Biosensor based on chitin membrane, method of preparation and kit

Abstract

The present disclosure provides a chitin film-based biosensor, a preparation method and a kit. Specifically, the biosensor comprises a chitin film layer, a first sensing area and a second sensing area; an attachment film layer arranged in the first sensing area; a wire layer arranged in the second sensing area; an electrochemical sensing layer arranged in the second sensing area and in communication with the wire layer; and a nucleic acid probe set, wherein the nucleic acid probe set comprises a first nucleic acid sequence, a second nucleic acid sequence, a third nucleic acid sequence and a fourth nucleic acid sequence. By simultaneously arranging the first sensing area and the second sensing area on the chitin film layer, and by means of the nucleic acid probe set, the fluorescence detection of the analyte in the first sensing area and the electrochemical detection of the analyte in the second sensing area, the high-sensitivity and multi-modal detection of the analyte is realized, and the biosensor 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 particularly relates to a chitin membrane-based biosensor, a preparation method and a kit. BACKGROUND

[0002] With the continuous development of biological detection technology, there is an increasing demand for biosensors with high sensitivity, high specificity and multi-modal detection. Traditional single-mode biosensors often have certain limitations in terms of detection accuracy and comprehensiveness. For example, electrochemical sensors have good quantitative analysis capability, but may lack specificity in molecular recognition. Fluorescent sensors have high sensitivity, but their stability in complex environments needs to be improved. Therefore, it is of great significance to develop a biosensor that can integrate the advantages of multiple detection modalities. SUMMARY

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

[0004] To achieve the above purpose, the present disclosure provides a chitin membrane-based biosensor, comprising:

[0005] a chitin membrane layer, comprising a first sensing area and a second sensing area;

[0006] an attachment membrane layer arranged in the first sensing area;

[0007] a wire layer arranged in the second sensing area;

[0008] an electrochemical sensing layer arranged in the second sensing area and in communication with the wire layer; and

[0009] a nucleic acid probe set, wherein

[0010] the nucleic acid probe set comprises 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 comprises an aptamer sequence of an analyte, a G-rich segment and a linker sequence, and there is a complementary sequence 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 linker sequence, respectively;

[0014] the fourth nucleic acid sequence modifies the electrochemical sensing layer; and the fourth nucleic acid sequence has a complementary sequence with the linker 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 to 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, further comprising a Fe3O4@ZIF-8 nanoprobe; the Fe3O4@ZIF-8 nanoprobe is surface-modified with a fifth nucleic acid sequence; the fifth nucleic acid sequence comprises an aptamer sequence of the analyte.

[0020] In some embodiments, the fifth nucleic acid sequence comprises a sequence having at least 70% identity 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 comprises an amino modification.

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

[0024] The material of the wire layer comprises 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 comprises semiconductor single-walled carbon nanotubes.

[0026] In some embodiments, the surface of the attachment film layer and the electrochemical sensing layer is provided with activated carboxyl groups.

[0027] Based on the same inventive concept, the present disclosure also provides a preparation method of a chitin film-based biosensor, comprising:

[0028] providing a chitin film layer; comprising a first sensing area and a second sensing area;

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

[0030] forming a wire layer and an electrochemical sensing layer on the second sensing area; the second sensing area is in communication with the wire layer;

[0031] assembling a first nucleic acid sequence, a second nucleic acid sequence and the third nucleic acid sequence on the attachment membrane layer;

[0032] binding a fourth nucleic acid sequence on the electrochemical sensing layer; wherein,

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

[0034] the second nucleic acid sequence comprises an aptamer sequence of a to-be-tested substance, a G-rich segment and a connecting sequence, and there is complementary sequence between the aptamer sequence and the first nucleic acid sequence;

[0035] there is complementary sequence between the third nucleic acid sequence and the aptamer sequence of the to-be-tested substance and the connecting sequence respectively;

[0036] the fourth nucleic acid sequence has complementary sequence with the connecting sequence or the aptamer sequence of the to-be-tested substance.

