A detection device based on nanozyme signal amplification and its use method and application

Abstract

The present invention discloses a detection device based on nanozyme signal amplification, and its use method and application, which belong to the field of protein detection technology. The present invention solves the problems of operational difficulties in collecting tear samples, greater stimulation to the tear film, and the elution of collected tears also affecting the results. The present invention prepares a matrix with a microcavity, and performs surface modification on a transparent film for embedding the matrix, and fixes a capture antibody to prepare a capture platform, and embeds the modified transparent film therein; a nanozyme with peroxidase-like activity is coupled to a detection antibody by carboxyl activation to prepare a nanozyme-enhanced detection probe; the detection probe is added to the capture platform that has been in contact with the object to be detected, incubated and then washed; then a hydrogen-donating substrate and peroxide are added for a color reaction, and a stop solution is added after sufficient reaction to stop the color reaction. The present invention has the performance of non-invasive, stable and accurate detection of proteins in 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:

[0005] 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;

[0006] The capture platform comprises a transparent film, to which a capture antibody that can specifically bind to the analyte is connected;

[0007] 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.

[0008] 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.

[0009] Preferably, the transparent film is a transparent film having active ester on the surface.

[0010] 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.

[0011] Preferably, the transparent film is one of a polyethylene film, a polyurethane film, a polypropylene film and a polycarbonate film.

[0012] 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.

[0013] Preferably, the detection probe is obtained by coupling a nanozyme having peroxidase-like activity and a detection antibody that can specifically detect the analyte.

[0014] Preferably, the nanozyme is an Au2Pt nanozyme synthesized by a one-pot process.

[0015] After adopting this technical solution, Au2Pt nanozyme has good activity and high catalytic efficiency. The one-pot synthesis method is relatively simple.

[0016] Preferably, the particle size of the nanozyme is 10-500 nm.

[0017] 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.

[0018] Preferably, there are several microcavities, and a capture platform is disposed in each microcavity.

[0019] After adopting this technical solution, several microcavities are set up to increase the capture efficiency, avoid contingency, and improve the detection accuracy.

[0020] Preferably, the substrate is a contact lens.

[0021] 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.

[0022] A method for using a detection device based on nanozyme signal amplification comprises the following steps:

[0023] Step A: placing the prepared matrix in the eye socket so that the matrix contacts the eyeball;

[0024] Step B: The tear fluid enters the microcavity on the substrate, and the analyte is captured by the capture platform in the microcavity;

[0025] Step C: taking the substrate out of the eye socket, removing excess liquid from the substrate, and washing the substrate;

[0026] Step D: adding detection probes to the capture platform and incubating;

[0027] Step E: washing after incubation;

[0028] Step F: adding nanozyme substrate color development solution to the capture platform for color development reaction;

[0029] Step G: After the color development reaction is completed, add the color stop solution.

[0030] A detection device based on nanozyme signal amplification is used for detecting human tear protein.

[0031] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0032] 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 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

[0033] Figure 1 It is the technical roadmap of the present invention;

[0034] Figure 2 It is a technical roadmap for surface modification of transparent film in the present invention;

[0035] Figure 3 A technical roadmap for preparing the substrate in the present invention;

[0036] 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;

[0037] 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;

[0038] Figure 6 This is the XPS characterization diagram of the C element of the modified polyethylene film of the present invention;

[0039] Figure 7 This is the XPS characterization diagram of the N element of the modified polyethylene film of the present invention;

[0040] Figure 8 This is the XPS characterization diagram of the O element after the polyethylene film is modified in the present invention;

[0041] Fig. 9 An image of the substrate prepared in the present invention;

[0042] Fig.10 This is a mold modeling diagram in the present invention;

[0043] 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;

[0044] Fig.12 The synthetic route of Au2Pt nanozyme in the present invention;

[0045] Fig.13 is a transmission electron microscope image of the Au2Pt nanozyme of the present invention;

[0046] Fig.14 This is the element mapping image of the Au2Pt nanozyme in the present invention;

[0047] 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;

[0048] Fig.16 This is the Zeta potential diagram before and after the Au2Pt nanozyme is coupled to the detection antibody in the present invention;

[0049] 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;

[0050] 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

[0051] 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.

[0052] Preparation Example 1

[0053]

Preparation of the matrix

[0054] Step 1: If Figure 3 As shown in Figure 2, a contact lens made of poly(2-hydroxyethyl methacrylate) was polymerized by thermal initiation: 1 mL of hydroxyethyl methacrylate (purity greater than 98%), 4 μL of ethylene glycol dimethacrylate (purity greater than 98%), 300 μL of water, and 40 μL of ammonium persulfate (purity greater than 98%) were mixed, and then 40 μL of tetramethylethylenediamine (purity greater than 98%) was added and mixed quickly, and then injected into a customized transparent resin mold (refer to Figure 3 , Fig.10 , Fig.11 It can be seen that the mold consists of a male mold and a female mold, and the surface of the male mold has six evenly distributed protrusions) and is sealed;

[0055] Step 2: react at 30-90 degrees Celsius (50 degrees Celsius in this embodiment) for 0.1-24 hours (1 hour in this embodiment), take out and soak in ultrapure water for 1-7 days (3 days in this embodiment), during which the water is changed regularly to remove uncrosslinked monomers;

[0056] Step 3: If Fig. 9 As shown, the obtained substrate has 6 evenly distributed microcavities with a diameter of 3 mm and a depth of 0.1 mm, which can be used to collect tear fluid. The size of the substrate refers to the size of existing contact lenses (13.5-14.5 mm), and in this embodiment, it is 14 mm.

