Preparation and application of positive electricity driving type composite nano-enzyme label

Abstract

The invention discloses preparation and application of a positive electricity driving type composite nano-enzyme label. According to the composite nano-enzyme microsphere, a PDA (polydopamine) particle is taken as a core, a PEI interlayer is constructed on the surface of the PDA particle by utilizing the self-assembly effect of a positive polymer PEI, chloroplatinic acid radical ions are adsorbed, and then the composite nano-enzyme particle is synthesized in situ under the action of a reducing agent. The preparation method comprises the following steps: firstly, carrying out surface modification on PDA nano-particles by using PEI so that the PDA nano-particles can adsorb chloroplatinate ions; then, a reducing agent sodium borohydride (NaBH4) is added, platinum (Pt) nano-particles are generated on the surfaces of the PDA nano-particles through in-situ reduction, and therefore the composite nano-enzyme microspheres PDA (at) Pt with the core-shell structure are constructed. The invention further discloses application of the colorimetric-catalytic dual-signal label prepared from the composite nano-enzyme microspheres in immunochromatography detection. The novel label is good in dispersity, adjustable in particle size, high in colorimetric and catalytic performance and easy to prepare in batches.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of preparation of new nanomaterials and nanocatalysis, in particular to a preparation and application of a positive electric driving type composite nanocatalysis label. BACKGROUND

[0002] In the field of point-of-care testing (POCT), lateral flow immunoassay (LFIA) is the most widely used commercial detection technology. However, LFIA based on colloidal gold has deficiencies in sensitivity and stability, which restricts its further development. Therefore, it is urgent to explore signal labels based on new nanomaterials and develop a high-sensitivity LFIA platform, especially to achieve ultra-sensitive detection through advanced signal amplification strategies, which has extremely important significance for early disease screening and pathogen monitoring.

[0003] Nanocatalysis is a functional nanoparticle with colorimetric properties and catalytic activity, which can realize the visual detection of trace analytes through the synergistic effect of catalysis and signal conversion. However, there are still two key challenges in the preparation of ideal nanocatalysis labels: first, single platinum (Pt) nanocatalysis particles have poor stability due to their small particle size; second, although composite nanocatalysis improves stability through structural design, its preparation process is relatively complex. These factors together limit the further application of nanocatalysis labels in POCT, and improving the preparation strategy of composite nanocatalysis and enhancing its colorimetric signal strength and catalytic activity are the key to improving the sensitivity of LFIA and expanding its application range.

[0004] Polydopamine (PDA) microspheres have the advantages of simple preparation, strong dispersibility, controllable particle size, strong colorimetric ability, etc., and can be easily recovered by centrifugation, making them suitable for immunochromatographic labels. A new composite nanocatalysis material is formed by in-situ synthesis of Pt nanocatalysis particles on the surface of PDA material, which not only has the colorimetric properties of PDA and Pt, but also has excellent catalytic performance of Pt nanocatalysis. It is a new type of nanocatalysis material with dual-mode and good dispersibility. This material can be used for colorimetric rapid qualitative screening and catalytic ultra-sensitive quantitative detection. Currently, the preparation of composite nanocatalysis materials mainly relies on a multi-step step-by-step method, and a one-step in-situ synthesis method based on polyethyleneimine (PEI) has not been reported.

[0005] In view of this, a preparation and application of a positive electric driving type composite nanocatalysis label are proposed. SUMMARY

[0006] The purpose of the present application is to solve the problems of the prior art and provide a novel composite nanoscale enzyme material with a colorimetric catalytic dual mode. The preparation process of the novel dual-mode composite nanoscale enzyme material is based on PEI-mediated electrostatic adsorption and in-situ reduction of Pt, and is simple, efficient and repeatable, and can be used to prepare composite nanoscale enzyme microspheres of different particle sizes. The present application also provides the above-mentioned dual-mode composite nanoscale enzyme microspheres as a colorimetric-catalytic dual-mode label for detection in an immunochromatographic system.

[0007] The technical route for solving the above technical problems is as follows: a dual-mode composite nanoscale enzyme material, comprising a size-controllable PDA core, a PEI positive interlayer and a Pt nanoscale enzyme shell. The PDA core particle size is 100-300 nm, the PEI self-assembly layer thickness is 1-10 nm, and the nanoscale enzyme particle is a Pt particle.