[0037] Based on the same inventive concept, the embodiments of the present disclosure also provide a detection kit, characterized by comprising the biosensor according to any one of the preceding embodiments.

[0038] In some embodiments, the kit further comprises a fluorescent substance.

[0039] As can be seen from the above, the present disclosure provides a chitin membrane-based biosensor, a preparation method and a kit, which simultaneously sets a first sensing area and a second sensing area on a chitin membrane layer, and realizes high-sensitivity and multi-modal detection of a to-be-tested substance by means of a nucleic acid probe group for fluorescence detection of the to-be-tested substance in the first sensing area and electrochemical detection of the to-be-tested substance in the second sensing area, thereby having a broad application prospect in the field of biological detection. BRIEF DESCRIPTION OF DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the present disclosure or the related art, the following will briefly introduce the drawings needed to be used in the embodiments or the related art description. Obviously, the drawings in the following description are only embodiments of the present disclosure, and for those skilled in the art, other drawings can also be obtained without creative labor based on these drawings.

[0041] Figure 1 a preparation flowchart of a biosensor provided by an embodiment of the present disclosure is shown;

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

[0043] Figure 3A linear relationship between electrochemical signal and OTA concentration is shown.

[0044] Figure 4 A linear relationship between absorbance and OTA concentration is shown. DETAILED DESCRIPTION

[0045] For the purpose of making the object, technical solutions and advantages of the present disclosure clearer, the present disclosure is further described in detail below with reference to specific embodiments and 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 be understood as the general meaning understood by those skilled in the art to which the present disclosure belongs. The terms "first", "second" and the like used in the embodiments of the present disclosure do not represent any order, number or importance, but are only used to distinguish different components. The terms "include" or "contain" and the like mean that the elements or objects before the terms cover the elements or objects listed after the terms and their equivalents, and do not exclude other elements or objects. "Up", "down", "left", "right" and the like are only used to represent relative positional relationship, when the absolute position of the described object is changed, the relative positional relationship may also be changed accordingly. Unless otherwise specified, "μM" herein refers to "μmol / L", and "mM" refers to "mmol / L".

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

[0048] Chitin, as a natural polysaccharide, has good biocompatibility, degradability and unique physical and chemical properties, and shows great application potential in many fields such as biomedicine and environmental science. It has a wide source and relatively low cost, and is one of the ideal base materials for constructing biosensors.

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

[0050] Ochratoxin A aptamer refers to an oligonucleotide sequence, such as single-stranded DNA or RNA, obtained through artificial screening, which can specifically recognize and bind to OTA with high affinity. The OTA aptamer forms a spatial complement with the OTA molecule through its unique three-dimensional conformation, achieving high-specificity binding. Its binding capacity is derived from the nucleotide sequence of the aptamer, which locks the target through hydrogen bonds, van der Waals forces, etc.

[0051] EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) is a water-soluble carbodiimide compound commonly used as a coupling agent for carboxylic acids and amines.

[0052] PBS (Phosphate Buffered Saline) is a buffer widely used in biochemical research. Its main components include NaHPO, KH PO, NaCl, and KCl. The main role of PBS is as a solvent for dissolving and protecting reagents, maintaining the stability of cells and biological molecules.

[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, belonging to the Zeolite-Imidazolate Frameworks (ZIFs) family.

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

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

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

[0057] SEBS (Styrene Ethylene Butylene Styrene) is a linear three-embedded copolymer material, which is composed of polystyrene as the terminal segment and ethylene-butene copolymer obtained by hydrogenation of polybutadiene as the intermediate elastic segment. SEBS does not contain unsaturated double bonds, so it has good stability and aging resistance.

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

[0059] SEBS-g-MAH (Maleic Anhydride Grafted Styrene-Ethylene-Butadiene-Styrene Block Copolymer) is a thermoplastic elastomer material modified by grafting maleic anhydride. It grafts maleic anhydride (MAH) onto the molecular chain of SEBS through chemical reaction.

