification process are shown below. Figure 12 As shown, the curve response current gradually decreases as the modification process proceeds, corresponding to the self-assembly process of the ordered interface. The current increase after methylene blue insertion reflects the charge transfer process.

[0239] 7. Characterization of Ordered Sensing Interfaces

[0240] The constructed ordered sensing interface is characterized using AFM, such as... Figure 13 As shown, a distinct band can be found in the middle of the DNA sheet structure, proving that the enzyme is directionally assembled with the DNA sheet structure. Some mica sheets do not show bands because the DNA sheet structure has a front and a back side; the side without bands is the back side of the DNA sheet structure.

[0241] Example 4

[0242] This embodiment provides a method for constructing an ordered sensing interface based on a DNA-enzyme composite nanostructure (e.g., Figure 9 (As shown) and applications, including the following:

[0243] 1. Preparation of enzyme substrate complex

[0244] 1.1 Preparation of fusion enzyme

[0245] The target enzyme gene, linker peptide (4GS), and TC1 gene were sequentially cloned into the pET32a vector using a one-step cloning kit (Vazyme) to obtain the pET32a-target enzyme-TC1 plasmid. This plasmid was transformed into *E. coli* BL21(DE3) to express the fusion enzyme. In this fusion enzyme, the target enzyme expressed by the target enzyme gene is sarcosine oxidase, and the sequence of the TC1 enzyme is shown in SEQ ID NO. 280. The TC1 enzyme naturally recognizes the substrate Handle B, the sequence of which is shown in SEQ ID NO. 278.

[0246] 1.2. Design Handle C

[0247] A Handle C was designed based on Handles A and B to connect the DNA sheet structure and the fusion enzyme, enabling the fusion enzyme to perform directional self-assembly on one side of the DNA sheet structure. The sequence of Handle C is shown in SEQ ID NO. 277. One end has the sequence of Handle B (semester shown in SEQ ID NO. 278), which can be specifically recognized by the TC1 enzyme. The other end has the sequence shown in SEQ ID NO. 279, which can be complementary to the fragment shown in SEQ ID NO. 276 in Handle A (distal binding). Figure 7 This is a schematic diagram of the distal binding of Handle A and the enzyme substrate complex as shown in Table 2.

[0248] 1.3 Preparation of enzyme substrate complex

[0249] The fusion enzyme (1 mg / mL) was co-incubated with 100 μM Handle C at a volume ratio of 100 μL:100 μL at 37 °C in the dark for 3 hours. Then, the enzyme was centrifuged 10 times using a 30 KD ultrafiltration tube to remove excess Handle C. The concentration of the enzyme-substrate complex after co-incubation was adjusted to 1.5 mg / mL and stored at 4 °C for later use.

[0250] 2. Pretreatment electrode

[0251] The gold electrode surface was polished with 0.3 μm and 0.05 μm Al2O3 for 5 minutes each, then ultrasonically cleaned with water, ethanol and acetone for 15 minutes each, and dried with nitrogen. Next, the electrode was immersed in a fresh piranha solution with a volume ratio of 30% hydrogen peroxide and concentrated sulfuric acid of 3:7 for 30 minutes, then rinsed with distilled water, dried with nitrogen, and placed in a nitrogen atmosphere for later use.

[0252] 3. Construction of an ordered sensing interface

[0253] (1) Mix 10 μL of the DNA sheet structure from Example 2 with 10 μL of 100 mM methylene blue solution and incubate at 37°C in the dark for 2 hours.

[0254] (2) Next, take 10 μL of the DNA sheet structure and methylene blue mixture from step (1) and place it on the electrode surface. Incubate at 4°C in the dark for 12 hours. Then rinse three times with Tris-NaCl-MgCl2 buffer solution and blow dry.

[0255] (3) Next, 10 μL of SH-PEG-SH was placed on the electrode surface and incubated at 4°C in the dark for 10 hours. The concentration of SH-PEG-SH was 10 mM. Then, it was rinsed three times with Tris-NaCl-MgCl2 buffer solution and dried.

