Biosensor based on chitin membrane, preparation method and kit

Abstract

The invention provides a chitin membrane-based biosensor, a preparation method and a kit. Specifically, the biosensor comprises a chitin film layer which comprises a first sensing area and a second sensing area; the adhesion film layer is arranged in the first sensing area; the wire layer is arranged in the second sensing area; the electrochemical sensing layer is arranged in the second sensing area and is communicated with the wire layer; the nucleic acid probe group comprises a first nucleic acid sequence, a second nucleic acid sequence, a third nucleic acid sequence and a fourth nucleic acid sequence. The first sensing area and the second sensing area are simultaneously arranged on the chitin film layer, and the nucleic acid probe group is used for performing fluorescence detection on the to-be-detected object in the first sensing area and performing electrochemical detection on the to-be-detected object in the second sensing area, so that high-sensitivity and multi-modal detection on the to-be-detected object is realized, and the sensor has a wide application prospect in the field of biological detection.

Description

Technical Field

[0001] The present disclosure relates to the technical field of biosensors, and in particular to a biosensor based on a chitin membrane, a preparation method, and a kit. Background Art

[0002] With the continuous advancement of bioassay technology, the demand for biosensors with high sensitivity, high specificity, and multimodal detection is growing. Traditional single-modality biosensors often have limitations in terms of detection accuracy and comprehensiveness. For example, electrochemical sensors have good quantitative analysis capabilities but may lack specificity in molecular recognition. Fluorescence sensors, while highly sensitive, have room for improvement in stability in complex environments. Therefore, developing biosensors that can integrate the advantages of multiple detection modalities is of great significance. Summary of the Invention

[0003] In view of this, the purpose of the present disclosure is to provide a biosensor based on chitin membrane, a preparation method and a kit.

[0004] Based on the above objectives, the present disclosure provides a chitin membrane-based biosensor, comprising:

[0005] The chitin membrane layer includes a first sensing area and a second sensing area;

[0006] an attachment film layer, disposed on the first sensing area;

[0007] a conductive line layer, disposed in the second sensing area;

[0008] an electrochemical sensing layer, disposed in the second sensing area and connected to the wire layer; and

[0009] A nucleic acid probe set, wherein

[0010] The nucleic acid probe set includes a first nucleic acid sequence, a second nucleic acid sequence, a third nucleic acid sequence and a fourth nucleic acid sequence;

[0011] The first nucleic acid sequence modifies the attachment membrane layer;

[0012] The second nucleic acid sequence includes an aptamer sequence, a G-rich segment, and a linker sequence of the analyte, and a complementary sequence exists between the aptamer sequence and the first nucleic acid sequence;

[0013] There are complementary sequences between the third nucleic acid sequence and the aptamer sequence of the analyte and the connecting sequence;

[0014] The fourth nucleic acid sequence modifies the electrochemical sensing layer; the fourth nucleic acid sequence and the connecting sequence have a complementary sequence.

[0015] In some embodiments, the analyte comprises ochratoxin; the first nucleic acid sequence comprises a sequence having at least 70% identity to SEQ ID No. 1;

[0016] The second nucleic acid sequence comprises a sequence having at least 70% identity to SEQ ID No. 2;

[0017] The third nucleic acid sequence comprises a sequence having at least 70% identity with SEQ ID No. 3;

[0018] The fourth nucleic acid sequence comprises a sequence having at least 70% identity to SEQ ID No.4.

[0019] In some embodiments, a Fe3O4@ZIF-8 nanoprobe is further included; the surface of the Fe3O4@ZIF-8 nanoprobe is modified with a fifth nucleic acid sequence; and the fifth nucleic acid sequence includes an aptamer sequence of the analyte.

[0020] In some embodiments, the fifth nucleic acid sequence comprises a sequence that is at least 70% identical to SEQ ID No.5.

[0021] In some embodiments, the first end of the first nucleic acid sequence comprises an amino modification; and / or

[0022] The second end of the fourth nucleic acid sequence includes an amino modification.

[0023] In some embodiments, the material of the attachment film layer includes SEBS-g-MAH; and / or

[0024] The material of the conductive line layer includes at least one of single-walled carbon nanotubes, hydroxylated single-walled carbon nanotubes and carboxylated single-walled carbon nanotubes; and / or

[0025] The material of the electrochemical sensing layer includes semiconductor single-walled carbon nanotubes.

[0026] In some embodiments, activated carboxyl groups are provided on the surfaces of the attachment film layer and the electrochemical sensing layer.

