A full-printing bio-detection chip and a preparation method and application thereof

Abstract

The application belongs to the field of material science and biomedical science, and particularly relates to a full-printing biological detection chip and a preparation method and application thereof. The full-printing biological detection chip comprises a substrate, a polymer coating layer formed on the substrate by inkjet printing, and a capture biological material arranged on the polymer coating layer. The substrate, the polymer coating layer, and the capture biological material form a sandwich structure, and the capture biological material can specifically react with a target object to be detected. The polymer coating layer is selected from polymer materials containing -COOH and / or -NH2, and the capture biological material is selected from materials containing -NH2. The sandwich immunodetection chip constructed by the application can realize rapid and high-sensitivity detection of the target object.

Description

Technical Field

[0001] This invention belongs to the fields of materials science and biomedicine, and in particular relates to a fully printed biodetection chip, its preparation method, and its application. Background Technology

[0002] In modern medical practice, biochips can detect and monitor diseases, enabling rapid and accurate assessments of patients' conditions, making them an important diagnostic and analytical tool. Currently, most biochips are constructed on a substrate surface using microelectronics or microfabrication techniques, creating a modular, integrated micro-biochemical analysis system. This method allows for efficient and rapid testing and analysis of target biomarkers, viruses, bacteria, and other bioactive substances.

[0003] The essence of a biochip is to mechanically immobilize bioactive substances (antibodies, aptamers, enzymes, etc.) on the surface of a solid-phase carrier, such as a plastic sheet, silicon wafer, glass, or NC membrane. Under certain conditions, the bioactive substances bind to or react with the sample to be detected. The sample is labeled using methods such as isotope methods, chemiluminescence methods, or enzyme-linked immunosorbent assays (ELISA). Then, a laser confocal microscope is used to detect the signal intensity displayed on the biochip after the reaction, and further quantitative analysis of the hybridization results is performed, thereby achieving high-throughput qualitative and quantitative detection.

[0004] The advantage of biochip technology lies in simplifying, integrating, and modularizing the complex and discontinuous analytical processes involved in life science research, such as sample preparation, chemical reactions, and analytical detection. This significantly reduces testing time and costs, which is of great importance for disease detection. With the increasing demand for point-of-care (POCT) and on-site detection, biochips that provide faster and more accurate test results have become a research hotspot. However, facing the ever-evolving clinical testing needs, existing biochips face significant challenges in areas such as multi-target detection and ultra-sensitive quantitative detection. Summary of the Invention

[0005] To improve the above-mentioned technical problems, the present invention provides a fully printed biodetection chip, including a substrate, on which a polymer coating is formed by inkjet printing, and a biocapture material is disposed on the polymer coating. The substrate, the polymer coating and the biocapture material constitute a sandwich structure, and the biocapture material can specifically react with the target analyte.

[0006] The polymer coating is selected from polymer materials containing -COOH and / or -NH2, and the bio-capturing material is selected from materials containing -NH2.

[0007] Preferably, the polymer material containing -COOH is selected from at least one of poly(methyl methacrylate-acrylic acid-styrene) emulsion, silica microsphere emulsion, polystyrene microsphere emulsion, dicarboxylated polyethylene glycol, carboxyl-terminated polylactic acid, etc., for example, poly(methyl methacrylate-acrylic acid-styrene) latex ball emulsion.

[0008] Preferably, the captured biological material is selected from at least one of enzymes, nucleic acids, antigens, antibodies, aptamers, binding proteins, phytohemagglutinins, and hormone receptors; more preferably, the captured biological material is selected from at least one of antibodies, aptamers, and polypeptides.

[0009] Preferably, the captured biomaterial is an antibody, such as a goat anti-rabbit IgG or ST2 nanobody.

[0010] Preferably, the captured biological material is an aptamer, such as the C6 Amino FAM aptamer.

[0011] Preferably, the captured biomaterial is a polypeptide, such as bradykinin.

[0012] According to an embodiment of the present invention, the captured biological material is labeled with a fluorescent molecule, preferably, the fluorescent molecule is selected from at least one of Rhodamine B, FITC, ROX, FAM, Cy-3, and Cy-5.

[0013] For example, the captured biological material is Cy-3 labeled goat anti-rabbit IgG or Cy-3 labeled ST2 nanobody.

[0014] According to an embodiment of the present invention, when the captured biomaterial comes into contact with the target analyte, the optical characteristics of the captured biomaterial change, such as ultraviolet, infrared, or fluorescence, for example, the fluorescence characteristics of the captured biomaterial change.

[0015] According to an embodiment of the present invention, the polymer coating adheres to the substrate, and the bio-capture material is bonded to the polymer coating.

[0016] Preferably, the bio-capture material is bonded to the polymer coating by chemical bonds, such as amide bonds.

[0017] Preferably, the polymer coating is bonded to the substrate; more preferably, the polymer coating is bonded to the substrate by chemical bonds, such as amide bonds or ester bonds.

[0018] According to an embodiment of the present invention, the polymer coating is a lattice structure, a linear structure, or a surface layer structure.