[0008] As a general inventive concept, the present application also provides a preparation method of a dual-mode composite nanoscale enzyme material, specifically comprising the following steps: (1) adding PDA particles to an aqueous solution, and making the PDA particles monodisperse through ultrasonic reaction to form a PDA aqueous solution; (2) adding the PDA aqueous solution obtained in step (1) to a PEI solution, and making PEI self-assemble on the surface of the PDA nanoparticles to form PDA-PEI particles with positive charges on the surface through ultrasonic reaction; (3) adding the PDA-PEI particles obtained in step (2) to a chloroplatinic acid (H2PtCl6) solution, and making the PDA-PEI particles adsorb a layer of chloroplatinate ions on the surface through intense ultrasonic reaction to obtain composite nanoparticles coated with chloroplatinate ions (pre-PDA@Pt); (4) adding the pre-PDA@Pt particles obtained in step (3) to a sodium borohydride (NaBH4) solution, and making Pt in-situ synthesized on the surface of the PDA nanoparticles through reduction reaction to obtain a dual-mode composite nanoscale enzyme material (PDA@Pt); (5) adding the PDA@Pt particles obtained in step (4) to an ethanol solution of mercaptoundecanoic acid (MUA), and making the PDA@Pt particles couple a layer of carboxyl functional groups on the surface through intense ultrasonic reaction to obtain a carboxyl-functionalized composite nanoscale enzyme material (PDA@Pt-COOH).

[0009] The preparation method of the above-mentioned composite nanoscale enzyme is characterized in that, in step (1), the preparation method of the PDA core particles adopts an improved redox method, the PDA is derived from dopamine hydrochloride (DA·HCl), the solvents are anhydrous ethanol and water, and the reducing agent is 28% ammonia water, and the reaction is carried out at room temperature for 16-24 h.

[0010] The composite nanoscale enzyme preparation method, in the step (2), the molecular weight of PEI is 5000-80000, the concentration of PEI solution is 0.2 mg / mL-5 mg / mL, preferably 1 mg / mL; the ultrasonic time is 10-60 min, preferably 20 min; The composite nanoscale enzyme preparation method, in the step (3), the chloroplatinic acid solution is an aqueous chloroplatinic acid solution, and the concentration ranges from 10 mM to 100 mM; the ultrasonic reaction time is 1-30 min, preferably 5 min; The composite nanoscale enzyme preparation method, in the step (4), the molecular weight of NaBH4 is 37.83, the concentration of NaBH4 solution is 1-100 mM, preferably 10 mM; the ultrasonic time is 1-30 min, preferably 5 min; The composite nanoscale enzyme preparation method, in the step (5), the carboxylation reagent is MUA, and the molecular weight is 218.36; the MUA concentration is 10 μM-50 μM; the ultrasonic reaction time is 20-60 min, preferably 40 min. The washing times after centrifugation are 1-2 times.

[0011] The composite nanoscale enzyme preparation method, in the step (1), the preparation method of the PDA core comprises the following steps: adding a DA-containing precursor and a reducing agent into an organic solvent, stirring uniformly, and then putting into a conical flask for redox reaction, centrifuging to collect the product after the reaction is completed, and obtaining PDA particles.

[0012] The composite nanoscale enzyme preparation method, preferably, the dopamine-containing precursor is dopamine hydrochloride (DA·HCl), the solvent is anhydrous ethanol and water, and the reducing agent is 28% ammonia water; the ratio of dopamine hydrochloride, 28% ammonia water, anhydrous ethanol, and deionized water is 300 mg:2 mL:20 mL:45 mL.

[0013] More preferably, the reaction time of the redox reaction is 16 h-24 h.

[0014] As a general inventive concept, the application also provides an application of a composite nanoscale enzyme microsphere as a dual-mode label of an immunochromatographic detection system. The specific steps are as follows: a specific antibody of influenza A H1N1 is modified on the surface of the dual-mode composite nanoscale enzyme microsphere as a multifunctional immunolabel; 40 μL of a human throat swab sample is added to 40 μL of a buffer, 2 μL of the modified composite nanoscale enzyme microsphere is added, and after mixing, it is added to the sample inlet of a test strip (the detection line is coated with a capture antibody of the influenza A virus), and after 12 minutes, the colorimetric signal at the detection line of the immunochromatographic test strip is read. After the reaction is completed, a color developing solution is added at the detection line of the test strip, and after 2 minutes, the colorimetric enhancement signal at the detection line of the test strip is read. The test strip can realize qualitative detection by colorimetry and super-sensitive detection by nanoscale enzyme catalytic enhancement.

