Detection device based on nano-enzyme signal amplification as well as use method and application of detection device

Abstract

The invention discloses a detection device based on nano-enzyme signal amplification as well as a use method and application thereof, and belongs to the technical field of protein detection. The problems that operation is difficult when a tear specimen is collected, stimulation to a tear film is large, and elution of collected tears can affect a result are solved. The method comprises the following steps: preparing a substrate with a microcavity, carrying out surface modification on a transparent film used for being embedded into the substrate, fixing a capture antibody to prepare a capture platform, and embedding the modified transparent film into the capture platform; nano-enzyme with peroxidase-like activity is coupled with a detection antibody in a carboxyl activation mode, and a nano-enzyme enhanced detection probe is prepared; adding the detection probe to a capture platform contacted with an object to be detected, incubating, and washing; then adding a hydrogen-donor substrate and peroxide to carry out a chromogenic reaction, and adding a stop solution to stop the chromogenic reaction after the reaction is sufficient. The method has the performance of noninvasively, stably and accurately detecting the protein in the tears.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein detection, and specifically relates to a detection device based on nanozyme signal amplification, and a use method and application thereof. Background Art

[0002] As one of the body fluids, tears contain rich biomolecular information, such as proteins, nucleic acids, and metabolites. Changes in these components can reflect the biological changes and pathological mechanisms of eye diseases and some systemic diseases. The detection of biomarkers in tears is of great significance for the timely treatment and control of diseases. Although tear proteins are of great significance for the diagnosis and detection of eye diseases, due to the limited amount of tears, collecting sufficient and high-quality tear proteins is still a challenge. At present, the collection of tear proteins for disease diagnosis and proteomics analysis in China mostly uses capillary pipettes and Schirmer test strips to collect tear specimens at the lacrimal caruncle of the conjunctival sac of the lower eyelid, but the operation is difficult and has a greater irritation to the tear film. In addition to the difficulties in sampling, the elution of the collected tears will also affect the results. Summary of the invention

[0003] In view of the problems in the prior art of collecting tear samples, such as the difficulty in operation, the great stimulation to the tear film, and the influence of elution of the collected tears on the results, the present invention provides a detection device based on nanozyme signal amplification, and its use method and application.

[0004] The technical solution adopted by the present invention is as follows: A detection device based on nanozyme signal amplification, comprising a substrate that can be placed in the eye, the substrate is provided with a microcavity that can accommodate tears, and the microcavity is provided with a capture platform; The capture platform comprises a transparent film, to which a capture antibody that can specifically bind to the analyte is connected; It also includes a detection probe, the preparation materials of which include a nanozyme with peroxidase-like activity and a detection antibody that can specifically detect the object to be detected.

[0005] After adopting this technical solution, the present invention makes a matrix that can be placed in the eye, and sets a microcavity with a capture platform on the matrix. When the matrix is ​​brought into the eye, the tears will automatically enter the microcavity and contact and combine with the capture antibody on the capture platform, and then the detection probe is used to combine with the analyte on the capture antibody, and then the color reaction of the detection probe can be used to detect whether the tear contains the analyte and the content of the analyte. Therefore, the detection device provided by the present invention realizes non-invasive, stable and accurate detection of the analyte in the tear.

[0006] Preferably, the transparent film is a transparent film having active ester on the surface.

[0007] After adopting this technical solution, the transparent film with active ester can be combined with the amino group on the antibody through the active ester on the surface, thereby connecting the transparent film with the capture antibody.

[0008] Preferably, the transparent film is one of a polyethylene film, a polyurethane film, a polypropylene film and a polycarbonate film.

[0009] After adopting this technical solution, the polyethylene film, polyurethane film, polypropylene film and polycarbonate film have stable structures and are easy to carry out surface modification.

[0010] Preferably, the detection probe is obtained by coupling a nanozyme having peroxidase-like activity and a detection antibody that can specifically detect the analyte.

[0011] Preferably, the nanozyme is an Au2Pt nanozyme synthesized by a one-pot process.

[0012] After adopting this technical solution, Au2Pt nanozyme has good activity and high catalytic efficiency. The one-pot synthesis method is relatively simple.

[0013] Preferably, the particle size of the nanozyme is 10-500 nm.

[0014] After adopting this technical solution, the smaller the particle size, the larger the specific surface area and the higher the activity, so the particle size of the nanozyme is selected to be 10-500nm.