[0019] According to an embodiment of the present invention, the bio-capturing material is disposed on the polymer coating in a lattice structure.

[0020] According to an embodiment of the present invention, the biodetection chip is further provided with a sealing layer, which covers the areas of the polymer coating and the substrate surface not covered by the captured biomaterial, so as to reduce non-specific adsorption during the detection process.

[0021] According to an embodiment of the present invention, soluble spots are formed on the sealing layer by inkjet printing, preferably, the soluble spots are surrounding the polymer coating.

[0022] The sealing layer is selected from skim milk powder or BSA.

[0023] According to an embodiment of the present invention, the soluble spot solution includes a detection biomaterial capable of specifically reacting with the captured biomaterial.

[0024] According to an embodiment of the present invention, the substrate is selected from materials capable of adhering to materials having -COOH groups. Preferably, the substrate material is selected from plastic sheets, silicon wafers, glass, NC films (nitrocellulose films), such as plastic sheets, exemplary of PET (polyethylene terephthalate), PVC (polyvinyl chloride), BOPS (polystyrene), PP (polypropylene), HIPS (high-impact polystyrene) plastic sheets, preferably PET plastic sheets.

[0025] According to an embodiment of the present invention, the substrate material is a hydrophobic material. As an example, the substrate is a PET plastic sheet substrate. The contact angle of the PET plastic sheet substrate is greater than 70°, preferably greater than 75°, more preferably greater than 80°, and even more preferably greater than 85°, for example, 71°, 72°, 74°, 76°, 78°, or 80°.

[0026] The purpose of a contact angle greater than 70° is to enable the printed ink droplets to form a "bulge" structure on the substrate material, preventing them from spreading too much and facilitating the enrichment of the printing material and subsequent reactions.

[0027] The present invention also provides a method for preparing the above-mentioned fully printed biodetection chip, comprising the following steps: forming a polymer coating on a substrate, and coupling a biocapture material on the polymer coating.

[0028] According to the technical solution of the present invention, forming a polymer coating on a substrate includes: setting a polymer material on the surface of the substrate and self-assembling to form a polymer coating, for example, inkjet printing the polymer material on the substrate and self-assembling to form the polymer coating.

[0029] According to the technical solution of the present invention, after the polymer material is inkjet printed on the substrate, a thermal sintering step is also included.

[0030] Preferably, the sintering temperature is the glass transition temperature of the polymer material.

[0031] Preferably, the temperature of the hot sintering is 100-150°C, more preferably 110-130°C, for example 120°C.

[0032] Preferably, the hot sintering time is 10-20 min; more preferably, the hot sintering time is 12-18 min; and even more preferably, the hot sintering time is 14-16 min.

[0033] According to an embodiment of the present invention, coupling the captured biomaterial onto the polymer coating comprises: immobilizing the captured biomaterial onto the polymer coating via a coupling reaction.

[0034] Preferably, the coupling reaction includes dropping the captured biological material onto the polymer coating, for example, by dot-matrix dropping.

[0035] Preferably, the concentration of the captured biological material is 10 μg / mL to 1 mg / mL; more preferably, the concentration of the captured biological material is 100 μg / mL to 1 mg / mL; and even more preferably, the concentration of the captured biological material is 300 μg / mL to 500 μg / mL. For example, the captured biological material is selected from Cy-3-labeled goat anti-rabbit IgG at a concentration of 1 mg / mL, C6Amino FAM aptamer at a concentration of 1 mg / mL, or Cly-5-labeled CRP aptamer I at a concentration of 10 μg / mL.

[0036] Preferably, the coupling reaction is carried out overnight under sealed conditions at a temperature of 4°C.

[0037] According to an embodiment of the present invention, the coupling reaction includes a step of activating the polymer coating first.

[0038] Preferably, activating the polymer coating involves immersing the substrate with the polymer coating in an activating agent to activate surface groups.

[0039] Preferably, the activating agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide at a volume ratio of 1:15 mg / mL and 10 mg / mL, respectively.

[0040] Preferably, the activation temperature is room temperature, and the activation time is 20-40 min, for example, 30 min.

[0041] According to an embodiment of the present invention, after the coupling reaction, a step of recovering the captured biological material is further included. Preferably, recovering the captured biological material includes rinsing the coupled substrate. More preferably, the rinsing solution is physiological saline, ultrapure water, or PBST solution, and the rinsing is performed 3-5 times, for example, 4 times using PBST solution.

[0042] According to an embodiment of the present invention, when the captured biological material is an antibody, aptamer, or polypeptide, the method further includes the steps of sealing the coupled biodetection chip and setting soluble plaques.

[0043] Preferably, the sealing includes the step of immersing the biodetector chip in a sealing solution. More preferably, the sealing solution is selected from solutions including skim milk solution or BSA solution, such as a 5% skim milk powder solution or a 5% BSA solution.