[0027] Based on the same inventive concept, the present disclosure also provides a method for preparing a chitin membrane-based biosensor, comprising:

[0028] Providing a chitosan film layer; comprising a first sensing area and a second sensing area;

[0029] forming an adhesion film layer on the first sensing area;

[0030] forming a wire layer and an electrochemical sensing layer on the second sensing area; wherein the second sensing area is connected to the wire layer;

[0031] Assembling the first nucleic acid sequence, the second nucleic acid sequence and the third nucleic acid sequence on the attachment film layer;

[0032] A fourth nucleic acid sequence is bound to the electrochemical sensing layer; wherein,

[0033] The first nucleic acid sequence modifies the attachment membrane layer;

[0034] The second nucleic acid sequence includes an aptamer sequence, a G-rich segment, and a linker sequence of the analyte, and a complementary sequence exists between the aptamer sequence and the first nucleic acid sequence;

[0035] There are complementary sequences between the third nucleic acid sequence and the aptamer sequence of the analyte and the connecting sequence;

[0036] The fourth nucleic acid sequence has a complementary sequence to the linker sequence or the adapter sequence of the analyte.

[0037] Based on the same inventive concept, an embodiment of the present disclosure further provides a detection kit, characterized in that it includes any of the aforementioned biosensors.

[0038] In some embodiments, a fluorescent substance is also included.

[0039] As can be seen from the above description, the present disclosure provides a chitin membrane-based biosensor, preparation method and kit, which simultaneously set a first sensing area and a second sensing area on the chitin membrane layer, and use a nucleic acid probe group to perform fluorescence detection of the analyte in the first sensing area and electrochemical detection of the analyte in the second sensing area, thereby achieving highly sensitive and multimodal detection of the analyte, and has broad application prospects in the field of biological detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the present disclosure or related technologies, the following briefly introduces the drawings required for use in the embodiments or related technical descriptions. Obviously, the drawings described below are only embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0041] Figure 1 A schematic diagram of a preparation process of a biosensor provided by an embodiment of the present disclosure is shown;

[0042] Figure 2 The linear relationship between fluorescence intensity and OTA concentration provided by the embodiment of the present disclosure is shown;

[0043] Figure 3The linear relationship between an electrochemical signal and OTA concentration provided by an embodiment of the present disclosure is shown;

[0044] Figure 4 The linear relationship between absorbance and OTA concentration provided by the embodiment of the present disclosure is shown. DETAILED DESCRIPTION

[0045] In order to make the objectives, technical solutions and advantages of the present disclosure more clearly understood, the present disclosure is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0046] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in the embodiments of the present disclosure should have the usual meanings understood by people with ordinary skills in the field to which the present disclosure belongs. The "first", "second" and similar words used in the embodiments of the present disclosure do not indicate any order, quantity or importance, but are only used to distinguish different components. "Include" or "comprising" and similar words mean that the elements or objects appearing before the word include the elements or objects listed after the word and their equivalents, without excluding other elements or objects. "Up", "down", "left", "right" and the like are only used to indicate relative positional relationships. When the absolute position of the described object changes, the relative positional relationship may also change accordingly. Unless otherwise specified, "μM" in this article refers to "μmol / L" and "mM" refers to "mmol / L".

[0047] In order to facilitate understanding of the technical solutions of the present disclosure, some technical terms involved in the present disclosure are introduced below.

[0048] Chitin, a natural polysaccharide, possesses excellent biocompatibility, biodegradability, and unique physical and chemical properties, showing great potential for application in numerous fields, including biomedicine and environmental science. Its wide availability and relatively low cost make it an ideal substrate for biosensors.

[0049] Ochratoxin A (OTA) is a secondary metabolite produced by Aspergillus and Penicillium fungi. It is commonly found in moldy grains, coffee beans, wine, dried fruits and other foods. It is nephrotoxic, carcinogenic and immunotoxic, so its rapid detection technology is crucial.

[0050] An OTA aptamer (Ochratoxin A Aptamer) is an oligonucleotide sequence, such as single-stranded DNA or RNA, obtained through artificial screening that specifically recognizes and binds to OTA with high affinity. Through its unique three-dimensional conformation, the OTA aptamer forms a spatial complementarity with the OTA molecule, achieving highly specific binding. Its binding ability stems from the aptamer's nucleotide sequence, which locks onto the target through hydrogen bonding, van der Waals forces, and other interactions.

[0051] Carbodiimide crosslinker (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, referred to as EDC) is a water-soluble carbodiimide compound, commonly used as a coupling agent for carboxylic acid and amino groups.

[0052] Phosphate-buffered saline (PBS) is a buffer widely used in biochemical research. Its main components include NaHPO, KHPO, NaCl, and KCl. PBS serves as a solvent, dissolving and preserving reagents and maintaining the stability of cells and biomolecules.

[0053] ZIF-8 (Zeolite-Imidazolate Framework-8) is a metal-organic framework (MOF) material composed of zinc ions (Zn2+) and 2-methylimidazole (2-MeIm) ligands, and belongs to the Zeolite-Imidazolate Frameworks (ZIFs) family.