[0015] Compared with the prior art, the application has the following advantages: (1) The dual-mode composite nanoscale enzyme material proposed in the application stands out with its excellent performance. From the perspective of structural composition, the material has a dense Pt shell, which can provide intuitive naked-eye readable colorimetric signals and colorimetric enhancement signals with enzyme-like catalytic activity; at the same time, the controllable PDA core can accurately control the size of the composite nanomaterial. In addition, the material also has good dispersibility, strong stability and easy storage characteristics, and the carboxylated shell provides an ideal site for antibody coupling. In summary, the material is undoubtedly one of the most advanced composite nanoscale enzyme microsphere materials in terms of comprehensive performance.

[0016] (2) The application proposes an in-situ synthesis technology driven by PEI electrostatic adsorption for preparing the composite nanoscale enzyme microsphere material. The preparation method is simple, efficient and repeatable, and has the potential for large-scale production.

[0017] (3) The PEI-driven in-situ synthesis technology proposed in the application is a general composite nanoscale enzyme preparation method. By replacing PDA particles of different particle sizes (100-300 nm), composite nanoscale enzyme microspheres of different particle sizes (120-320 nm) can be prepared.

[0018] (4) The dual-mode composite nanoscale enzyme microsphere material proposed in the application has broad application prospects, including applications in the fields of biosensing, on-site rapid detection, etc. By modifying detection antibodies on the surface of the dual-mode composite nanoscale enzyme microsphere, it can be used as a high-performance dual-mode composite nanoscale enzyme immunolabel for rapid and super-sensitive detection of target substances in biological samples.

[0019] (5) The composite nanoscale enzyme microsphere material as a dual-mode label is used for immunochromatographic detection, has a naked-eye readable colorimetric signal and a colorimetric signal enhanced by catalysis.

[0020] In conclusion, the PEI-driven in-situ synthesis technology of the application realizes controllable preparation of the dual-mode composite nanoscale enzyme microsphere material, and the preparation procedure is simple, efficient and good in repeatability. The prepared dual-mode composite nanoscale enzyme microsphere material has good dispersity and colorimetric catalytic performance, and is uniform in particle size and controllable in surface carboxyl modification, and has a wide application prospect in biological sample detection. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The figure is a preparation method of the dual-mode composite nanoscale enzyme microsphere material of the application.

[0022] Figure 2 The figures are transmission electron microscope (TEM) and element scanning diagrams of each component in the preparation process of the dual-mode composite nanoscale enzyme microsphere material, wherein a diagram is a TEM diagram of PDA particles, b diagram is a TEM diagram of PDA-PEI particles, c diagram is a TEM diagram of PDA@Pt particles, d diagram is a high-definition transmission electron microscope diagram of PDA@Pt particles, e diagram is a HAADF diagram of PDA@Pt, and f diagram is an element surface scanning diagram of PDA@Pt.

[0023] Figure 3 The figures are characterization diagrams in the preparation process of the dual-mode composite nanoscale enzyme material, wherein a diagram is a Zeta potential diagram of each component, b diagram is a wide scanning X-ray photoelectron spectroscopy (XPS) diagram of PDA@Pt particles, and c diagram is a corresponding high-resolution C 1s, N1s, O 1s, 4f and Pt 4f diagram.

[0024] Figure 4 The figures are catalytic performance characterization diagrams of the dual-mode composite nanoscale enzyme microsphere material, wherein a diagram is a UV-vis spectrum and a real object photo of different components after catalysis, and b diagram is a UV-vis spectrum and a real object photo of PDA@Pt with different concentrations after catalysis.

[0025] Figure 5 The figure is a preparation process of antibody modification on the surface of the composite nanoscale enzyme microsphere material of Example 2 of the application.