[0256] (4) Next, take 10 μL of enzyme substrate complex, incubate at 37°C for 3 hours, then rinse 3 times with buffer solution, blow dry with nitrogen and place in a nitrogen atmosphere for later use.

[0257] The Tris-NaCl-MgCl2 buffer solution contains water, 10 mM Tris, 400 mM NaCl, and 10 mM MgCl2, with a pH of 7.5.

[0258] 4. Ordered sensor interface test

[0259] A three-electrode electrochemical testing system was constructed using the ordered sensing interface prepared in step 3. Tests were conducted in a sarcosine buffer solution system (Tris-NaCl-MgCl2 buffer solution containing water, 10 mM Tris, 400 mM NaCl, and 10 mM MgCl2, pH = 7.5) with a concentration gradient from 0 μM to 100 μM. Methylene blue was added to the buffer solution as an electron mediator, with a working concentration of 10 μM. The detection method was DPV, and the working voltage range was -0.4 V to 0.1 V. The results of the interface response current changes during the modification process are shown below. Figure 14 As shown, the curve response current gradually decreases as the modification process proceeds, corresponding to the self-assembly process of the orientation interface. The current increase after methylene blue insertion reflects the charge transfer process.

[0260] 5. Characterization of ordered sensing interfaces

[0261] The constructed ordered sensing interface is characterized using AFM, such as... Figure 15 As shown, a distinct band can be found in the middle of the DNA sheet structure, proving that the enzyme is directionally assembled with the DNA sheet structure. Some mica sheets do not show bands because the DNA sheet structure has a front and a back side; the side without bands is the back side of the DNA sheet structure.

[0262] The beneficial effects achieved by the embodiments of this application include: (1a) preparing a novel fusion enzyme-DNA sheet structure sensor; (2) providing a scheme for constructing an ordered interface related to the DNA sheet structure; and (3) introducing methylene blue molecules into the DNA sheet structure to construct an electron transfer channel and improve the electron transfer rate of the enzyme electrode interface.