[0054] Bovine serum albumin (BSA) is a major protein in bovine serum. It has a molecular weight of approximately 66.5 kDa, is composed of 607 amino acids, and has an isoelectric point of 4.7. It is widely used in biochemical experiments, for example as a protein standard, stabilizer, carrier protein, and in cell culture.

[0055] 3,3',5,5'-Tetramethylbenzidine (TMB) is a commonly used chromogenic substrate.

[0056] Thioflavin T (ThT) is a fluorescent dye that has weak fluorescence in aqueous solution. However, when it binds to specific nucleic acid or protein structures, the fluorescence intensity is significantly enhanced.

[0057] SEBS (Styrene Ethylene Butylene Styrene) is a linear triblock copolymer material with polystyrene as the terminal segment and an ethylene-butene copolymer derived from hydrogenated polybutadiene as the middle elastic block. SEBS does not contain unsaturated double bonds, resulting in excellent stability and aging resistance.

[0058] Maleic anhydride (MAH) is an important organic chemical raw material with a wide range of industrial applications.

[0059] SEBS-g-MAH (maleic anhydride grafted styrene-ethylene-butadiene-styrene block copolymer) is a thermoplastic elastomer material modified by maleic anhydride grafting. Maleic anhydride (MAH) is grafted onto the molecular chain of SEBS through a chemical reaction.

[0060] As described in the background technology section, single-modality biosensors can no longer meet the needs of highly sensitive and specific detection of biomolecules in complex samples.

[0061] In view of this, the embodiments of the present disclosure provide a chitin membrane-based biosensor, a preparation method, and a kit. By simultaneously setting a first sensing area and a second sensing area on the chitin membrane layer, a nucleic acid probe group is used to perform fluorescence detection of the analyte in the first sensing area and electrochemical detection of the analyte in the second sensing area, thereby achieving highly sensitive and multimodal detection of the analyte, which has broad application prospects in the field of biological detection.

[0062] In order to make the technical solution of the present disclosure clearer and easier to understand, the chitin membrane-based biosensor, preparation method and kit provided by the present disclosure are described in detail below with reference to the accompanying drawings and specific examples.

[0063] In a first aspect, embodiments of the present disclosure provide a biosensor based on a chitin membrane. Figure 1 FIG. 1 is a schematic diagram showing a preparation process of a biosensor provided by an embodiment of the present disclosure. Figure 1 As shown, the biosensor includes:

[0064] The chitin film layer 101 includes a first sensing region 103 and a second sensing region 102; an attachment film layer 201 disposed on the first sensing region 103; a wire layer 203 disposed on the second sensing region 102; an electrochemical sensing layer 202 disposed on the second sensing region 102 and connected to the wire layer 203; and a nucleic acid probe assembly. Activated carboxyl groups are provided on the surfaces of the attachment film layer 201 and the electrochemical sensing layer 202.

[0065] Optionally, the material of the adhesion film layer 201 includes SEBS-g-MAH. Optionally, the material of the wire layer 203 includes at least one of single-walled carbon nanotubes (SWCNTs), hydroxylated single-walled carbon nanotubes (SWCNTs-OH), and carboxylated single-walled carbon nanotubes (SWCNTs-COOH). Optionally, the material of the electrochemical sensing layer 202 includes semiconductor single-walled carbon nanotubes (s-SWCNTs).

[0066] In some embodiments, the nucleic acid probe set includes a first nucleic acid sequence 301 , a second nucleic acid sequence, a third nucleic acid sequence, and a fourth nucleic acid sequence.

[0067] The first nucleic acid sequence 301 modifies the attachment film 201; here, the first nucleic acid sequence includes a second end (e.g., a 5' end) and a first end (e.g., a 3' end). Optionally, the 3' end of the first nucleic acid sequence 301 may include an amino modification, such as NH2-C6, where the NH2 can bind to carboxyl groups on the surface of the attachment film 201, thereby modifying the attachment film 201. Optionally, unbound carboxyl groups can be blocked using BSA 401 to prevent nonspecific binding.

[0068] The second nucleic acid sequence 302 includes an aptamer sequence, a G-rich segment, and a linker sequence of the analyte, and a complementary sequence exists between the aptamer sequence and the first nucleic acid sequence 301 .

[0069] It should be noted that the first nucleic acid sequence 301 can be a complementary sequence to a portion of the sequence in the aptamer sequence, and this disclosure is not limited to this. Based on the complementary pairing of the aptamer sequence and the first nucleic acid sequence, the second nucleic acid sequence 302 can be assembled on the first nucleic acid sequence 301. The G-rich segment can form a G-quadruplex, which helps to change the fluorescence intensity of the fluorescent substance. For example, Thioflavin T can specifically embed into the groove of the G-quadruplex. This binding will change the electronic environment of Thioflavin T, so that its fluorescence intensity is significantly enhanced.