[0015] Preferably, there are several microcavities, and a capture platform is disposed in each microcavity.

[0016] After adopting this technical solution, several microcavities are set up to increase the capture efficiency, avoid contingency, and improve the detection accuracy.

[0017] Preferably, the substrate is a contact lens.

[0018] After adopting this technical solution, the contact lens that can be put into the eye is used as a base, which reduces the probability of causing resistance emotions in users.

[0019] A method for using a detection device based on nanozyme signal amplification comprises the following steps: Step A: placing the prepared matrix in the eye socket so that the matrix contacts the eyeball; Step B: The tear fluid enters the microcavity on the substrate, and the analyte is captured by the capture platform in the microcavity; Step C: taking the substrate out of the eye socket, removing excess liquid from the substrate, and washing the substrate; Step D: adding detection probes to the capture platform and incubating; Step E: washing after incubation; Step F: adding nanozyme substrate color development solution to the capture platform for color development reaction; Step G: After the color development reaction is completed, add the color stop solution.

[0020] A detection device based on nanozyme signal amplification is used for detecting human tear protein.

[0021] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: The present invention makes a matrix that can be placed in the eye, and sets a microcavity with a capture platform on the matrix. When the matrix is ​​brought into the eye, the tears will automatically enter the microcavity and contact and combine with the capture antibody on the capture platform, and then the detection probe is used to combine with the analyte on the capture antibody, and then the color reaction of the detection probe can be used to detect whether the tear contains the analyte and the content of the analyte can be detected. Therefore, the detection device provided by the present invention realizes the non-invasive, stable and accurate detection of the analyte in the tear. At the same time, the present invention can also place a capture platform for capturing different analytes in each microcavity, so as to realize the detection of multiple analytes at the same time and improve the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the technical roadmap of the present invention; Figure 2 It is a technical roadmap for surface modification of transparent film in the present invention; Figure 3 A technical roadmap for preparing the substrate in the present invention; Figure 4 This is an image of the polyethylene film coupled to the antibody before modification and then coupled to the fluorescently labeled secondary antibody under a fluorescence microscope in the present invention; Figure 5 This is an image of the modified polyethylene film coupled to the antibody and then coupled to the fluorescently labeled secondary antibody under a fluorescence microscope in the present invention; Figure 6 This is the XPS characterization diagram of the C element of the modified polyethylene film of the present invention; Figure 7 This is the XPS characterization diagram of the N element of the modified polyethylene film of the present invention; Figure 8 This is the XPS characterization diagram of the O element after the polyethylene film is modified in the present invention; Fig. 9 An image of the substrate prepared in the present invention; Fig.10 This is a mold modeling diagram of the present invention; Fig.11 This is a 3D printed physical picture of the mold modeling in the present invention, wherein Fig.11 a is the physical picture of the concave die, Fig.11 b is the physical picture of the punch; Fig.12 The synthetic route of Au2Pt nanozyme in the present invention; Fig.13 is a transmission electron microscope image of the Au2Pt nanozyme of the present invention; Fig.14 This is the element mapping image of the Au2Pt nanozyme in the present invention; Fig.15 This is a hydration particle size diagram before and after the Au2Pt nanozyme is coupled to the detection antibody in the present invention; Fig.16 This is the Zeta potential diagram before and after the Au2Pt nanozyme is coupled to the detection antibody in the present invention; Fig.17 It is a calibration diagram of the color absorbance of Au2Pt nanozyme and the concentration of matrix metalloproteinase-9 in the present invention; Fig.18 This is a calibration diagram of the colorimetric absorbance of the Au2Pt nanozyme and the tumor necrosis factor concentration in the present invention. DETAILED DESCRIPTION

[0023] To make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments. The components of the embodiments of the present application generally described and shown in the drawings here can be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present application provided in the drawings is not intended to limit the scope of the application claimed for protection, but merely represents the selected embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present application.

[0024] Preparation Example 1

Preparation of the matrix

[0025] In this preparation example, a contact lens is used as a substrate, and a preparation method of a contact lens is provided, which is a prior art. For details not described, please refer to the prior art. In other embodiments, other substrates may also be used, as long as they can be placed in the eye.