[0263] Table 1. Handle A for proximal binding of other nucleic acids

[0264] Serial Number Staple chain name DNA sequence SEQ ID NO.1 EE-H03-5p-Y-001 (5')AGTTTCGGCCATCCGGTAAACAGGAGAAGAACTCAAACTATAAAAACGC SEQ ID NO.2 EE-H03-5p-Y-002 (5')AGTTTCGGCCATCCGGTGGAACGGTTTTGATTAGTAATAACGCTCAATC SEQ ID NO.3 EE-H03-5p-Y-003 (5')AGTTTCGGCCATCCGGTTTTATAATTCACGCAAATTAACCGGGCAGATT SEQ ID NO.4 EE-H03-5p-Y-004 (5')AGTTTCGGCCATCCGGTTCCGAACTGACGCATTTCACATAAGGCCAGTG SEQ ID NO.5 EE-H03-5p-Y-005 (5')AGTTTCGGCCATCCGGTAGTAAACATGGGCACGAATATAGGGGGTGGAT SEQ ID NO.6 EE-H03-5p-Y-006 (5')AGTTTCGGCCATCCGGTTACCTCGATGCGGCCCTGCCATCTATGCGCAC SEQ ID NO.7 EE-H03-5p-Y-007 (5')AGTTTCGGCCATCCGGTCCGAGCTCTCGCCCTGGAGTGACTATTGTCAA SEQ ID NO.8 EE-H09-5p-Y-001 (5')AGTTTCGGCCATCCGGTGGATTTAGAATTCATCAATATAATATCAAAAT SEQ ID NO.9 EE-H09-5p-Y-002 (5')AGTTTCGGCCATCCGGTCGACAACTTATCATCATATTCCTGAGAAATTG SEQ ID NO.10 EE-H09-5p-Y-003 (5')AGTTTCGGCCATCCGGTGTTATTAAGGAACAAAGAAACCACAGATGAAT SEQ ID NO.11 EE-H09-5p-Y-004 (5')AGTTTCGGCCATCCGGTACAGGAAGTTGATAATCAGAAAAGATATTTTA SEQ ID NO.12 EE-H09-5p-Y-005 (5')AGTTTCGGCCATCCGGTTGTAAACGAAAACTAGCATGTCAAAGGTAAAG SEQ ID NO.13 EE-H09-5p-Y-006 (5')AGTTTCGGCCATCCGGTTAAATTTTAGCAAAAGAGAATCAGACAGTC SEQ ID NO.14 EE-H09-5p-Y-007 (5')AGTTTCGGCCATCCGGTCCAATAGGAAGGCTATCAGGTCATAACCGTTC SEQ ID NO.15 EE-H15-5p-Y-001 (5')AGTTTCGGCCATCCGGTCAATCGCAATGCGTTATACAAACATATTTA SEQ ID NO.16 EE-H15-5p-Y-002 (5')AGTTTCGGCCATCCGGTTTCAAATATCATAATTACTAGAAAAGAGGCAT SEQ ID NO.17 EE-H15-5p-Y-003 (5')AGTTTCGGCCATCCGGTGACCTAAATAAGGCGTTAAATAAATAAAAGTA SEQ ID NO.18 EE-H15-5p-Y-004 (5')AGTTTCGGCCATCCGGTGCGTTTTAGAAGCCCGAAAGACTTCCAGACGA SEQ ID NO.19 EE-H15-5p-Y-005 (5')AGTTTCGGCCATCCGGTACCGGAAGGCAAAGCGGATTGCATGGCTTTTG SEQ ID NO.20 EE-H15-5p-Y-006 (5')AGTTTCGGCCATCCGGTAGTACCTCAGGTCTTTACCCTGTAATAGTA SEQ ID NO.21 EE-H15-5p-Y-007 (5')AGTTTCGGCCATCCGGTGGTCATTTTCAGAAAACGAGAATGCAATACTG SEQ ID NO.22 EE-H21-5p-Y-001 (5')AGTTTCGGCCATCCGGTTAACGAGCTTAACTGAACCCCTGATTGAGTT SEQ ID NO.23 EE-H21-5p-Y-002 (5')AGTTTCGGCCATCCGGTGTTACAAAAAAAACAGGGAAGCCAATGAAA SEQ ID NO.24 EE-H21-5p-Y-003 (5’)AGTTTCGGCCATCCGGTCAATCCAATGAAAATAGCAGCCTTCCCTTTTT SEQ ID NO.25 EE-H21-5p-Y-004 (5’)AGTTTCGGCCATCCGGTTTGTGTCGAGATTTGTATCATCGCGTTAAAGG SEQ ID NO.26 EE-H21-5p-Y-005 (5’)AGTTTCGGCCATCCGGTACTTAGCCATTATACCAAGCGCGATCAGCAGC SEQ ID NO.27 EE-H21-5p-Y-006 (5’)AGTTTCGGCCATCCGGTATAAGGGATACACTAAAACACTCAAGCAACGG SEQ ID NO.28 EE-H21-5p-Y-007 (5’)AGTTTCGGCCATCCGGTAGGACAGAGCACCAACCTAAAACGGACTTTTT SEQ ID NO.29 EE-H27-5p-Y-001 (5’)AGTTTCGGCCATCCGGTCACCGTAAACCCTCAGAACCGCCACGCCAGCA SEQ ID NO.30 EE-H27-5p-Y-002 (5’)AGTTTCGGCCATCCGGTCCTTTAGCCCACCACCGGAACCGCGACGATTG SEQ ID NO.31 EE-H27-5p-Y-003 (5’)AGTTTCGGCCATCCGGTCGGCATTTCTTTTCATAATCAAAAAATCCTCA SEQ ID NO.32 EE-H27-5p-Y-004 (5’)AGTTTCGGCCATCCGGTATAGGTGTGGGTTGATATAAGTATTCTCTGAA SEQ ID NO.33 EE-H27-5p-Y-005 (5’)AGTTTCGGCCATCCGGTTACCGCCACTCAGTACCAGGCGGAACATGGCT SEQ ID NO.34 EE-H27-5p-Y-006 (5’)AGTTTCGGCCATCCGGTCCGCCACCTCAAGAGAAGGATTAGTAATAAGT SEQ ID NO.35 EE-H27-5p-Y-007 (5’)AGTTTCGGCCATCCGGTCAGGGATAAAACATGAAAGTATTATAACAGTG

[0265] Table 2. Handle A for remote binding of other nucleic acids

[0266]

[0267]

[0268] Table 3 shows the segments in Handle A that are complementary to the scaffold chain, corresponding to Tables 1 and 2.