[0060] As described in the background section, single-mode biosensors have been difficult to meet the needs of high-sensitivity and high-specificity detection of biomolecules in complex samples.

[0061] Therefore, the present disclosure provides a chitin film-based biosensor, a preparation method and a kit. By simultaneously arranging a first sensing area and a second sensing area on the chitin film layer, and by means of the nucleic acid probe set, the first sensing area performs fluorescence detection on the measured object, and the second sensing area performs electrochemical detection on the measured object, thereby realizing high-sensitivity and multi-modal detection of the measured object, and having a wide application prospect in the field of biological detection.

[0062] In order to make the technical solutions of the present disclosure clearer and easier to understand, the chitin film-based biosensor, the preparation method and the kit provided by the present disclosure will be described in detail below in combination with the drawings and specific embodiments.

[0063] In a first aspect, the present disclosure provides a chitin film-based biosensor. Figure 1 A preparation flowchart of a biosensor provided by the present disclosure is shown. As shown in Figure 1 The biosensor comprises:

[0064] The chitin film layer 101 comprises a first sensing area 103 and a second sensing area 102; the adhesion film layer 201 is arranged in the first sensing area 103; the wire layer 203 is arranged in the second sensing area 102; the electrochemical sensing layer 202 is arranged in the second sensing area 102 and is in communication with the wire layer 203; and the nucleic acid probe set. Here, the surface of the adhesion film layer 201 and the electrochemical sensing layer 202 is provided with activated carboxyl groups.

[0065] Optionally, the material of the adhesion film layer 201 comprises SEBS-g-MAH. Optionally, the material of the wire layer 203 comprises 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 comprises semiconducting single-walled carbon nanotubes (s-SWCNTs).

[0066] In some embodiments, the nucleic acid probe set comprises 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 adhesion film layer 201. Here, the first nucleic acid sequence comprises a second end (e.g., 5' end) and a first end (e.g., 3' end). Optionally, the 3' end of the first nucleic acid sequence 301 can comprise an amino modification, e.g., NH2-C6, wherein NH2 can bind to the carboxyl groups on the surface of the adhesion film layer 201, thereby modifying the adhesion film layer 201. Optionally, unbound carboxyl groups can be blocked using BSA 401 to prevent non-specific binding.

[0068] The second nucleic acid sequence 302 comprises an aptamer sequence of the analyte, a G-rich segment, and a linker sequence, and there is a complementary sequence 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 of part of the aptamer sequence, which is not limited in the present disclosure. Based on the complementary pairing between 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 insert into the groove of the G-quadruplex, and this binding changes the electronic environment of thioflavin T, resulting in a significant increase in its fluorescence intensity.

[0070] The third nucleic acid sequence 303 has a complementary sequence with the aptamer sequence of the analyte and the linker sequence, respectively. Here, the third nucleic acid sequence 303 can be complementary to the linker sequence of the second nucleic acid sequence 302, and the third nucleic acid sequence 303 can be combined with the second nucleic acid sequence 302. In addition, the third nucleic acid sequence 303 has a complementary sequence of the aptamer sequence of the analyte, and another second nucleic acid sequence 302 can be combined with the third nucleic acid sequence 303. Based on such 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, forming a signal amplification element, effectively improving the detection sensitivity, and making it possible to detect trace biomolecules.

[0071] 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., 5' end) and a first end (e.g., 3' end). Optionally, the 5' end of the fourth nucleic acid sequence 304 can include an amino modification, such as NH2-C6; the NH2can bind to a carboxyl group on the surface of the electrochemical sensing layer 202, thereby modifying the electrochemical sensing layer 202. Optionally, unbound carboxyl groups can be blocked using BSA 401 to prevent non-specific binding. Further, the fourth nucleic acid sequence 304 has a complementary sequence to the linking sequence of the second nucleic acid sequence, whereby the second nucleic acid sequence 304 can also be assembled to the fourth nucleic acid sequence 304.