[0026] Figure 6 The figure is an experimental flowchart of the composite nanoscale enzyme microsphere label of Example 3 of the application as a dual-mode label combined with an immunochromatographic system to detect influenza A H1N1.

[0027] Figure 7The colorimetric and catalytic result photos and gray value intensity of the dual-mode composite nano-enzyme label-based immunochromatographic system of embodiment 3 of the present application for detecting H1N1 antigen.

[0028] Figure 8 The results of the dual-mode composite nano-enzyme label-based immunochromatographic system of embodiment 3 of the present application for detecting clinical samples. DETAILED DESCRIPTION

[0029] The following examples are intended to further illustrate the present application.

[0030] Example 1 The composite nano-enzyme material prepared by the present application comprises a PDA core, a PEI interlayer and a layer of Pt particle shell. The particle size of the PDA particles is 230 nm, the thickness of the PEI interlayer is 5 nm, the particle size of the Pt particles is 5 nm, the final particle size of the synthesized composite nano-enzyme is about 240 nm, and the composite nano-enzyme has both colorimetric signal and catalytic enhancement signal, and has good dispersibility.

[0031] A preparation method of the dual-mode composite nano-enzyme material of the present embodiment, as shown in Figure 1 , comprises the following steps: (1) Preparation of 230 nm PDA core with good dispersibility: Add 45 mL of deionized water, 20 mL of anhydrous ethanol and 2 mL of 28% ammonia water into a 200 mL wide-mouth bottle in turn, add a clean stirring rod, and magnetically stir for 20 min. Add 300 mg of DA·HCl at one time, and stir at room temperature for 24 h. After the reaction is completed, centrifuge at 10000 rpm / 7 min to obtain PDA precipitate, wash twice with anhydrous ethanol, and then place the product in a vacuum oven for vacuum drying at 60°C for 6 h to obtain PDA particle dry powder for standby use.

[0032] (2) Preparation of PDA-PEI particles: Weigh about 10 mg of 230 nm PDA particles and dissolve them in 50 mL of freshly prepared PEI solution (1 mg / mL), and ultrasonically react for 20 min. Centrifuge at 10000 rpm / 7 min, and resuspend in 10 mL of deionized water and 10 mL of anhydrous ethanol for standby use after washing twice with deionized water.

[0033] (3) Preparation of pre-PDA@Pt particles: Add 300 μL of 20 mM chloroplatinic acid solution to the PDA-PEI solution (20 mL), and ultrasonically for 5 min. The PDA-PEI with strong positive electricity adsorbs a large amount of chloroplatinate ions to form pre-PDA@Pt particles.

[0034] (4) Preparation of PDA@Pt particles: A 0.1M sodium borohydride solution (200 μL) was added to the prepared pre-PDA@Pt solution (20 mL) and ultrasonically reacted for 5 minutes. Centrifugation was performed at 8000 rpm for 7 minutes, and the particles were washed twice with deionized water and resuspended in 5 mL of deionized water for use.

[0035] Figure 2 a is the PDA core particle prepared in step (1) of this example, Figure 2 b is the PDA-PEI particle prepared in step (2), Figure 2 c is a transmission electron microscope (TEM) image of the PDA@Pt particle prepared in step (4). As can be seen from the above TEM results, the composite nanoscale enzyme material has a uniform particle size, and the Pt shell is wrapped around the surface of the PDA particle, so that the product has both colorimetric and catalytic signals, from Figure 2 e and f in the figure, it can be seen that the Pt nanoparticles are uniformly distributed on the surface of the PDA.

[0036] The characterization results of the composite nanoscale enzyme microsphere material prepared in this example are shown in Figure 3 The bifunctional composite nanoscale enzyme material proposed in the present application is prepared by in-situ synthesis driven by the electrostatic adsorption of PEI. As can be seen from the change in the Zeta potential of each component in Figure 3 a, with the aid of the positive charge adsorption property of PEI, chloroplatinic acid radicals can be effectively adsorbed on the surface of the PDA core and in-situ synthesis can be achieved. Figure 3 b-c are the wide XPS spectrum of the PDA@Pt particle and the corresponding high-resolution C 1s, N 1s, O 1s, 4f and Pt 4f spectrum. As can be seen from the figure, the binding energy of each atom is consistent with the valence state reported in the literature, confirming the successful reduction and attachment of Pt on the surface of PDA.