[0269] Sequence number Corresponding Staple strand name DNA sequence SEQ ID NO.71 Corresponding to EE-H03-5p-Y-001 TAAACAGGAGAAGAACTCAAACTATAAAAACGC SEQ ID NO.72 Corresponding to EE-H03-5p-Y-002 TGGAACGGTTTTGATTAGTAATAACGCTCAATC SEQ ID NO.73 Corresponding to EE-H03-5p-Y-003 TTTTATAATTCACGCAAATTAACCGGGCAGATT SEQ ID NO.74 Corresponding to EE-H03-5p-Y-004 TTCCGAACTGACGCATTTCACATAAGGCCAGTG SEQ ID NO.75 Corresponding to EE-H03-5p-Y-005 TAGTAAACATGGGCACGAATATAGGGGGTGGAT SEQ ID NO.76 Corresponding to EE-H03-5p-Y-006 TTACCTCGATGCGGCCCTGCCATCTATGCGCAC SEQ ID NO.77 Corresponding to EE-H03-5p-Y-007 TCCGAGCTCTCGCCCTGGAGTGACTATTGTCAA SEQ ID NO.78 Corresponding to EE-H09-5p-Y-001 TGGATTTAGAATTCATCAATATAATATCAAAAT SEQ ID NO.79 Corresponding to EE-H09-5p-Y-002 TCGACAACTTATCATCATATTCCTGAGAAATTG SEQ ID NO.80 Corresponding to EE-H09-5p-Y-003 TGTTATTAAGGAACAAAGAAACCACAGATGAAT SEQ ID NO.81 Corresponding to EE-H09-5p-Y-004 TACAGGAAGTTGATAATCAGAAAAGATATTTTA SEQ ID NO.82 Corresponding to EE-H09-5p-Y-005 TTGTAAACGAAAACTAGCATGTCAAAGGTAAAG SEQ ID NO.83 Corresponding to EE-H09-5p-Y-006 TTAAATTTTAGCAAACAAGAGAATCAGACAGTC SEQ ID NO.84 Corresponding to EE-H09-5p-Y-007 TCCAATAGGAAGGCTATCAGGTCATAACCGTTC SEQ ID NO.85 Corresponding to EE-H15-5p-Y-001 TCAATCGCAATATGCGTTATACAAACATATTTA SEQ ID NO.86 Corresponding to EE-H15-5p-Y-002 TTTCAAATATCATAATTACTAGAAAAGAGGCAT SEQ ID NO.87 Corresponding to EE-H15-5p-Y-003 TGACCTAAAATAAGGCGTTAAATAAATAAAGTA SEQ ID NO.88 Corresponding to EE-H15-5p-Y-004 TGCGTTTTAGAAGCCCGAAAGACTTCCAGACGA SEQ ID NO.89 Corresponding to EE-H15-5p-Y-005 TACCGGAAGGCAAAGCGGATTGCATGGCTTTTG SEQ ID NO.90 Corresponding to EE-H15-5p-Y-006 TAGAGTACCTCAGGTCTTTACCCTGTAATAGTA SEQ ID NO.91 Corresponding to EE-H15-5p-Y-007 TGGTCATTTTCAGAAAACGAGAATGCAATACTG SEQ ID NO.92 Corresponding to EE-H21-5p-Y-001 TTAACGAGCTTAACTGAACACCCTGATTGAGTT SEQ ID NO.93 Corresponding to EE-H21-5p-Y-002 TGTTACAAAATAAAAACAGGGAAGCCAATGAAA SEQ ID NO.94 Corresponding to EE-H21-5p-Y-003 TCAATCCAATGAAAATAGCAGCCTTCCCTTTTTT SEQ ID NO.95 Corresponding to EE-H21-5p-Y-004 TTTGTGTCGAGATTTGTATCATCGCGTTAAAGG SEQ ID NO.96 Corresponding to EE-H21-5p-Y-005 TACTTAGCCATTATACCAAGCGCGATCAGCAGC SEQ ID NO.97 Corresponding to EE-H21-5p-Y-006 TATAAGGGATACACTAAAACACTCAAGCAACGG SEQ ID NO.98 Corresponding to EE-H21-5p-Y-007 TAGGACAGAGCACCAACCTAAAACGGACTTTTT SEQ ID NO.99 Corresponding to EE-H27-5p-Y-001 TCACCGTAAACCCTCAGAACCGCCACGCCAGCA SEQ ID NO.100 Corresponding to EE-H27-5p-Y-002 TCCTTTAGCCCACCACCGGAACCGCGACGATTG SEQ ID NO.101 Corresponding to EE-H27-5p-Y-003 TCGGCATTTCTTTTCATAATCAAAAAATCCTCA SEQ ID NO.102 Corresponding to EE-H27-5p-Y-004 TATAGGTGTGGGTTGATATAAGTATTCTTCTGAA SEQ ID NO.103 Corresponding to EE-H27-5p-Y-005 TTACCGCCACTCAGTACCAGGCGGAACATGGCT SEQ ID NO.104 Corresponding to EE-H27-5p-Y-006 TCCGCCACCTCAAGAGAAGGATTAGTAATAAGT SEQ ID NO.105 Corresponding to EE-H27-5p-Y-007 TCAGGGATAAAACATGAAAGTATTATAACAGTG