[0072] Based on the biosensor described above, two modalities of detection can be performed on the analyte. Specifically, first, the first nucleic acid sequence 301 modifies the attachment membrane layer 201 and is blocked using 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 blocked using BSA; next, the "linear" nucleic acid probe is assembled to the first nucleic acid sequence 301; then, the analyte solution is added, and the analyte solution contacts the attachment membrane layer 201 and the electrochemical sensing layer 202. Due to the specific binding between the analyte and the aptamer sequence of the second nucleic acid sequence 302, the second nucleic acid sequence 302 is detached from the attachment membrane layer 201, and it should be understood that other nucleic acid sequences (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) connected to the side of the second nucleic acid sequence 302 away from the first nucleic acid sequence 301 are also detached from the attachment membrane layer 201; again, due to the complementary sequence between the fourth nucleic acid sequence 304 of the electrochemical sensing layer 202 and the second nucleic acid sequence 302, the detached analyte and the second nucleic acid sequence 302 complex is captured by the electrochemical sensing layer 202; then, a fluorescent substance solution is added on the first sensing area, and the fluorescent substance (e.g., thioflavin T) can bind to the G-rich segment in the second nucleic acid sequence 302 to generate a fluorescent signal; finally, the fluorescent signal and the electrochemical signal in the region of the electrochemical sensing layer 202 are detected. Using the pre-determined concentration curve of the fluorescent signal and the analyte, the concentration curve of the electrochemical signal and the analyte, and the fluorescent signal and the electrochemical signal detected above, the concentration of the analyte can be calculated, thereby achieving two-modality high-sensitivity detection of the analyte.

[0073] In some embodiments, the analyte includes ochratoxin; and the first nucleic acid sequence 301 includes a sequence having at least 70% identity to SEQ ID No. 1. Illustratively, 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 with SEQ ID No. 2. Exemplarily, Stem-1: 5'-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA TTT TGG AAG GAG AGA GAG GGT TTT GGA AGG AGA GAG AGG GTT TTG GAA GGA GAG AGA GGG TTT TGT CAC GAG TCA 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. Exemplarily, 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. Exemplarily, 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 modal detection of the analyte, i.e. visible light detection.

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

[0079] Here, when the second nucleic acid sequence 302 is detached from the attachment film layer 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 be complementary to the first nucleic acid sequence 301 or the third nucleic acid sequence 303, so that the Fe3O4@ZIF-8 nanoprobe is combined to the region of the attachment film layer 201. Since the Fe3O4@ZIF-8 nanoprobe has peroxidase-like activity, after adding H2O2 and TMB color developing solution, due to the catalytic activity of the Fe3O4@ZIF-8 nanoprobe, a blue change, for example, a wavelength of 652 nm, will occur, and such color change can be visible to the naked eye, and can also be detected under the condition of 652 nm.

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

[0081] For example, the fifth nucleic acid sequence includes a sequence that has at least 70% identity with 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 analyte, 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 may include amino modifications.

[0083] Secondly, this disclosure also provides a method for preparing a chitin membrane-based biosensor. It should be noted that, unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions.

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

[0085] First, a chitosan film layer 101 is provided, including a first sensing region 103 and a second sensing region 102. Here, the first sensing region 103 and the second sensing region 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 chitin membrane 101 includes chitin purification and the preparation of chitin hydrosol.

[0087] For example, chitin purification can be performed using the following steps: Weigh 10-20g of chitin, place it in a blue-mouth bottle, add 150-200mL of 5% NaOH aqueous solution and soak for 12-24h. After centrifugation, wash with deionized water (10000rpm, 5min), repeat 3 times until the solution is neutral. Then add 150-200mL of 7% HCl aqueous solution and soak for 24h. After centrifugation, wash with deionized water (10000rpm, 5min), repeat 3 times until the solution is neutral. Then dry in a 60℃ oven for 24h and store at room temperature for later use.