[0044] Preferably, the sealing temperature is 25-37℃ and the sealing time is 30-120 min; more preferably, the sealing temperature is 28-35℃ and the sealing time is 50-100 min; even more preferably, the sealing temperature is 30-35℃ and the sealing time is 60-80 min, for example, sealing at 37℃ for 1 hour.

[0045] Preferably, after the sealing is completed, the step further includes rinsing the sealed biodetector chip. More preferably, the rinsing solution is physiological saline, ultrapure water or PBST solution, and the rinsing is performed 3-5 times, for example, rinsing 4 times with PBST solution.

[0046] According to an embodiment of the present invention, setting a soluble spot includes fixing a soluble spot solution around the polymer coating on a sealed biodetection chip.

[0047] Preferably, the fixation is performed by in-situ inkjet printing a patterned array using Microplatter II. The soluble spot solution includes trehalose and detection biomaterials, which can pair with the capture biomaterials. For example, when the target analyte is CRP, the aptamer is Cy-5 labeled CRP aptamer I, and the detection biomaterial is Cy-3 labeled CRP aptamer II.

[0048] Preferably, the concentration of the detection biological material is 1 μg / ml to 100 μg / ml; more preferably, the concentration of the detection biological material is 10 μg / ml to 80 μg / ml; and even more preferably, the concentration of the detection biological material is 20 μg / ml to 50 μg / ml, for example, 10 μg / ml, 20 μg / ml, or 30 μg / ml.

[0049] Preferably, the concentration of trehalose is 0.01–1 mg / mL, more preferably, the concentration of trehalose is 0.1–0.6 mg / mL, and even more preferably, the concentration of trehalose is 0.3–0.4 mg / mL, for example, 1 mg / mL.

[0050] Preferably, the volume ratio of the aptamer to trehalose is 1:1 to 3, for example, 1:1.

[0051] The present invention also provides a method for using the above-mentioned biodetection chip, including adding the analyte to the biodetection chip for incubation and then detecting the optical changes of the biodetection chip.

[0052] According to an embodiment of the present invention, the incubation is carried out at room temperature, and the incubation time is 5-15 min, preferably 8-12 min, for example 10 min.

[0053] According to an embodiment of the invention, the optical changes are detected, for example, under a fluorescence microscope.

[0054] According to an embodiment of the present invention, after incubation and before detecting optical changes in the biochip, a step of rinsing the biochip is further included. Preferably, the rinsing solution is PBST solution, and the rinsing is performed 3-5 times.

[0055] The present invention also provides a reagent kit comprising the above-mentioned biodetection chip, wherein the biodetection chip is disposed within the reagent kit.

[0056] According to an embodiment of the present invention, the kit further includes independent diluent, rinsing solution, dropper, and storage medium.

[0057] The present invention also provides an application of the above-mentioned biological detection chip for biological detection, wherein the sample to be detected is, for example, human whole blood, serum, animal whole blood, or serum.

[0058] Beneficial effects

[0059] 1. This invention uses inkjet printing to print a polymer coating rich in -COOH functional groups onto a substrate material, immobilizes a capture biomaterial in situ on the polymer coating, and prints a detection biomaterial around it to construct a sandwich immunodetection chip, thereby achieving rapid and highly sensitive detection of the target analyte.

[0060] 2. The biological platform built by this invention has universal applicability, and achieves visual detection by immobilizing biological materials (antibodies, aptamers, and peptides, etc.) through simple coupling reactions.

[0061] 3. The polymer of the present invention is a carrier that can be coupled with a variety of biological materials (antibodies, aptamers, and peptides, etc.), which can realize both single-target detection and multi-target detection, and is an integrated, modular, high-sensitivity, and high-throughput detection platform.

[0062] 4. The fully printable soluble stain detection chip in this invention only requires a drop of blood from the fingertip to perform the detection, eliminating the need for cumbersome detection procedures and specialized personnel in traditional detection methods.

[0063] 5. The method for fabricating the fully printed soluble spot detection chip of this invention is simple, environmentally friendly, can be mass-produced, and is easy to commercialize. Attached Figure Description

[0064] Figure 1 This is a photograph of the polymer coating material dissolved in DMF solution in Example 1 of the present invention.

[0065] Figure 2 This is a schematic diagram of the structure of the biodetection chip (a PET substrate with a polymer coating having -COOH functional groups coupled with Cy-3-labeled IgG antibodies) prepared in Example 3.

[0066] Figure 3 The images show fluorescence images of the immobilized capture biomaterial (Cy3-labeled goat anti-rabbit IgG) and the directly added biomaterial in Example 3.

[0067] Figure 4 This is a schematic diagram of the structure of the C6Amino FAM aptamer biodetector chip coupled to a PET substrate with a polymer coating having -COOH functional groups, prepared in Example 4.

[0068] Figure 5 The images show fluorescence of the immobilized capture biomaterial (C6 Amino FAM aptamer) and the directly added biomaterial in Example 4.

[0069] Figure 6 This is a schematic diagram of the structure of the fully printed soluble spot detection chip in Example 5.

[0070] Figure 7 This is a physical image of the biodetection chip in Example 7.