[0270] Table 4. Other scaffold chain joints that are complementary to the scaffold chain

[0271]

[0272]

[0273]

[0274] Table 5: Genomic DNA of long single-stranded viruses

[0275]

[0276] Table 6. sDNA with thiol free ends

[0277]

[0278]

[0279] SEQ ID NO.275: Free end (5')AGTTTCGGCCATCCGG.

[0280] SEQ ID NO.276: Free end (3')GGCCTACCGGCTTTGA.

[0281] SEQ ID NO.277 (Handle C): CGTATATATTACAGACCGGATGGCCGAAACT (substrate chain).

[0282] SEQ ID NO. 278 (Handle B): CGTATAATATTAC.

[0283] SEQ ID NO. 279: CCGGATGGCCGAAACT.

[0284] SEQ ID NO.280:

[0285] MHHHHHHGGGGSSTHFDVIVVGAGSMGMAAGYYLAKQGVKTLLVDSFDPPHTNGSHHGDTRIIRHAYGEGREYVPFALRAQELWYELEKETHHKIF

[0286] TQTGVLVFGAKGESDFVAETMEAAKIHSLEHELFEGKQLTERWAGVEVPENYEAIFEPNSGVLFSENCIQAYRELAEAHGAKVLTYAPVEDFEVSEDLV

[0287] KIQTAKGLYTANKLIVSMGAWNSKLLSKLGVEIPLQPYRQVVAYFECNEEKYSNNVHYPAFMVEVANGIYYGFPSFGGSGLKIGYHTYGQEIDPDTINR

[0288] EFGAYPEDEANLRKFLEKYMPEANGEFKKGAVCMYTKTPDEHFVIDLHPKYSNVVIGAGFSGHGFKFSSAVGETLAQLAVTGKTEHDISIFSLNRDAL

[0289] KKEAAKPRSGRFSIKAKNYFLTYPKCDLTKENALSQITNLQTPTNKLFIKICRELHENGEPHLHILIQFEGKYNCTNQRFFDLVSPTRSAHFHPNIQGAKSSSDVKSYIDKDGDVLEWGTFQIDGR.

[0290] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0291] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. An ordered sensing interface based on a DNA-enzyme composite nanostructure, characterized in that, The ordered sensing interface includes: an electrode, and a DNA-enzyme composite nanostructure connected to the electrode. The DNA-enzyme composite nanostructure includes a DNA electron transport module and an enzyme catalysis module; The DNA electron transport module includes a DNA sheet structure containing a mediator; The enzyme catalytic module includes an enzyme substrate complex, which is attached to the DNA sheet structure. The enzyme substrate complex, the mediator-containing DNA sheet structure, and the electrode are spatially ordered in the order of enzyme substrate complex - mediator-containing DNA sheet structure - electrode.