[0088] Exemplarily, the chitin hydrogel can be prepared by the following steps: weighing 0.6-1.2 g of purified chitin, adding 20 mL of a solution of NaOH / urea / water (11:4:85), and finally obtaining a chitin solution with a concentration of 3%-6%. After mixing, freeze at-40°C for 3-4 h, take out and continuously stir with a glass rod to thaw, and then freeze at 40°C for 3-4 h again. Repeat the freezing-thawing three times, remove the undissolved precipitate by centrifugation (8000 rpm, 15 min, 0°C), and collect the supernatant into a clean beaker for ice bath. Stir on a magnetic stirrer, then add epoxy chloropropane (ECH) 0.5 mL dropwise within 30 min, and continue to stir for 2 h. After the end, eliminate the residual gas in the liquid by centrifugation (8000 rpm, 5 min, 0°C). Use electrospinning, casting, spraying, extrusion, blow molding and other methods to spread the chitin solution on the glass substrate to form a uniform film, and the thickness of the film can be controlled. Put anhydrous ethanol at 4°C, then immerse the glass substrate in anhydrous ethanol, and then stand for several hours to obtain a chitin hydrogel. Finally, roll or place the glass plate on the surface of the chitin hydrogel to dry and form a flat chitin fiber paper. Perform plasma treatment on the surface of the chitin fiber paper to obtain a hydrophilic region on the surface for fixing wires and sensitive materials.

[0089] It should be noted that the preparation method of the chitin film layer 101 is exemplary, and the present disclosure does not limit it. Those skilled in the art can prepare the chitin film layer 101 of the present disclosure by using a method capable of forming a chitin film layer.

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

[0091] Next, an adhesion film layer 201 is formed on the first sensing area 103. Here, the first sensing area 103 can be a circular area with a diameter of 2-4 cm.

[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 in communication with the wire layer 203.

[0093] Next, the preparation method of the wire layer 203 and the electrochemical sensing layer 202 will be exemplarily described by taking carbon nanotubes as an example.

[0094] Weigh single-walled carbon nanotubes (SWCNTs) or hydroxylated single-walled carbon nanotubes (SWCNTs-OH) or carboxylated single-walled carbon nanotubes (SWCNTs-COOH), pyrene carboxylic acid (PCA) in a blue bottle, add ultrapure water, the ratio is 2:1:4, after ultrasonic treatment for 5h, get uniform SWCNTs ink. Use inkjet printing, screen printing, pen straight writing, 3D printing and other technologies to fix SWCNTs on the surface of the chitin film to form the wire layer 203.

[0095] Weigh semiconductor single-walled carbon nanotubes (s-SWCNTs) and add N,N-dimethylformamide (DMF), the ratio is 1:50-100, ultrasonic treatment in ice water mixture for 2 hours to get sensitive ink. Use inkjet printing, screen printing, pen straight writing, 3D printing and other technologies to fix s-SWCNTs between the two SWCNTs wires on the chitin film to form the electrochemical sensing layer 202, and use specific reagents (such as PBASE, PCA, EDC, etc.) for surface treatment to make the surface exist activated carboxyl groups to combine with nucleic acids with amino groups. Among them, PBASE is the abbreviation of N-hydroxysuccinimide ester 1-pyrene butyric acid.

[0096] Optionally, before fixing SWCNTs or s-SWCNTs on the surface of the chitin film, titanium carbide powder can also be dispersed in a specific solvent, uniformly coated on the second sensing area 102 of the chitin film after ultrasonic treatment, and heat annealing treatment is adopted to densify the titanium carbide film. Here, the specific solvent is N,N-dimethylformamide, dimethyl sulfoxide, acetone, aqueous solution, etc. including surfactants.

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

[0098] As Figure 1As shown, the assembly method of the "linear" nucleic acid probe can adopt the following method: incubate the solution of the first nucleic acid sequence 301 on the surface of the attachment membrane layer 201 for 30-60 min; then use BSA 401 to block the unbound carboxyl groups on the surface of the attachment membrane layer 201 to prevent non-specific binding; next, incubate the second nucleic acid sequence 302 on the surface of the attachment membrane layer 201 for 30-60 min; then incubate the third nucleic acid sequence 303 on the surface of the attachment membrane layer 201 for 30-60 min. Repeat the steps of incubating the second nucleic acid sequence 302 and the third nucleic acid sequence 303, and the "linear" nucleic acid probe can be obtained.