[0070] Complementary sequences exist between the third nucleic acid sequence 303 and the aptamer sequence and linker sequence of the analyte, respectively. Here, the third nucleic acid sequence 303 is complementary to the linker sequence of the second nucleic acid sequence 302, allowing the third nucleic acid sequence 303 to bind to the second nucleic acid sequence 302. Furthermore, because the third nucleic acid sequence 303 contains a complementary sequence to the aptamer sequence of the analyte, another second nucleic acid sequence 302 can be bound to the third nucleic acid sequence 303. Based on this sequence design, multiple second nucleic acid sequences 302 and multiple third nucleic acid sequences 303 can be assembled on each first nucleic acid sequence 301 to form a signal amplification element, effectively improving detection sensitivity and providing the possibility for the detection of trace biomolecules.

[0071] Among them, the fourth nucleic acid sequence 304 modifies the electrochemical sensing layer 202. Here, the fourth nucleic acid sequence 304 includes a second end (e.g., a 5' end) and a first end (e.g., a 3' end). Optionally, the 5' end of the fourth nucleic acid sequence 304 may include an amino modification, such as NH2-C6; NH2 can bind to the carboxyl groups on the surface of the electrochemical sensing layer 202 to achieve modification of the electrochemical sensing layer 202. Optionally, BSA401 can be used to block the unbound carboxyl groups to prevent nonspecific binding. Furthermore, the fourth nucleic acid sequence 304 has a complementary sequence to the connecting sequence of the second nucleic acid sequence, so that the second nucleic acid sequence 304 can also be assembled on the fourth nucleic acid sequence 304.

[0072] Based on the above-mentioned biosensor, two modes of detection can be performed on the analyte. Specifically, first, the first nucleic acid sequence 301 modifies the attachment film layer 201 and is sealed with BSA, the second nucleic acid sequence 302 and the third nucleic acid sequence 303 are assembled into a "linear" nucleic acid probe, and the fourth nucleic acid sequence 304 modifies the electrochemical sensing layer 202 and is sealed with BSA; next, the "linear" nucleic acid probe is assembled on the first nucleic acid sequence 301; then, the analyte solution is added, and the analyte solution contacts the attachment film layer 201 and the electrochemical sensing layer 202. Due to the specific binding of the aptamer sequence of the analyte and the second nucleic acid sequence 302, the second nucleic acid sequence 302 is separated from the attachment film layer 201. It should be understood that the second nucleic acid sequence 302 is connected to the second nucleic acid sequence 302 away from the first nucleic acid sequence. The other nucleic acid sequences on one side of the nucleic acid sequence 301 (e.g., the third nucleic acid sequence 303, the second nucleic acid sequence 302 connected to the other end of the third nucleic acid sequence 303) are then separated from the attachment film 201; and because the fourth nucleic acid sequence 304 of the electrochemical sensing layer 202 has a complementary sequence to the second nucleic acid sequence 302, the separated analyte and the second nucleic acid sequence 302 complex are captured by the electrochemical sensing layer 202; then, a fluorescent substance solution is dripped onto the first sensing area, and the fluorescent substance (e.g., Thioflavin T) can bind to the G-rich section in the second nucleic acid sequence 302 to generate a fluorescent signal; finally, the fluorescent signal and the electrochemical signal in the electrochemical sensing layer 202 area are detected. Using the predetermined fluorescence signal and the concentration curve of the analyte, the electrochemical signal and the concentration curve of the analyte, combined with the aforementioned detected fluorescence signal and electrochemical signal, the concentration of the analyte can be calculated, thereby achieving high-sensitivity detection of the analyte in two modes.

[0073] In some embodiments, the analyte includes ochratoxin, and the first nucleic acid sequence 301 includes a sequence having at least 70% identity with SEQ ID No. 1. For example, Root: 5'-CCT TTA CGC CAC CCA CAC CCG ATC-NH2-C6-3'—SEQ ID No. 1.

[0074] The second nucleic acid sequence 302 comprises a sequence having at least 70% identity to SEQ ID No. 2. For example, Stem-1: 5′-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA TTT TGG AAG GAG AGAGAG GGT TTT GGA AGG AGA GAG AGG GTT TTG GAA GGA GAG AGA GGG TTT TGT CAC GAGTCA CTA TAG AGA-3′—SEQ ID No. 2.

[0075] The third nucleic acid sequence 303 comprises a sequence having at least 70% identity with SEQ ID No. 3. For example, Stem-2: 5′-CCT TTA CGC CAC CCA CAC CCG ATC TCT CTA TAG TGA CTC GTG AC-3′—SEQ ID No. 3.

[0076] The fourth nucleic acid sequence 304 comprises a sequence having at least 70% identity with SEQ ID No. 4. For example, FETRoot: 5′-NH2-C6-TCT CTA TAG TGA CTC GTG AC-3′—SEQ ID No. 4.