[0071] Figure 8 A confocal diagram is shown to verify the recognition function of the CRP sandwich-type immune detection chip built for the fully printed soluble plaque detection chip in Example 5.

[0072] Figure 9 This is a standard linear detection curve of the biochip recognizing the inflammatory factor CRP in Example 6. Detailed Implementation

[0073] The structure, preparation method, and application of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0074] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0075] Example 1: Preparation of polymer materials rich in -COOH functional groups

[0076] 1 mg of polylactic acid (PLGA), a polymer material rich in -COOH functional groups, was dissolved in 5 mL of LDM (dimethylformamide) solution and heated at 60 °C with magnetic stirring for 30 min to form a viscous solution.

[0077] Figure 1 The image shows the polymer dissolved in DMF solution. As can be seen from the image, the solution after dissolution is a viscous liquid.

[0078] Example 2: Preparation of polymer coatings rich in -COOH functional groups by inkjet printing

[0079] The polymer solution rich in -COOH functional groups prepared in Example 1 was patterned and array-printed onto a PET substrate using a Microplatter II printer. The substrate was then placed in a 60°C oven for heating and sintering for 10-20 minutes to obtain a cured polymer coating rich in -COOH functional groups.

[0080] Polymer coating printing can be surface printing, line printing, or dot array printing, which can be achieved by patterning the desired pattern using the SnonDraw software of the Microplatter II printer.

[0081] Example 3: Preparation of Immobilized Capture Biomaterial (Antibody as an Example) Biochip

[0082] An antibody-containing biochip was constructed using Cy-3-labeled goat anti-rabbit IgG (excitation wavelength 554 nm, emission wavelength 568 nm) and a PET substrate with a polymer coating containing -COOH functional groups prepared in Example 2.

[0083] See Figure 2 As shown, the specific steps are as follows:

[0084] (1) Prepare 5 mg / mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 10 mg / mL of N-hydroxysuccinimide (NHS). The cross-linking reaction uses a volume ratio of 1:1 and should be prepared and used immediately.

[0085] (2) The PET substrate with polymer coating having -COOH functional groups was immersed in 5 mL of EDC / NHS mixed solution and the surface groups were activated by immersion for 30 min at room temperature.

[0086] (3) Take out the PET substrate with the polymer coating of -COOH functional group and place it in a culture dish. After drying, add 1 mg / mL Cy-3 labeled goat anti-rabbit IgG to each dot on the activated polymer coating. The volume of each dot is about 0.3 μL. Seal the culture dish and place it in a refrigerator at 4°C overnight to form a microarray.

[0087] (4) Remove the culture dish and wash the PET substrate with the polymer coating having -COOH functional groups with PBST (PBS buffer (phosphate buffer), pH=7.3, Tween-20, 0.05%) 3 to 5 times to remove excess uncoupled Cy-3 labeled goat anti-rabbit IgG, thus obtaining the antibody-containing biochip prepared in this example.

[0088] Because the conjugation process should be carried out at 4°C, antibody activity can be guaranteed, and the eluted unconjugated Cy-3-labeled goat anti-rabbit IgG can be collected, recovered, and reused.

[0089] The antibody-containing biochip prepared in this embodiment was placed under a fluorescence microscope, and a green filter with an excitation band of 512-552nm and an emission band of 565-615nm was selected for observation and fluorescence image capture.

[0090] Meanwhile, another PET substrate with a polymer coating containing -COOH functional groups prepared in Example 2 was used without coupling reaction treatment. Cy-3 labeled goat anti-rabbit IgG was directly dropped onto the matrix, and a fluorescence image was captured.

[0091] The fluorescence intensity of the PET substrate with a polymer coating containing -COOH functional groups that has not undergone coupling reaction is compared with that of the antibody-containing biochip prepared in this embodiment to calculate the average coupling rate of antibody fixation.

[0092] See Figure 2 It is known that because a large number of -COOH functional groups are exposed on the surface of the polymer coating, EDC / NHS is used as a coupling agent to react with -NH2 in the antibody protein to form amide bonds, thereby immobilizing the antibody on the PET substrate with the polymer coating containing -COOH functional groups.

[0093] Figure 3 The images show fluorescence images of the immobilized capture biomaterial (Cy3-labeled goat anti-rabbit IgG) and the directly added biomaterial in Example 3. The left image shows the fluorescence image of the capture biomaterial dropped directly onto the biochip, with a fluorescence intensity value of 2330 automatically read by confocal imaging software. The right image shows the fluorescence image of the immobilized capture biomaterial on the biochip through chemical reaction bonding, with a corresponding fluorescence intensity value of 1860. The antibody immobilization coupling efficiency was calculated to be 80%.

[0094] Example 4: Preparation of a biochip using immobilized and captured biomaterials (aptamers as an example)

[0095] A biochip based on the aptamer system was constructed using C6 Amino FAM aptamers (excitation wavelength 480 nm, emission wavelength 524 nm) and a PET substrate with a polymer coating having -COOH functional groups.