[0099] In some alternative embodiments, the assembly method of the "linear" nucleic acid probe can also assemble the first nucleic acid sequence 301, the second nucleic acid sequence 302 and the third nucleic acid sequence 303 first, and then assemble the first nucleic acid sequence 301 on the attachment membrane layer 201.

[0100] Finally, the fourth nucleic acid sequence 304 is combined on the electrochemical sensing layer 202. Exemplarily, incubate the solution of the fourth nucleic acid sequence 304 on the surface of the electrochemical sensing layer 202 for 30-60 min; then use BSA 401 to block the unbound carboxyl groups on the surface of the electrochemical sensing layer 202 to prevent non-specific binding.

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

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

[0103] Exemplarily, mix Fe3O4, Zn(NO3)2, 2-methylimidazole and other materials, and react in a 70°C water bath for 20 min to obtain Fe3O4@ZIF-8 nano-materials. The surface of the Fe3O4@ZIF-8 nano-materials can be connected to the aptamer nucleic acid sequence of the analyte, such as the fifth nucleic acid sequence.

[0104] In a third aspect, the embodiments of the present disclosure also provide a detection kit, which comprises the biosensor of any of the foregoing.

[0105] The detection kit of the above embodiments has the beneficial effects of the corresponding biosensor embodiments, which will not be repeated here.

[0106] Example 1

[0107] The test substance of the embodiment is OTA. The corresponding materials and reagents include, but are not limited to: chitin film, SEBS-g-MAH, EDC, a plurality of nucleic acid sequences synthesized according to a preset nucleic acid sequence, BSA, etc.

[0108] First, the chitin film is pretreated by soaking it in a mild activator solution (such as an ice ethanol solution) and treating it in an ultrasonic cleaner for more than 20 minutes (such as 25 minutes). After taking it out, it is rinsed clean with deionized water and air dried. Then, in a clean environment, the SEBS-g-MAH solution is accurately added to the circular area of the chitin film, and a uniform film is prepared using a spin coating method, with the film thickness controlled at 0.1-0.5 mm, which can be 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, etc. The prepared film 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 film is transferred to a hybridization solution containing SEQ ID No. 1 and hybridized at 42°C for 1 hour. After hybridization is complete, the film is soaked in a 1% BSA solution and blocked at room temperature for 45 minutes.

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

[0110] Then, the titanium carbide powder is first dispersed in a specific solvent, uniformly coated on the second sensor area of the chitin film after ultrasonic treatment, and densified by heat annealing treatment. Subsequently, the single-walled carbon nanotubes are uniformly dispersed and laid on the titanium carbide film as the wire layer 203, and the semiconductor single-walled carbon nanotubes are uniformly dispersed and laid on the titanium carbide film as the electrochemical sensing layer 202.

[0111] Finally, the SEQ ID No. 4 solution is added to the electrochemical sensing layer 202 and incubated at 30°C for 1 hour to firmly bind it.

[0112] It should be noted that the steps not explained in this embodiment, such as the formation of the chitin film, can be referred to the foregoing and will not be described again.

[0113] Example 2 performance testing

[0114] A series of OTA standard solutions with different concentrations are configured, with the concentration range being: 5x10 -7 -0.5 μg / mL.

[0115] The prepared biosensor was placed on a detection platform, and different concentrations of OTA standard solution were added to the surface of the sensor using a microsyringe, ensuring that the solution covered the first sensing area and the second sensing area at the same time, and incubated at room temperature for 30 min.

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

[0117] At the same time, the change of the electrochemical signal of the second sensing area was detected using an electrochemical workstation, two wire parts were connected by an aluminum clamp, and the current-potential curve was recorded by linear voltammetry.