[0077] In addition to the above-mentioned fluorescence detection and electrochemical detection, the embodiments of the present disclosure also provide a third modality detection of the object to be detected - visible light detection.

[0078] In some embodiments, the biosensor further comprises a Fe3O4@ZIF-8 nanoprobe; the surface of the Fe3O4@ZIF-8 nanoprobe is modified with a fifth nucleic acid sequence; and the fifth nucleic acid sequence comprises an aptamer sequence of the analyte.

[0079] Here, when the second nucleic acid sequence 302 detaches from the attachment film 201, the end of the first nucleic acid sequence 301 or the third nucleic acid sequence 303 is exposed, and the fifth nucleic acid sequence can complementarily pair with the first nucleic acid sequence 301 or the third nucleic acid sequence 303, allowing the Fe3O4@ZIF-8 nanoprobe to bind to the region of the attachment film 201. Because the Fe3O4@ZIF-8 nanoprobe has peroxidase-like activity, after the addition of H2O2 and TMB color developing solution, the catalytic activity of the Fe3O4@ZIF-8 nanoprobe causes a blue color change, for example, to a wavelength of 652 nm. This color change is visible to the naked eye and can also be detected under 652 nm conditions.

[0080] It should be understood that the higher the content of the analyte, the more the second nucleic acid sequence 302 is separated from the attachment film layer 201, the more ends of the first nucleic acid sequence 301 or the third nucleic acid sequence 303 are exposed, and more Fe3O4@ZIF-8 nanoprobes can be bound to the attachment film layer 201, the faster the blue color can be observed, and the stronger the blue light, thereby realizing the third modal detection of the analyte.

[0081] Illustratively, the fifth nucleic acid sequence comprises a sequence having at least 70% identity to SEQ ID No. 5. For example, 5'-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA-3'—SEQ ID No. 5.

[0082] It should be noted that, depending on the different analytes, the first nucleic acid sequence 301 to the fifth nucleic acid sequence can be designed separately, and this disclosure does not limit this. It should be understood that the first nucleic acid sequence 301 and the fourth nucleic acid sequence 304 can include amino modifications.

[0083] In a second aspect, the present disclosure also provides a method for preparing a chitin membrane-based biosensor. It should be noted that the experimental methods used in the following examples are conventional methods unless otherwise specified, and are performed in accordance with the techniques or conditions described in literature in the field or in accordance with product specifications.

[0084] like Figure 1 As shown, the preparation method includes:

[0085] First, a chitosan film layer 101 is provided, comprising a first sensing area 103 and a second sensing area 102. Here, the first sensing area 103 and the second sensing area 102 are a partitioning method of the chitosan film layer 101, and such partitioning does not change the material of the chitosan film layer 101 itself.

[0086] In some embodiments, the preparation of the chitosan film layer 101 includes purification of chitosan and preparation of chitosan hydrosol.

[0087] Exemplarily, chitosan purification can be carried out by the following steps: weigh 10-20 g of chitosan, place it in a blue-mouth bottle, add 150-200 mL of 5% NaOH aqueous solution and soak for 12-24 hours, centrifuge, add deionized water to wash (10000 rpm, 5 min), repeat 3 times until the solution is neutral, then add 150-200 mL of 7% HCl aqueous solution and soak for 24 hours, centrifuge, add deionized water to wash (10000 rpm, 5 min), repeat 3 times until the solution is neutral, and then place it in a 60°C oven to dry for 24 hours, and store it at room temperature for use.

[0088] Exemplarily, the chitosan hydrosol can be prepared by the following steps: weighing 0.6-1.2 g of purified chitosan, adding 20 mL of NaOH / Urea / water solution (11:4:85), and finally obtaining a chitosan solution with a concentration of 3%-6%. After mixing, place it at -40 ° C and freeze it for 3-4 hours. After taking it out, stir it with a glass rod and thaw it continuously. Then, freeze it at 40 ° C for 3-4 hours. Repeat freezing and thawing three times. Centrifuge (8000 rpm, 15 min, 0 ° C) to remove undissolved precipitates. The collected supernatant is transferred to a clean beaker for ice bath, stirred on a magnetic stirrer, and then 0.5 mL of epichlorohydrin (ECH) is added dropwise within 30 minutes. Stirring is continued for 2 hours. After the end, the residual gas in the liquid is eliminated by centrifugation (8000 rpm, 5 min, 0 ° C). The chitosan solution is spread onto a glass substrate into a uniform film using electrospinning, casting, spraying, extrusion, and blow molding. The film's thickness can be controlled. Anhydrous ethanol is placed at 4°C, and then the glass substrate is immersed in the anhydrous ethanol and allowed to stand for several hours to form a chitosan hydrogel. The surface is then rolled or placed on a glass plate, and dried to form a smooth chitosan fiber paper. The surface of the chitosan fiber paper is plasma treated to create hydrophilic areas for securing wires and sensitive materials.