[0057] 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.

[0058] Preparation Example 2

[0059] Preparation of capture platform

[0060] Step 1: If 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;

[0061] 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;

[0062] 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;

[0063] 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;

[0064] 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;

[0065] 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.

[0066] 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 4Before 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.

[0067] Preparation Example 3

[0068] [Assembly of substrate and capture platform]

[0069] 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.

[0070] Preparation Example 4

[0071]

Preparation of Au2Pt nanozyme

[0072] Step 1: If Fig.12 As shown, 15 mg of L-proline was dissolved in 2 ml of ultrapure water, 400 μl of 10 mmol / l chloroauric acid and 400 μl of 10 mmol / l potassium chloroplatinate were added, and after mixing, 100 μl of 20 mg / ml polyvinyl pyrrolidone was added.

[0073] Step 2: Add 800 μL of 20 mg / mL L-ascorbic acid to the above solution, vortex quickly to mix and heat at 65°C for 5 minutes. After the reaction is completed, resuspend in 2 mL of ultrapure water and store at 4°C for later use. Fig.13 and Fig.14 As shown, from Fig.13 and Fig.14 It can be seen that Fig.13 This is the transmission electron microscope image of the nanozyme, which proves the morphology and good dispersibility of the nanozyme. Fig.14 This is the elemental mapping diagram, which proves the elemental distribution of the nanozyme.

[0074] Preparation Example 5

[0075]

Preparation of detection probe

[0076] Step 1: Take 50 μL of the Au2Pt nanozyme prepared in Preparation Example 4 and dissolve it in 150 μL of 2-morpholineethanesulfonic acid buffer, add 2 μL of 0.1 mol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1 μL of 0.1 mol / L N-hydroxysuccinimide for activation, react for 15 minutes, wash and centrifuge, and disperse the obtained product in 50 μL of 0.01 mol / L phosphate Tween buffer;

[0077] Step 2: Take the activated nanozyme and connect it to the detection antibody of matrix metalloproteinase-9 in phosphate-Tween buffer, incubate it on a shaker at 4 degrees Celsius for 2 hours, and centrifuge and wash it after the reaction is completed;

[0078] Step 3: After the coupling is completed, centrifuge and wash, add phosphate Tween buffer containing 5% bovine serum albumin to block the remaining activated groups, and finally resuspend the product in phosphate Tween buffer containing 1% bovine serum albumin to obtain the detection probe. The hydration particle size diagram before and after Au2Pt nanozyme coupling detection antibody and the Zeta potential diagram before and after Au2Pt nanozyme coupling detection antibody are shown in Figure 2. Fig.15 and Fig.16 As shown, from Fig.15 and Fig.16 It can be seen that Fig.15 The results of dynamic light scattering show that the particle size of the nanozyme increased after being coupled with the antibody. Fig.16 The result of zeta potential is that the antibody is positively charged under neutral conditions, and after connecting with the nanozyme, the electronegativity of the nanozyme is weakened. Both characterizations prove the success of the coupling of the antibody and the nanozyme.

[0079] Example 1

[0080] 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;

[0081] The capture platform comprises a transparent film, on which there is a capture antibody that can specifically bind to the analyte;

[0082] 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.

[0083] Example 2

[0084] like Figure 1 As shown, a method for using a detection device based on nanozyme signal amplification in Example 1 comprises the following steps:

[0085] Step A: placing the contact lens described in Example 1 in the eye socket so that the contact lens contacts the eyeball;

[0086] 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;

[0087] 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;

[0088] 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;

[0089] Step E: After incubation, wash with phosphate-Tween buffer, repeat 3 times;

[0090] Step F: adding nanozyme substrate color development solution to the capture platform for color development reaction;

[0091] 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 .

[0092] 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 .

[0093] 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, characterized in that: 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; Also included is a detection probe, wherein the raw materials for preparing the detection probe include a nanozyme having peroxidase-like activity and a detection antibody capable of specifically detecting the object to be detected; The transparent film is a transparent film having active ester on the surface, and the transparent film is one of a polyethylene film, a polyurethane film, a polypropylene film, and a polycarbonate film; The specific steps for obtaining a transparent film with active esters are as follows: Step 1: Soak the transparent film in a mixed solution of dopamine hydrochloride and 3,4-dihydroxybenzoic acid to deposit a layer of hydroxyl and carboxyl polyethylene film on the surface; Step 2: soaking the polyethylene film with a layer of hydroxyl and carboxyl groups deposited on the surface in a polyethyleneimine solution to obtain a polyethylene film connected with polyethyleneimine; Step 3: dissolving N-hydroxysuccinimide-polyethylene glycol-N-hydroxysuccinimide in 2-morpholineethanesulfonic acid buffer to obtain a mixed solution, and immersing a polyethylene film connected with polyethyleneimine in the mixed solution to obtain a polyethylene film with active ester; The detection probe is obtained by coupling a nanozyme having peroxidase-like activity and a detection antibody capable of specifically detecting the analyte; There are several microcavities, and each microcavity is provided with a capture platform; The substrate is a contact lens; The microcavity is integrally formed with the substrate; The nanozyme is an Au2Pt nanozyme synthesized by a one-pot method.

2. A detection device based on nanozyme signal amplification according to claim 1, characterized in that: The particle size of nanozymes is 10-500nm.

3. A method for using the detection device based on nanozyme signal amplification according to any one of claims 1 to 2, characterized in that: 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: After wearing for 1-2 hours, remove the substrate from the eye socket, remove excess liquid on the substrate, and wash 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.

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