[0037] The catalytic performance of the composite nanoscale enzyme microsphere material prepared in this example is shown in Figure 4 As can be seen from Figure 4 a, the composite nanoscale enzyme PDA@Pt has peroxidase activity, and the catalytic activity is stronger as the particle concentration increases Figure 4 b). The above results show that the composite nanoscale enzyme microsphere has excellent catalytic ability and is an ideal signal amplification tag.

[0038] Example 2 The surface of the composite nanoscale enzyme microsphere material proposed in the present application is a carboxylated site, and the carboxyl group on the surface can be used to couple antibodies, so as to realize the surface functionalization of the nanomaterial. The steps for modifying the surface of the composite nanoscale enzyme microsphere with antibodies are shown in Figure 5 and include the following steps: (1) Preparation of carboxyl-functionalized PDA@Pt particles: The prepared PDA@Pt solution (5 mL) was added to a 35 mL MUA (40 μM) solution and ultrasonicated vigorously for 60 min to form PDA@Pt-COOH particles. The particles were centrifuged at 8000 rpm for 7 min and washed once with ethanol before being dried for use.

[0039] (2) Activation of PDA@Pt-COOH particles: The 1 mg PDA@Pt-COOH powder was dissolved in 1 mL 2-(N-morpholino)ethanesulfonic acid solution (0.1 M, pH 5.5), and then 100 μL carbodiimide solution (0.01 M) and 20 μL N-hydroxysuccinimide solution (0.1 M) were added to activate the carboxyl groups on the surface of the PDA@Pt-COOH by ultrasonic reaction for 15 min; the PDA@Pt-COOH was then recovered by centrifugation and resuspended in 200 μL PBST solution (0.01 M, pH 7.4); 15 μg of H1N1 detection antibody was added, and after 2 h of reaction at room temperature, 100 μL of BSA (10%) was added for further blocking reaction for 1 h; the product was then recovered by centrifugation and washed once with 0.05% PBST, resuspended in 100 μL of 0.05% PBST, and 100 μL of gold-labeled diluent was added; after mixing, the mixture was stored at 4°C, and the test strip was assembled for use.

[0040] Example 3 The antibody-modified bifunctional composite nano-enzyme label of the present application can be used as a high-performance colorimetric-catalytic nano-label for an immunochromatographic system. In this example, the H1N1 antibody-modified bifunctional composite nano-enzyme label was combined with an immunochromatographic system to detect different concentrations of H1N1 virus in human throat swabs.

[0041] Figure 6 The experimental flowchart for the composite nano-enzyme label as a colorimetric-catalytic bifunctional label combined with an immunochromatographic system to rapidly detect viruses in human throat swab samples.

[0042] Figure 7 The test analysis results for detecting H1N1 antigen in human throat swab samples based on the composite nano-enzyme microsphere immunochromatographic system. Figure 7 a is the colorimetric result of the test strip for detecting H1N1 antigen in human throat swab samples. As can be seen from the figure, the colorimetric intensity on the test line of the test strip gradually weakens as the antigen concentration decreases, and the colorimetric signal observed by the naked eye is 0.5 ng / mL; Figure 7 b is the catalytic (colorimetric enhancement) result of the test strip for detecting H1N1 antigen in human throat swab samples. As can be seen from the figure, the colorimetric intensity on the test line of the test strip gradually weakens as the antigen concentration decreases, and the colorimetric signal observed by the naked eye is 0.005 ng / mL.Figure 7 c-d The gray value signal based on the bifunctional composite nanoscale enzyme label was established according to the colorimetric signal intensity at T line under colorimetric and catalytic modes. The error line is obtained by measuring five times, and it can be seen from the figure that the detection limit of the catalytic mode is 100 times higher in sensitivity than that of the colorimetric mode.

[0043] Figure 8 The results of the clinical sample detection based on the bifunctional composite nanoscale enzyme label of the embodiment 3 of the present application are shown. The results show that the detection results are highly consistent with PCR, proving that the bifunctional composite nanoscale enzyme label has stable performance, and the immunochromatography system based on the bifunctional composite nanoscale enzyme label has good accuracy.