[0089] It should be noted that the above-mentioned method for preparing the chitosan film layer 101 is merely exemplary and is not limited in the present disclosure. A person skilled in the art can prepare the chitosan film layer 101 of the present disclosure in a manner that can form a chitosan film layer.

[0090] Next, an adhesive film layer 201 is formed on the first sensing area 103. Here, the first sensing area 103 may be a circular area with a diameter of 2-4 cm.

[0091] The following describes the preparation method of the attachment film layer 201 using SEBS-g-MAH as an example. SEBS-g-MAH is dissolved in a solution such as tetrahydrofuran (THF) or cyclohexane. The circular area is covered with the SEBS-g-MAH solution, and the area is plasma-treated. The carboxyl groups of SEBS-g-MAH are activated using EDC, enabling binding reactions with nucleic acid chains. For example, EDC is dissolved in PBS (pH 6.0) and incubated on the SEBS-g-MAH membrane surface for 30 minutes to activate its carboxyl groups.

[0092] Then, a wire layer 203 and an electrochemical sensing layer 202 are formed on the second sensing area 102 ; the second sensing area 102 is connected to the wire layer 203 .

[0093] The following uses carbon nanotubes as an example to illustrate the preparation method of the wire layer 203 and the electrochemical sensor layer 202.

[0094] Single-walled carbon nanotubes (SWCNTs), hydroxylated single-walled carbon nanotubes (SWCNTs-OH), or carboxylated single-walled carbon nanotubes (SWCNTs-COOH), and pyrenecarboxylic acid (PCA) were weighed into a blue-mouthed bottle and added to ultrapure water at a ratio of 2:1:4. After sonication for 5 hours, a uniformly dispersed SWCNT ink was obtained. The SWCNTs were then attached to the surface of the chitin film using techniques such as inkjet printing, screen printing, direct pen writing, and 3D printing to form a conductive layer 203.

[0095] Semiconducting single-walled carbon nanotubes (s-SWCNTs) are weighed and added to N,N-dimethylformamide (DMF) at a ratio of 1:50-100. The mixture is then sonicated in an ice-water solution for 2 hours to produce a sensitive ink. The s-SWCNTs are then fixed between two SWCNT conductors on a chitin membrane using techniques such as inkjet printing, screen printing, pen writing, and 3D printing to form an electrochemical sensing layer 202. Specific reagents (such as PBASE, PCA, and EDC) are then used for surface treatment to create activated carboxyl groups on the surface that bind to amino-containing nucleic acids. PBASE stands for 1-pyrenebutyric acid N-hydroxysuccinimide ester.

[0096] Optionally, before attaching the SWCNTs or s-SWCNTs to the chitin membrane surface, titanium carbide powder may be dispersed in a specific solvent, ultrasonically treated, and then uniformly coated on the second sensing region 102 of the chitin membrane. Thermal annealing is then performed to densify the titanium carbide membrane. The specific solvent may include N,N-dimethylformamide, dimethyl sulfoxide, acetone, or an aqueous solution containing a surfactant.

[0097] Next, a first nucleic acid sequence 301, a second nucleic acid sequence 302, and a third nucleic acid sequence 303 are assembled on the attachment film 201. Here, the first nucleic acid sequence 301 modifies the attachment film 201; the second nucleic acid sequence 302 includes the aptamer sequence, G-rich segment, and linker sequence of the analyte, and there is a complementary sequence between the aptamer sequence and the first nucleic acid sequence 301; the third nucleic acid sequence 303 has a complementary sequence with the aptamer sequence and linker sequence of the analyte, respectively; and the fourth nucleic acid sequence 304 has a complementary sequence with the linker sequence or the aptamer sequence of the analyte.

[0098] like Figure 1As shown, the assembly method of the "line" nucleic acid probe can be as follows: a solution of the first nucleic acid sequence 301 is incubated on the surface of the attachment film 201 for 30-60 minutes; unbound carboxyl groups on the surface of the attachment film 201 are then blocked with BSA 401 to prevent nonspecific binding; a second nucleic acid sequence 302 is then incubated on the surface of the attachment film 201 for 30-60 minutes; and a third nucleic acid sequence 303 is then incubated on the surface of the attachment film 201 for 30-60 minutes. The steps of incubating the second nucleic acid sequence 302 and the third nucleic acid sequence 303 are repeated to obtain the "line" nucleic acid probe.

[0099] In some alternative embodiments, the “linear” nucleic acid probe assembly method may also first assemble the first nucleic acid sequence 301 , the second nucleic acid sequence 302 and the third nucleic acid sequence 303 , and finally assemble the first nucleic acid sequence 301 on the attachment film layer 201 .