[0096] See Figure 4 As shown, the specific steps are as follows:

[0097] (1) Prepare 5 mg / mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 10 mg / mL of N-hydroxysuccinimide (NHS). The cross-linking reaction uses a volume ratio of 1:1 and should be prepared and used immediately.

[0098] (2) The PET substrate with the polymer coating of -COOH functional group prepared in Example 2 was immersed in 5 mL of EDC / NHS mixed solution and activated by immersion at room temperature for 30 min.

[0099] (3) Take out the PET substrate with the polymer coating of -COOH functional group and place it in a culture dish. After drying, add 1 mg / mL C6 Amino FAM aptamer to each dot on the activated PET substrate with the polymer coating of -COOH functional group. The volume of each dot is about 0.3 μL. Seal the culture dish and place it in a refrigerator at 4°C overnight.

[0100] (4) Remove the culture dish and wash the PET substrate with the polymer coating having -COOH functional groups with PBST (PBS buffer, pH=7.3, Tween-20, 0.05%) 3-5 times to remove excess uncoupled C6Amino FAM aptamers, thus obtaining the biochip containing aptamers prepared in this example.

[0101] The coupling process should be carried out at 4°C to ensure the activity of the aptamers. Excess aptamers eluted from the microarray can be collected, recycled, and reused.

[0102] The PET substrate with a polymer coating containing -COOH functional groups was placed under a fluorescence microscope. A filter with an excitation band of 465-495 nm and an emission band of 515-555 nm was selected for observation and fluorescence image capture.

[0103] Meanwhile, another PET substrate with a polymer coating having -COOH functional groups was used without coupling reaction. C6 Amino FAM aptamers were directly dropped onto the matrix, and fluorescence images were captured.

[0104] The fluorescence intensity of the PET substrate with the uncoupled polymer coating containing -COOH functional groups was compared with that of the biochip containing aptamers prepared in this embodiment to calculate the average coupling rate of aptamer fixation. After repeating the experiment 20 times, the average coupling rate of aptamer fixation was calculated.

[0105] See Figure 4 It is known that because a large number of -COOH functional groups are exposed on the surface of the polymer coating, the aptamer can be fixed on the photonic crystal microarray by using EDC / NHS as a coupling agent to react with the -NH2 modified at the end of the aptamer to form amide bonds.

[0106] Figure 5 The images show fluorescence images of the immobilized capture biomaterial (C6 Amino FAM aptamer) and the directly added biomaterial in Example 4. The left image shows the fluorescence image of the capture biomaterial dropped directly onto the biochip, with a fluorescence intensity value of 3620 automatically read by confocal imaging software. The right image shows the fluorescence image of the immobilized capture biomaterial on the biochip through chemical reaction bonding, with a corresponding fluorescence intensity value of 2960. The antibody immobilization coupling efficiency was calculated to be 82%.

[0107] Example 5: CRP Biochip (Fully Printed Soluble Plaque Detection Chip)

[0108] Taking the CRP aptamer system as an example, CRP antigen, Cy-5 labeled CRP aptamer I, and Cy-3 labeled CRP aptamer II are selected. Since the target analyte is the inflammatory factor CRP, Cy-5 labeled CRP aptamer I is used in this embodiment to visually verify that the antibody is immobilized on the polymer coating and at the same time, the immobilization efficiency can be calculated.

[0109] See Figure 6 As shown, the specific experimental steps are as follows:

[0110] (1) The polymer solution rich in -COOH functional groups prepared in Example 1 was patterned and array-printed onto a PET substrate using a Microplatter II printer. Then, it was placed in a 60°C oven for heating and sintering. The heating time was generally controlled at 10-20 min to obtain a PET chip substrate with a polymer coating rich in -COOH functional groups that had been cured.

[0111] (2) Prepare 5 mg / mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 10 mg / mL of N-hydroxysuccinimide (NHS). The cross-linking reaction is carried out at a volume ratio of 1:1. Prepare and use immediately.

[0112] (3) Immerse the PET substrate with the polymer coating having -COOH functional groups in the EDC / NHS mixed solution and activate it by immersion at room temperature for 30 min.

[0113] (4) Take out the PET substrate with the polymer coating of -COOH functional group and place it in a culture dish. After drying, add 10 μg / mL Cy-5 labeled CRP aptamer I to the activated PET substrate one by one, with a volume of about 0.3 μL per spot. Seal the culture dish and place it in a refrigerator at 4°C overnight.

[0114] (5) Remove the culture dish and wash the PET substrate with the polymer coating having -COOH functional groups with PBST (PBS buffer, pH=7.3, Tween-20, 0.05%) 3-5 times to remove excess uncoupled Cy-5 labeled CRP aptamer I.

[0115] (6) The above biochip is sealed by preparing 5% skim milk powder and completely immersing it in the biochip at 37°C for 1 hour to occupy the uncoupled area of ​​the biochip and reduce non-specific adsorption. After sealing, the biochip is washed 3-5 times with PBST (PBS buffer, pH=7.3, Tween-20, 0.05%) to remove excess skim milk powder solution.