[0118] After the fluorescence signal measurement was completed, Fe3O4@ZIF-8 nano-probes were added to the surface of the first sensing area, and after incubation at room temperature for 10 min, TMB and H2O2 solution were added, and after incubation at 42℃ for 10 min, the absorbance was detected at 652 nm.

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

[0120] The experimental results show that, as shown in Figures 2-4 , the chitin film-based three-mode biosensor of the present application 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, and has a good linear relationship in a wide concentration range, which can meet the needs of OTA detection in actual biological samples.

[0121] It should be understood by those skilled in the art that the discussion of any of the above embodiments is only exemplary and is not intended to limit the scope of the present disclosure (including claims) to these examples; under the idea of the present disclosure, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other changes of different aspects of the embodiments of the present disclosure as described above. In order to be brief, they are not provided in detail.

[0122] Embodiments of the present disclosure are intended to cover all such alternatives, modifications, and variations as fall within the broad scope of the appended claims. Accordingly, any one or more of the omitted, modified, equivalently replaced, improved, and the like, as long as within the spirit and principle of the embodiments of the present disclosure, should be included in the scope of protection of the present disclosure.

Claims

1. Biosensor based on a chitin film, characterized in that, The biosensor comprises: a chitin film layer comprising a first sensing area and a second sensing area; an attachment film layer disposed on the first sensing area; a wire layer disposed on the second sensing area; an electrochemical sensing layer disposed on the second sensing area and in communication with the wire layer; and a nucleic acid probe set, wherein the nucleic acid probe set comprises 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 film layer; the second nucleic acid sequence comprises an aptamer sequence of an analyte, a G-rich segment and a linker sequence, and there is a complementary sequence between the aptamer sequence and the first nucleic acid sequence; there is a complementary sequence between the third nucleic acid sequence and the aptamer sequence of the analyte and the linker sequence, respectively; the fourth nucleic acid sequence modifies the electrochemical sensing layer; and the fourth nucleic acid sequence has a complementary sequence with the linker sequence. the analyte comprises ochratoxin; and the first nucleic acid sequence comprises a sequence having at least 70% identity with SEQ ID No. 1; 2. The biosensor of claim 1, wherein, the second nucleic acid sequence comprises a sequence having at least 70% identity with 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 with SEQ ID No.

4. 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 an analyte.

3. The biosensor according to any one of claims 1 to 2, wherein The fifth nucleic acid sequence comprises a sequence having at least 70% identity with SEQ ID No.

5.

4. The biosensor of claim 3, wherein, The material of the attachment film layer comprises SEBS-g-MAH; and / or 5. The biosensor of claim 1, wherein, The material of the wire layer comprises 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 comprises semiconductor single-walled carbon nanotubes. The surface of the attachment film layer and the electrochemical sensing layer is provided with activated carboxyl groups.

6. The biosensor of claim 1, wherein, The first end of the first nucleic acid sequence comprises an amino modification; and / or 7. The biosensor of claim 6, wherein, The second end of the fourth nucleic acid sequence comprises an amino modification. The biosensor comprises:

8. A method for preparing a biosensor based on a chitin film, characterized by, providing a chitin film layer; comprising a first sensing area and a second sensing area; forming an attachment film layer on the first sensing area; forming a wire layer and an electrochemical sensing layer on the second sensing area; the second sensing area is in communication with the wire layer; assembling a first nucleic acid sequence, a second nucleic acid sequence and a third nucleic acid sequence on the attachment film layer; binding a fourth nucleic acid sequence on the electrochemical sensing layer; wherein the first nucleic acid sequence modifies the attachment film layer; the second nucleic acid sequence comprises an aptamer sequence of an analyte, a G-rich segment and a linker sequence, and there is a complementary sequence between the aptamer sequence and the first nucleic acid sequence; there is a complementary sequence between the third nucleic acid sequence and the aptamer sequence of the analyte and the linker sequence, respectively; the fourth nucleic acid sequence has a complementary sequence with the linker sequence. The biosensor comprises any one of claims 1-7.

9. A test kit comprising, ​ 10. The test kit according to claim 9, characterized in that Also included are fluorescent substances.

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