[0100] Finally, the fourth nucleic acid sequence 304 is bound to the electrochemical sensing layer 202. For example, the solution of the fourth nucleic acid sequence 304 is incubated on the surface of the electrochemical sensing layer 202 for 30-60 minutes, and then BSA 401 is used to block the unbound carboxyl groups on the surface of the electrochemical sensing layer 202 to prevent nonspecific binding.

[0101] It should be noted that the above steps can be adjusted as needed, and this disclosure does not limit this.

[0102] In some embodiments, the preparation method further comprises preparing Fe3O4@ZIF-8 nanoprobes.

[0103] For example, Fe3O4, Zn(NO3)2, and 2-methylimidazole were mixed and reacted in a 70°C water bath for 20 minutes to obtain a Fe3O4@ZIF-8 nanomaterial. The surface of the Fe3O4@ZIF-8 nanomaterial can be linked to an aptamer nucleic acid sequence of the analyte, such as the fifth nucleic acid sequence.

[0104] In a third aspect, embodiments of the present disclosure further provide a detection kit comprising any of the aforementioned biosensors. Furthermore, the detection kit further comprises a fluorescent substance (eg, Thioflavin T), a potassium solution, a PBS buffer, and the like.

[0105] The detection kit of the above embodiment has the beneficial effects of the corresponding biosensor embodiment, which will not be described in detail here.

[0106] Example 1

[0107] The analyte in this embodiment is OTA, and the corresponding materials and reagents include, but are not limited to: chitin membrane, SEBS-g-MAH, EDC, multiple nucleic acid sequences synthesized according to a preset nucleic acid sequence, BSA, etc.

[0108] First, the chitin membrane is pretreated by soaking it in a mild activator solution (e.g., ice ethanol solution), treating it in an ultrasonic cleaner for more than 20 minutes (e.g., 25 minutes), taking it out and rinsing it with deionized water and drying it. Then, in a clean environment, the SEBS-g-MAH solution is accurately dripped onto the circular area of ​​the chitin membrane and a uniform thin film is prepared by spin coating. The film thickness is controlled at 0.1mm-0.5mm, specifically 0.1mm, 0.2mm, 0.3mm, 0.4mm, 0.5mm, etc. The prepared membrane is placed in a buffer solution containing an appropriate amount of EDC and incubated at 37°C for 30 minutes for carboxylic acid activation. Immediately after activation, the membrane is transferred to a hybridization solution containing SEQ ID No.1 and hybridized at 42°C for 1 hour. After hybridization is completed, the membrane is immersed in a 1% BSA solution and blocked at room temperature for 45 minutes.

[0109] Next, the nucleic acid sequence is assembled. During the hybridization process, the ionic strength and temperature of the solution are strictly controlled to ensure accurate structure formation. For example, the concentration of sodium ions in the solution is 0.1M; the concentration of magnesium ions is 0.05M.

[0110] Next, titanium carbide powder is dispersed in a specific solvent and, after ultrasonic treatment, evenly coated on the second sensor region of the chitin membrane. Thermal annealing is then used to densify the titanium carbide membrane. Subsequently, single-walled carbon nanotubes are evenly dispersed and laid on the titanium carbide membrane to form the conductive layer 203, and semiconducting single-walled carbon nanotubes are evenly dispersed and laid on the titanium carbide membrane to form the electrochemical sensing layer 202.

[0111] Finally, the SEQ ID No. 4 solution was dropped onto the electrochemical sensing layer 202 and incubated at 30° C. for 1 hour to allow for firm binding.

[0112] It should be noted that, for steps not described in this embodiment, such as the formation of the chitin film, reference can be made to the above description and will not be repeated here.

[0113] Example 2 Performance Test

[0114] Prepare a series of OTA standard solutions with different concentrations, the concentration range is: 5×10 -7 -0.5μg / mL.

[0115] The prepared biosensor was placed on the detection platform, and OTA standard solutions of different concentrations were added dropwise onto the sensor surface using a microsyringe to ensure that the solutions covered both the first and second sensing areas, and incubated at room temperature for 30 min.

[0116] The first sensing area and the second sensing area were cleaned, and Thioflavin T with a final concentration of 50 μM was incubated on the surface of the first sensing area for 5 minutes, and then the fluorescence intensity was detected with an excitation wavelength of 425 nm.

[0117] At the same time, an electrochemical workstation was used to detect changes in the electrochemical signal of the second sensing area. Aluminum clips were used to connect the two wire parts, and the linear voltammetry method was used to record the current-potential curve.

[0118] After the fluorescence signal was measured, Fe3O4@ZIF-8 nanoprobes were added to the surface of the first sensing area and incubated at room temperature for 10 min. TMB and H2O2 solutions were then added and incubated at 42°C for 10 min. The absorbance was then detected at 652 nm.