[0116] (7) Microplatter II printing: In-situ inkjet printing of 10 μL Cy-3 labeled CRP aptamer II soluble spot solution (a mixed solution of Cy-3 labeled CRP aptamer II and trehalose, volume ratio 1:1, trehalose concentration 1 mg / mL) to print a patterned array onto the periphery of the polymer coating of fixed aptamer I.

[0117] (8) Add 10 μL of CRP antigen (100 μg / mL) to the detection chip in step (7) and incubate at room temperature for 10 min.

[0118] (9) Remove the culture dish and wash the biochip treated in step (8) 3-5 times with PBST (PBS buffer, pH=7.3, Tween-20, 0.05%) to remove the excess unrecognized CRP antigen and Cy-3 labeled CRP aptamer II mixed solution on the biochip, and you will get the biochip that has been recognized.

[0119] The identified biochips were placed under a confocal microscope and excited with 561nm and 640nm excitation light to observe and capture fluorescence images.

[0120] Control groups were set up with no target detection substance CRP antigen added and no human IgG that cannot recognize each other added to replace CRP antigen, respectively, as control group 1 and control group 2 of this embodiment. The above experimental steps (1) to (8) were repeated.

[0121] See Figure 6 The diagram illustrates the preparation of a sandwich-type immunoassay chip for recognizing the inflammatory factor CRP using a Cy-3-labeled CRP aptamer II. This chip exposes numerous -COOH functional groups on the surface of a polymer-coated PET substrate. Using EDC / NHS as a coupling agent, these groups react with the -NH2 groups at the aptamer ends to form amide bonds, thus immobilizing Cy-5-labeled CRP aptamer I on the polymer-coated PET substrate with -COOH functional groups. Unoccupied active sites are then blocked to reduce non-specific adsorption and eliminate false positives. Finally, the chip verifies the recognition function of the mixture of the inflammatory factor CRP and Cy-3-labeled CRP aptamer II with aptamer II immobilized on the polymer-coated PET substrate.

[0122] Figure 7 This is a physical diagram of the fabrication process of the fully printed soluble spot detection chip in this embodiment (using a dot array as an example).

[0123] Figure 8 A confocal diagram was constructed to verify the recognition function of the CRP sandwich-type immune detection chip for the fully printed soluble plaque detection chip in this embodiment. The chips that completed the recognition in this embodiment are shown in Figure 1 (numbered 1, 2, and 3 in the figure), control group 1 without the target detection substance CRP antigen is shown in Figure 4, 5, and 6 in the figure, and control group 2 with human IgG that cannot be mutually recognized is shown in Figure 7, 8, and 9 in the figure.

[0124] from Figure 8As can be seen, Cy-5-labeled CRP aptamer I (excitation wavelength: 640nm) exhibits red fluorescence (Figures 3, 6, and 9), while Cy-3-labeled CRP aptamer II (excitation wavelength: 640nm) exhibits green fluorescence (Figures 2, 5, and 8). The combined image (Merge image) shows an orange fluorescence pattern with red and green fluorescence superimposed (Figures 1, 4, and 7). The fluorescence imaging of the control groups shows that control groups 1 and 2 show no green fluorescence in the 561nm excitation channel, indicating that this biochip can not only immobilize biological antibodies but also maintain their original recognition function.

[0125] Example 6

[0126] The method for rapid detection of the inflammatory factor CRP using a biochip with the lowest detection limit is as follows:

[0127] Taking the CRP aptamer system as an example, CRP antigen, Cy-5-labeled CRP aptamer I, and Cy-3-labeled CRP aptamer II are selected because of the lowest detection limit of the target analyte, the inflammatory factor CRP.

[0128] The specific steps are as follows:

[0129] (1) The polymer solution rich in -COOH functional groups prepared in Example 1 was patterned and array-printed onto a PET substrate using a Microplatter II printer. Then, it was placed in a 60°C oven for heating and sintering. The heating time was controlled at 10-20 min to obtain a PET chip substrate with a polymer coating rich in -COOH functional groups that was cured.

[0130] (2) Prepare 5 mg / mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 10 mg / mL of N-hydroxysuccinimide (NHS). The cross-linking reaction is carried out at a volume ratio of 1:1. Prepare and use immediately.

[0131] (3) The PET chip substrate with the polymer coating rich in -COOH functional groups prepared in Example 2 was immersed in the EDC / NHS mixed solution and immersed at room temperature for 30 min to activate the polymer coating rich in -COOH functional groups.

[0132] (3) Take out the PET chip substrate with polymer coating rich in -COOH functional groups and place it in a culture dish. After drying, add 10 μg / mL Cly-5 labeled CRP aptamer I to each dot array on the activated PET chip substrate with polymer dot array coating rich in -COOH functional groups. The volume of each dot array is about 0.3 μL. Seal the culture dish and place it in a refrigerator at 4°C overnight.

[0133] (4) Remove the culture dish and wash the dot array 3-5 times with PBST (PBS buffer, pH=7.3, Tween-20, 0.05% by volume) to remove excess uncoupled Cy-5 labeled CRP aptamer I, thus obtaining the biochip containing Cy-5 labeled CRP aptamer I prepared in this embodiment.