[0026] Preparation Example 2 Preparation of capture platform Step 1: Figure 2 As shown in the figure, a polyethylene film was cut into a disc with a radius of 1.5 mm and a thickness of 0.04 mm using a puncher, and the disc was immersed in a mixed solution of dopamine hydrochloride and 3,4-dihydroxybenzoic acid (the total amount of the mixed solution was 20 mL, and the dopamine hydrochloride contained was 2 mg mL -1 , 3,4-dihydroxybenzoic acid 1 mg mL -1 ), and soaking overnight at room temperature under aerobic conditions to deposit a layer of hydroxyl and carboxyl groups on the surface of the polyethylene film to obtain an activated polyethylene film; Step 2: Dissolve 20 mg of polyethyleneimine in 10 ml of ultrapure water, adjust the pH to 5.5 with dilute hydrochloric acid, and soak the activated polyethylene film in the water for 5 hours to obtain a polyethylene film connected with polyethyleneimine; Step 3: dissolve 20 mg of N-hydroxysuccinimide-polyethylene glycol-N-hydroxysuccinimide in 10 ml of 2-morpholineethanesulfonic acid buffer, and soak the polyethylene film treated in the previous step for 1 hour to obtain a polyethylene film with active ester; Step 4: adding a capture antibody of matrix metalloproteinase-9 to the surface of the polyethylene film capable of bioconjugation and incubating overnight at 4 degrees Celsius; since the matrix metalloproteinase-9 in tears is detected in this embodiment, the added capture antibody is a capture antibody of matrix metalloproteinase-9, and in other embodiments, the added capture antibody can be adjusted according to different objects to be detected; Step 5: discard the liquid on the surface of the polyethylene film and wash with 0.01 mol / L phosphate Tween buffer, repeating 3 times; Step 6: Add 0.01 mol phosphate buffer containing 2% bovine serum albumin and incubate at 37 degrees Celsius for 2 hours. Wash with phosphate-Tween buffer and set aside.

[0027] The thickness of the capture platform is smaller than the depth of the microcavity, which can avoid direct contact between the capture platform and the eyes and reduce the probability of displacement of the capture platform. The fluorescence microscopy images of the polyethylene film before and after modification, coupled with the antibody and then coupled with the fluorescently labeled secondary antibody are shown in Figure 2. Figure 4 and Figure 5 As shown, from Figure 4 and Figure 5 It can be seen that the PE membrane before and after surface modification is coupled with the capture antibody, and then coupled with the fluorescent secondary antibody corresponding to the capture antibody. Figure 4 Before surface modification, no obvious fluorescence can be seen, proving that the capture antibody cannot be fixed on the unmodified PE membrane. Figure 5 After surface modification, there is uniform fluorescence intensity, proving that the capture antibody is uniformly modified on the PE membrane. The XPS characterization of the polyethylene membrane after surface modification is shown in the figure below. Figure 6-8 As shown, from Figure 6-8 It can also be seen that the polyethylene film has been fully modified by the above-mentioned method.

[0028] Preparation Example 3 [Assembly of substrate and capture platform] The capture platform obtained in Preparation Example 2 is embedded into the microcavity of the substrate to obtain an assembled substrate such as Fig. 9 As shown, the capture platform can be prefabricated and stored at -20 degrees Celsius. In order to improve the connection effect between the capture platform and the substrate, in other embodiments, the capture platform can also be connected to the microcavity by biocompatible glue, and the biocompatible glue can be cyanoacrylate glue, polyethylene glycol hydrogel, chitosan glue, etc., and the amount used is 0.5 μL glue per membrane. In this embodiment, the capture platform for capturing matrix metalloproteinase-9 is placed in all microcavities. Such an arrangement can form a mutual comparison and improve the accuracy of detection. In other embodiments, a capture platform for capturing different analytes can also be placed in each microcavity to achieve simultaneous detection of multiple analytes and improve detection efficiency.

[0029] Preparation Example 4

Preparation of Au2Pt nanozyme

[0030] Preparation Example 5

Preparation of detection probe

[0031] Example 1

[0032] A detection device based on nanozyme signal amplification, comprising a contact lens insertable into the eye as obtained in Preparation Example 1, wherein the contact lens is provided with a microcavity for accommodating tears, and the microcavity is provided with a capture platform as obtained in Preparation Example 2; The capture platform comprises a transparent film, on which there is a capture antibody that can specifically bind to the analyte; It also includes the detection probe obtained as in Preparation Example 5, wherein the raw materials for preparing the detection probe include a nanozyme having peroxidase-like activity and a detection antibody that can specifically detect the object to be detected.