[0119] According to the test results, the fluorescence signal intensity-OTA concentration curve was drawn (such as Figure 2 As shown), electrochemical signal-OTA concentration curve (as shown Figure 3 As shown) and the absorbance-OTA concentration curve (as shown Figure 4 The performance indicators of the sensor, such as sensitivity, linear range and detection limit, were analyzed.

[0120] The experimental results show that Figures 2 to 4 As shown, the chitin membrane-based trimodal biosensor of the present invention exhibits high sensitivity in OTA detection. For example, the detection limit of fluorescence detection can reach 0.36 pg / mL, the detection limit of electrochemical detection can reach 0.32 pg / mL, and the detection limit of visible light detection can reach 0.47 pg / mL. It also has a good linear relationship in a wide concentration range, which can meet the needs of OTA detection in actual biological samples.

[0121] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present disclosure (including the claims) is limited to these examples. Within the scope of the present disclosure, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present disclosure as described above, which are not provided in detail for the sake of simplicity.

[0122] The embodiments of the present disclosure are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the embodiments of the present disclosure should be included in the scope of protection of the present disclosure.

Claims

1. A biosensor based on a chitin membrane, characterized in that: include: The chitin membrane layer includes a first sensing area and a second sensing area; an attachment film layer, disposed on the first sensing area; a conductive line layer, disposed in the second sensing area; an electrochemical sensing layer, disposed in the second sensing area and connected to the wire layer; as well as A nucleic acid probe set, wherein The nucleic acid probe set includes a first nucleic acid sequence, a second nucleic acid sequence, a third nucleic acid sequence and a fourth nucleic acid sequence; The first nucleic acid sequence modifies the attachment membrane layer; The second nucleic acid sequence includes an aptamer sequence, a G-rich segment, and a linker sequence of the analyte, and a complementary sequence exists between the aptamer sequence and the first nucleic acid sequence; There are complementary sequences between the third nucleic acid sequence and the aptamer sequence of the analyte and the connecting sequence; The fourth nucleic acid sequence modifies the electrochemical sensing layer; the fourth nucleic acid sequence and the connecting sequence have a complementary sequence.

2. The biosensor according to claim 1, wherein The analyte includes ochratoxin; the first nucleic acid sequence includes a sequence having at least 70% identity with SEQ ID No. 1; The second nucleic acid sequence comprises a sequence having at least 70% identity to SEQ ID No. 2; The third nucleic acid sequence comprises a sequence having at least 70% identity with SEQ ID No. 3; The fourth nucleic acid sequence comprises a sequence having at least 70% identity to SEQ ID No.

4.

3. The biosensor according to any one of claims 1 to 2, characterized in that It also includes a Fe3O4@ZIF-8 nanoprobe; the surface of the Fe3O4@ZIF-8 nanoprobe is modified with a fifth nucleic acid sequence; the fifth nucleic acid sequence includes an aptamer sequence of the object to be detected.

4. The biosensor according to claim 3, wherein The fifth nucleic acid sequence comprises a sequence having at least 70% identity to SEQ ID No.

5.

5. The biosensor according to claim 1, wherein The material of the adhesion film layer includes SEBS-g-MAH; and / or The material of the conductive line layer includes at least one of single-walled carbon nanotubes, hydroxylated single-walled carbon nanotubes and carboxylated single-walled carbon nanotubes; and / or The material of the electrochemical sensing layer includes semiconductor single-walled carbon nanotubes.

6. The biosensor according to claim 1, wherein Activated carboxyl groups are provided on the surfaces of the attachment film layer and the electrochemical sensing layer.

7. The biosensor according to claim 6, wherein The first end of the first nucleic acid sequence comprises an amino modification; and / or The second end of the fourth nucleic acid sequence includes an amino modification.

8. A method for preparing a biosensor based on a chitin membrane, characterized in that: include: Providing a chitin film layer; including a first sensing area and a second sensing area; forming an adhesion film layer on the first sensing area; forming a wire layer and an electrochemical sensing layer on the second sensing area; wherein the second sensing area is connected to the wire layer; Assembling the first nucleic acid sequence, the second nucleic acid sequence and the third nucleic acid sequence on the attachment film layer; A fourth nucleic acid sequence is bound to the electrochemical sensing layer; wherein, The first nucleic acid sequence modifies the attachment membrane layer; The second nucleic acid sequence includes an aptamer sequence, a G-rich segment, and a linker sequence of the analyte, and a complementary sequence exists between the aptamer sequence and the first nucleic acid sequence; There are complementary sequences between the third nucleic acid sequence and the aptamer sequence of the analyte and the connecting sequence; The fourth nucleic acid sequence and the connecting sequence have a complementary sequence.

9. A detection kit, characterized in that A biosensor comprising the biosensor according to any one of claims 1 to 7.

10. The detection kit according to claim 9, characterized in that Also included are fluorescent substances.

Images