[0134] (5) The biochip from step (4) is sealed by preparing 5% skim milk powder and completely immersing it in the solution for 37°C for 1 hour to occupy the uncoupled area on the dot array and reduce non-specific adsorption. After sealing, the biochip is washed with PBST (PBS buffer, pH=7.3, Tween-20, 0.05% by volume) 3-5 times to remove excess skim milk powder solution.

[0135] (6) Microplatter II printing: In-situ inkjet printing of 10 μL Cy-3 labeled CRP aptamer II soluble spot solution (a mixed solution of Cy-3 labeled CRP aptamer II and trehalose, volume ratio 1:1, trehalose concentration 1 mg / mL) to print a patterned array onto the periphery of the polymer coating of fixed aptamer I.

[0136] (7) Dilute the CRP antigen (original solution concentration 100 μg / mL) 10 times and 10 times respectively. 2 times, 10 3 times, 10 4 times, 10 5 times, 10 6 times, 10 7 times, 10 8 After multiplication, a series of CRP antigen solutions were obtained (concentrations of 10 μg / mL, 1 μg / mL, 0.1 μg / mL, 10 μg / mL, etc.). -2 μg / mL, 10 -3 μg / mL, 10 -4 μg / mL, 10 -5 μg / mL, 10 -6 (μg / mL).

[0137] (8) Add 10 μL of the CRP standard antigen samples of different concentrations prepared in step (7) to the detection chip in step (6) and incubate for 10 min at room temperature.

[0138] (9) Remove the culture dish and wash the detection chip 3 to 5 times with PBST (PBS buffer, pH=7.3, Tween-20, 0.05%) to remove excess unrecognized CRP antigen and Cy-3 labeled CRP aptamer II mixed solution on the detection chip.

[0139] (10) Place the detection chip identified in step (9) under a confocal microscope, select 561nm excitation light for excitation, observe and capture fluorescence images.

[0140] Negative control group: A group without the target analyte CRP antigen was set up as a negative control group. Because the interference of non-specific adsorption cannot be eliminated in the experiment, a control group with CRP antigen needs to be added as background for each experiment. The preparation method of the control group is the same as above, except that CRP antigen is not included in step (6).

[0141] Figure 9 The standard linearity curve of the CRP sandwich immunoassay prepared in this embodiment for recognizing the inflammatory factor CRP (the dilution gradient of the target analyte CRP antigen corresponding to the biochip and the absence of CRP antigen, with an initial antigen concentration of 1 μg / mL).

[0142] Example 7

[0143] A universal, fully printed biochip reagent kit and its preparation are detailed below:

[0144] 1. Reagent kit

[0145] The universal fully printed biochip kit in this embodiment includes a detection chip, diluent, rinsing solution, dropper, and storage medium.

[0146] 2. Preparation of the reagent kit

[0147] 1) Fabrication of the detection chip:

[0148] (1) A polymer solution rich in -COOH functional groups is prepared and patterned and array-printed onto a PET substrate using a Microplatter II printer. Then, it is placed in a 60℃ oven for heating and sintering. The heating time is generally controlled at 10-20 min to obtain a PET chip substrate with a polymer coating rich in -COOH functional groups that has been cured.

[0149] (2) Prepare 5 mg / mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 10 mg / mL of N-hydroxysuccinimide (NHS). The cross-linking reaction uses a volume ratio of 1:1 and should be prepared and used immediately.

[0150] (3) The PET chip substrate with the polymer coating rich in -COOH functional groups prepared in Example 2 was immersed in the EDC / NHS mixed solution and the polymer coating rich in -COOH functional groups was activated at room temperature for 30 min.

[0151] (3) Take out the PET chip substrate with polymer coating rich in -COOH functional groups and place it in a culture dish. After drying, add the capture-type biological material drop by drop onto the activated PET chip substrate with polymer dot array coating rich in -COOH functional groups one by one. The volume of each dot array is about 0.3 μL. Seal the culture dish and place it in a refrigerator at 4°C overnight.

[0152] (4) Remove the culture dish and wash the dot array 3-5 times with PBST (PBS buffer, pH=7.3, Tween-20, 0.05% by volume) to remove excess uncoupled capture biomaterials, thus obtaining the biochip containing capture biomaterials prepared in this embodiment.

[0153] (5) The biochip from step (4) is sealed by preparing 5% skim milk powder and completely immersing it in the solution for 37°C for 1 hour to occupy the uncoupled area on the dot array and reduce non-specific adsorption. After sealing, the biochip is washed with PBST (PBS buffer, pH=7.3, Tween-20, 0.05% by volume) 3-5 times to remove excess skim milk powder solution.

[0154] (6) Microplatter II in-situ inkjet printing of 10 μL of soluble detection biomaterial (a mixed solution of detection biomaterial and trehalose, volume ratio 1:1, trehalose concentration 1 mg / mL) prints a patterned array onto the periphery of the polymer coating that immobilizes the capture biomaterial.