2. The ordered sensing interface based on the DNA-enzyme composite nanostructure according to claim 1, characterized in that, The mediator includes an electronic mediator; The main body of the DNA electron transport module is a DNA sheet structure containing the electron medium, which is used to directionally anchor the enzyme catalytic module on the electrode and realize directional electron transport.

3. The ordered sensing interface based on the DNA-enzyme composite nanostructure according to claim 1, characterized in that, The main body of the enzyme catalysis module is the enzyme substrate complex, which is used to catalyze the substrate to produce an electroactive substance. The electrons generated by the catalytic reaction are directionally transferred to the electrode through the DNA electron transport module.

4. The ordered sensing interface based on the DNA-enzyme composite nanostructure according to any one of claims 1 to 3, characterized in that, The mediator includes a cationic electron mediator; Optionally, the cationic electron mediator includes one or more of methylene blue, methyl chloride, ferrocene and their derivatives.

5. The ordered sensing interface based on a DNA-enzyme composite nanostructure according to any one of claims 1 to 3, characterized in that, The enzyme-substrate complex comprises a fusion enzyme and a substrate chain. Optionally, the fusion enzyme includes a catalytic enzyme and a HUH-tagged enzyme; Optionally, the substrate chain includes nucleic acids; Optionally, the catalytic enzyme includes one or more of sarcosine oxidase, uricase oxidase, and lactate dehydrogenase; Optionally, the HUH tag enzyme includes one or more of TC1, Trwc, Tn608, TopoI, DCV, Int-Tn, HI0217, TraI36, RayT AAV5-ReP, FBNYV-Rep, and SIRVI-Rep.

6. The ordered sensing interface based on the DNA-enzyme composite nanostructure according to claim 5, characterized in that, One end of the substrate chain is specifically recognized by the HUH tag enzyme and undergoes autocatalytic ligation, while the other end binds to the DNA electron transport module through complementary base pairing.

7. The ordered sensing interface based on a DNA-enzyme composite nanostructure according to any one of claims 1 to 3 and 6, characterized in that, The electrode satisfies one or more of the conditions shown in 1) and 2): 1) The electrode contains gold; Optionally, the gold-containing electrode is a gold electrode, a gold-plated electrode, or a gold adsorption electrode; And, 2) the electrode includes one or more of a planar electrode and a printed electrode.

8. The method for constructing an ordered sensing interface based on a DNA-enzyme composite nanostructure according to any one of claims 1 to 7, characterized in that, The construction method includes the steps of constructing an ordered sensing interface using the electrode, the DNA sheet structure, the mediator, and the enzyme substrate complex.

9. The method for constructing an ordered sensing interface based on a DNA-enzyme composite nanostructure according to claim 8, characterized in that, The construction method includes the following steps: (1) The electrodes are pretreated as follows: the electrodes are polished, cleaned, soaked and rinsed in piranha solution, and then dried. (2) Prepare the DNA sheet structure and the fusion enzyme; (3) First, modify the DNA sheet structure on the surface of the electrode, and then embed the mediator on the DNA sheet structure; Alternatively, the mediator can be embedded into the DNA sheet structure first, and then the DNA sheet structure can be modified on the electrode; (3) Seal the blank sites on the electrode interface; and, (4) The substrate chain and the fusion enzyme are sequentially linked to the DNA sheet structure, or the enzyme-substrate complex is linked to the DNA sheet structure.

10. A testing device, characterized in that, The detection device includes an ordered sensing interface as described in any one of claims 1 to 7.

11. A method for detecting a target substrate in a sample, characterized in that, The detection method includes the following steps: The target substrate in the sample to be tested is detected using a three-electrode chemical detection system comprising an ordered sensing interface as described in any one of claims 1 to 7 or the detection device as described in claim 10. The enzyme substrate complex specifically recognizes the target substrate and undergoes a catalytic reaction.

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