[0044] The above description is only preferred embodiments of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and to implement it. Any modification, replacement, improvement, etc. belongs to the protection scope of the present application.

Claims

1. A preparation method of a positive-electricity-driven composite nanoszyme material, characterized in that, The application comprises the following steps: (1) providing a polydopamine (PDA) nanoparticle aqueous solution with a negative surface charge; (2) mixing the PDA aqueous solution of step (1) with a polyethyleneimine (PEI) solution, self-assembling PEI on the surface of the PDA particles through ultrasonic reaction to form positively charged PDA particles (PDA-PEI), and then centrifuging and washing; (3) mixing the PDA-PEI solution of step (2) with a chloroplatinic acid solution, adsorbing chloroplatinic ions on the surface of the PDA-PEI particles through ultrasonic reaction to form a composite nanoscale enzyme precursor (pre-PDA@Pt); (4) adding the pre-PDA@Pt solution of step (3) into a sodium borohydride (NaBH4) aqueous solution, performing in-situ reduction reaction through ultrasonic treatment to synthesize PDA@Pt composite nanoscale enzyme particles, and then centrifuging and washing; (5) mixing the PDA@Pt particles of step (4) with a 11-mercapto-undecanoic acid (MUA) solution, realizing carboxyl functionalization through ultrasonic reaction to obtain PDA@Pt-COOH particles, and then washing and resuspending.

2. The production method according to claim 1, characterized by, In step (1), the PDA particles are prepared by a modified redox method, specifically including: mixing dopamine hydrochloride, anhydrous ethanol, deionized water and 28% ammonia water, and reacting at room temperature for 16-24 hours, wherein the amount ratio of dopamine hydrochloride, 28% ammonia water, anhydrous ethanol and deionized water is 300 mg:2 mL:20 mL:45 mL.

3. The preparation method according to claim 1, characterized in that, In step (2), the molecular weight of the PEI is 5000-80000, the solution concentration is 0.2 mg / mL-5 mg / mL, and preferably 1 mg / mL; the ultrasonic time is 10-60 minutes, and preferably 20 minutes.

4. The method of claim 1, wherein, In step (3), the chloroplatinic acid is a chloroplatinic acid solution, the solution concentration is 10-100 mM, and the ultrasonic time is 1-30 minutes, and preferably 5 minutes.

5. The preparation method according to claim 1, characterized in that, In step (4), the concentration of the NaBH4 solution is 1-100 mM, and preferably 10 mM; the ultrasonic time is 1-30 minutes, and preferably 5 minutes.

6. The method of claim 1, wherein, In step (5), the concentration of the MUA is 10 μM-50 μM; the ultrasonic time is 20-60 minutes, and preferably 40 minutes; and the number of washing times after centrifugation is 1-2 times.

7. The composite nanoscale enzyme material prepared by the method of any one of claims 1-6, wherein, The composite nanoscale enzyme material has a core-shell structure, including: a PDA inner core with a particle size of 100-300 nm; a PEI interlayer with a thickness of 1-10 nm covering the surface of the inner core; a Pt outer shell covering the surface of the PEI interlayer; and the overall particle size of the composite nanoscale enzyme material is 120-320 nm.

8. The composite nanoszyme material of claim 7, wherein, The surface of the composite nanoscale enzyme material is further modified with a specific antibody, and the antibody is coupled by forming an amide bond between the carboxyl group of the MUA and the amino terminal of the antibody.

9. Use of the antibody-functionalized composite nanozyme material of claim 8 in the preparation of an immunochromatographic test reagent, characterized in that, The application comprises the following steps: (1) mixing a sample to be tested with the antibody functionalized composite nanoscale enzyme material to obtain a mixed solution; (2) adding the mixed solution into the sample adding port of the immunochromatography test strip, and the test line (T line) of the test strip is fixed with a capture antibody capable of binding to the target; (3) after the reaction, reading the colorimetric signal at the test line for qualitative or semi-quantitative detection; (4) adding a color developing solution at the test line, and reading the colorimetric signal enhanced by catalysis for super-sensitive quantitative detection.

10. Use according to claim 9, characterized in that, The target is the influenza A H1N1 virus, the specific antibody is the anti-influenza A H1N1 antibody, and the capture antibody is also the anti-influenza A H1N1 antibody.

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