[0155] 2) How to use the detection chip:

[0156] (1) Add the target analyte (e.g., human whole blood, serum, etc.) to the detection chip and incubate for 10 minutes at room temperature.

[0157] (2) Wash the biochip 3 to 5 times with PBST (PBS buffer, pH=7.3, Tween-20, 0.05%) to remove excess unidentified samples on the biochip.

[0158] (3) Place the biochip identified in step (2) under a fluorescence microscope, select the excitation light required for the specific label fluorescence, and observe the fluorescence image.

[0159] 3) Storage of the reagent kit

[0160] The kit should be stored at 4°C.

[0161] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a full-print bio-detection chip, characterized in that, The method comprises the following steps: printing a polymer material on a substrate by inkjet printing, self-assembling to form a polymer coating, coupling a capturing biomaterial on the polymer coating, the substrate, the polymer coating and the capturing biomaterial forming a sandwich structure, and the capturing biomaterial being capable of specifically reacting with a target object to be detected. The polymer coating is selected from a polymer material containing -COOH, and the capturing biomaterial is selected from a material containing -NH2; the polymer material containing -COOH is selected from at least one of a poly(methyl methacrylate-acrylic acid-styrene) emulsion, a silica microsphere emulsion, a polystyrene microsphere emulsion, a double-carboxyl polyethylene glycol, and a carboxyl-terminated polylactic acid.

2. The method of claim 1, wherein the method further comprises the step of: The capturing biomaterial is selected from at least one of an enzyme, a nucleic acid, an antigen, an antibody, an aptamer, a binding protein, a plant coagulant, and a hormone receptor.

3. The method of claim 1, wherein the method further comprises the step of: The capturing biomaterial is selected from at least one of an antibody, an aptamer, and a polypeptide. ​ 4. The method of claim 1, wherein the method further comprises the step of: The substrate is selected from a material capable of adhering to a group containing -COOH. ​ 5. The method for preparing a full-print bio-detection chip according to claim 1, characterized in that, The substrate material is selected from a plastic sheet, a silicon wafer, glass, and an NC film.

6. The method of claim 1, wherein the printing is performed by a 3D printer. The concentration of the capturing biomaterial is 10 μg / mL-1 mg / mL.

7. The method of claim 6, wherein the printing is performed by a thermal inkjet printer. The concentration of the capturing biomaterial is 100 μg / mL-1 mg / mL.

8. The method of claim 1-7, wherein the method further comprises, The coupling reaction is preceded by a step of activating the polymer coating, which comprises immersing the substrate with the polymer coating in an activating reagent to activate the surface groups. The coupling reaction is followed by a step of recovering the capturing biomaterial.

9. The method of claim 1-7, wherein the method further comprises, When the capturing biomaterial is an antibody, an aptamer, or a polypeptide, the method further comprises a step of blocking the bio-detection chip after the coupling and setting a soluble spot.

10. The method of claim 9, wherein the printing is performed by a method selected from the group consisting of inkjet printing, thermal transfer printing, and laser printing. The blocking comprises a step of immersing the bio-detection chip in a blocking solution at a temperature of 25-37°C for 30-120 min, and the setting of the soluble spot comprises fixing a soluble spot solution on the periphery of the polymer coating of the blocked bio-detection chip.

11. The method of claim 10, wherein the printing is performed by a method selected from the group consisting of inkjet printing, thermal transfer printing, and laser printing. The fixing is performed by in-situ inkjet printing patterning array printing using a Microplatter II, the soluble spot solution comprises trehalose and a detection biomaterial, and the detection biomaterial is capable of pairing with the capturing biomaterial.

12. The method of claim 11, wherein the printing is performed by a method selected from the group consisting of inkjet printing, thermal transfer printing, and laser printing. The concentration of the detection biomaterial is 1 μg / ml-100 μg / ml.

13. The method of claim 12, wherein the printing is performed by a method selected from the group consisting of inkjet printing, thermal transfer printing, and laser printing. The concentration of the trehalose is 0.01-1 mg / mL, and the volume ratio of the aptamer to the trehalose is 1:1-3.

14. A method for using a bio-detection chip prepared by the method for preparing a fully-printed bio-detection chip according to any one of claims 1-13, which comprises adding a target object to be detected to the bio-detection chip for incubation, and detecting optical changes of the bio-detection chip.

15. The method of use of claim 14, wherein, The incubation is performed at room temperature, and the incubation time is 5-15 min.

16. The method of use of claim 14, wherein, The detection of the optical changes is performed under a fluorescence microscope. After the incubation and before the detection of the optical changes of the bio-detection chip, the method further comprises a step of washing the bio-detection chip.

17. A kit, which comprises a bio-detection chip prepared by the method for preparing a fully-printed bio-detection chip according to any one of claims 1-13.

18. The kit of claim 17, wherein The kit is also provided with diluents, flushing liquids, pipettes and storage media, which are independent of each other. The kit is also provided with diluents, flushing liquids, pipettes and storage media, which are independent of each other.

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