[0033] Example 2

[0034] like Figure 1 As shown, a method for using a detection device based on nanozyme signal amplification in Example 1 comprises the following steps: Step A: placing the contact lens described in Example 1 in the eye socket so that the contact lens contacts the eyeball; Step B: Tear fluid enters the microcavity on the contact lens, and the object to be tested is captured by the capture platform in the microcavity; Step C: After 1-2 hours, remove the contact lens from the eye socket, remove excess liquid on the contact lens, and wash it with phosphate-tween buffer, repeating 3 times; Step D: Add 100uL of the detection probe prepared in Example 1 with a final concentration of 0.4ug mL-1 to the capture platform and incubate at 37 degrees Celsius for 1 hour; Step E: After incubation, wash with phosphate-Tween buffer, repeat 3 times; Step F: adding nanozyme substrate color development solution to the capture platform for color development reaction; Step G: After the color development reaction is completed, add the nanozyme substrate color development solution (the components of the nanozyme substrate color development solution are 1.7 mL of acetic acid-sodium acetate buffer with a pH of 3.7, 200 uL of hydrogen peroxide and 100 uL of 3,3',5,5'-tetramethylbenzidine), protect from light and color for 3 minutes, add the color stop solution after the reaction is completed, use a pipette to transfer the liquid to the ELISA plate to detect the absorbance value at a wavelength of 450 nanometers, and judge whether the analyte is contained and the content of the analyte based on the color and absorbance value of the liquid in the microcavity. The calibration graph of the color development absorbance of Au2Pt nanozyme and the concentration of matrix metalloproteinase-9 is shown in the figure Fig.17 As shown. Fig.17 It can be seen that in the range of 0-250ng mL -1 The detection limit of matrix metalloproteinase-9 was calculated to be 17.06809 ng mL according to the formula of LOD=3σ / k. -1 .

[0035] In other embodiments, from Fig.18 It can be seen that the detection limit of the present invention for tumor necrosis factor is 11.99769 ng mL -1 .

[0036] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A detection device based on nanozyme signal amplification, Features: It comprises a substrate that can be placed in the eye, the substrate is provided with a microcavity that can accommodate tear fluid, and the microcavity is provided with a capture platform; The capture platform comprises a transparent film, to which a capture antibody that can specifically bind to the analyte is connected; It also includes a detection probe, the preparation materials of which include a nanozyme with peroxidase-like activity and a detection antibody that can specifically detect the object to be detected.

2. A detection device based on nanozyme signal amplification according to claim 1, Features: The transparent film is a transparent film having active ester on the surface.

3. A detection device based on nanozyme signal amplification according to claim 2, Features: The transparent film is one of a polyethylene film, a polyurethane film, a polypropylene film and a polycarbonate film.

4. A detection device based on nanozyme signal amplification according to any one of claims 1 to 3, Features: The detection probe is obtained by coupling a nanozyme having peroxidase-like activity and a detection antibody capable of specifically detecting the object to be detected.

5. A detection device based on nanozyme signal amplification according to any one of claims 1 to 3, Features: The nanozyme is a one-pot synthesized Au 2 Pt nanozymes.

6. A detection device based on nanozyme signal amplification according to claim 4, Features: The particle size of nanozymes is 10-500nm.

7. The detection device based on nanozyme signal amplification according to any one of claims 1 to 3, Features: There are several microcavities, and a capture platform is arranged in each microcavity.

8. The detection device based on nanozyme signal amplification according to any one of claims 1 to 3, Features: The substrate is a contact lens.

9. A method for using the detection device based on nanozyme signal amplification according to any one of claims 1 to 8, Features: The following steps are involved: Step A: placing the prepared matrix in the eye socket so that the matrix contacts the eyeball; Step B: The tear fluid enters the microcavity on the substrate, and the analyte is captured by the capture platform in the microcavity; Step C: taking the substrate out of the eye socket, removing excess liquid from the substrate, and washing the substrate; Step D: adding detection probes to the capture platform and incubating; Step E: washing after incubation; Step F: adding nanozyme substrate color development solution to the capture platform for color development reaction; Step G: After the color development reaction is completed, add the color stop solution.

10. Use of the detection device based on nanozyme signal amplification as described in any one of claims 1 to 8 in detecting human tear protein.

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