Cu-tcpp / au / pt nanoszyme for lateral flow immunochromatography, and preparation method and application thereof

Abstract

The application belongs to the technical field of lateral flow immunochromatographic detection, and particularly relates to Cu-TCPP / Au / Pt nanoscale enzyme for lateral flow immunochromatography, a preparation method and application thereof. The Cu-TCPP / Au / Pt nanoscale enzyme is obtained by in-situ reduction of gold and platinum nanoparticles loaded on two-dimensional Cu-TCPP metal organic framework nanosheets in sequence. The nanoscale enzyme has excellent peroxidase-like activity, can efficiently catalyze H2O2 to oxidize 3-amino-9-ethylcarbazole color developing substrate to generate a strong color signal. The nanoscale enzyme can be used as a signal tag to prepare a probe for lateral flow immunochromatographic test strip development; the test strip is used for detecting alpha-fetal protein, has both colorimetric signal and catalytic signal output modes, and the visual detection limit of the colorimetric LFIA is 5 ng / mL, the visual detection limit of the catalytic signal is 0.5 ng / mL, and the calculated detection limit of the catalytic LFIA is 0.25 ng / mL. The lateral flow immunochromatographic detection of alpha-fetal protein is simple in operation, high in sensitivity, and suitable for clinical instant diagnosis and primary medical detection.

Description

Technical Field

[0001] This invention belongs to the field of lateral flow immunochromatographic detection technology, specifically relating to a Cu-TCPP / Au / Pt nanozyme, its preparation method, and its application in lateral flow immunochromatography. Background Technology

[0002] Alpha-fetoprotein (AFP) is the most commonly used serum tumor marker for primary liver cancer. Its high sensitivity and rapid detection have significant clinical value for early screening, efficacy evaluation, and recurrence monitoring of liver cancer. Lateral flow immunochromatography (LFIA) has become the mainstream technology for point-of-care testing (POCT) due to its simple operation, rapid detection, low cost, and lack of complex instruments. However, traditional LFIA using colloidal gold nanoparticles (AuNPs) as signal tags has low sensitivity and poor concentration signal response, which can easily lead to false negatives or missed diagnoses, making it difficult to monitor AFP concentration.

[0003] To improve the sensitivity of LFIA (Low-Frequency Interventional Imaging), researchers have developed nanozymes with peroxidase-like activity as signal tags. While noble metal particles exhibit good enzymatic activity, they are prone to aggregation, leading to reduced catalytic activity. Metal-organic frameworks (MOFs), due to their high specific surface area and ease of functionalization, have become ideal supports for loading noble metal nanoparticles. Therefore, developing a MOF-supported bimetallic nanozyme to amplify the detection signal using its enzymatic activity and to construct a highly sensitive LFIA platform has significant research value and promising clinical application prospects. Summary of the Invention

[0004] The purpose of this invention is to provide a highly sensitive and easy-to-operate MOF-loaded bimetallic nanozyme Cu-TCPP / Au / Pt, its preparation method, and its application in lateral flow immunochromatographic detection.

[0005] This invention first provides a method for preparing Cu-TCPP / Au / Pt nanozymes for lateral flow immunochromatography. The Cu-TCPP / Au / Pt nanozymes are obtained by sequential in-situ reduction of gold and platinum nanoparticles loaded onto two-dimensional Cu-TCPP metal-organic framework (MOF) nanosheets. The specific steps are as follows:

[0006] (1) A copper source, medium-tetra(4-carboxyphenyl)porphyrin (TCPP) and a surfactant were mixed in a solvent and subjected to a solvothermal reaction to obtain Cu-TCPP nanosheets;

[0007] (2) On this basis, HAuCl4·3H2O and H2PtCl6·6H2O are added in sequence to carry out in-situ reduction reaction to obtain the Cu-TCPP / Au / Pt nanozyme.

[0008] Furthermore:

[0009] In step (1), the copper source is copper nitrate, the surfactant is polyvinylpyrrolidone, and the solvent is a mixture of ethanol and N,N-dimethylformamide.

[0010] The mass ratio of the copper source to centrifugal tetra(4-carboxyphenyl)porphyrin (TCPP) is 0.8:1 to 1:1 ((0.8~1):1), the mass ratio of the copper source to polyvinylpyrrolidone is 1:2.6 to 1:2.9 (1:(2.6~2.9)), and the mass ratio of TCPP to polyvinylpyrrolidone is 1:2.4 to 1:2.6 (1:(2.4~2.6)).

[0011] The solvothermal reaction is carried out at a temperature of 70-80°C for 3-5 hours.

[0012] In step (2), the reducing agent used in the in-situ reduction reaction is sodium borohydride. The molar ratio of HAuCl4·3H2O to the copper source is 1.6:1 to 1.8:1 ((1.6 to 1.8):1), and the molar ratio of H2PtCl6·6H2O to the copper source is 0.6:1 to 0.8:1 ((0.6 to 0.8):1).

[0013] The Cu-TCPP MOF nanozyme prepared in this invention has gold and platinum nanoparticles loaded in situ on its surface. This nanozyme exhibits excellent peroxidase-like activity. When used as a signal tag in lateral flow immunochromatographic test strips for AFP detection, it significantly amplifies the detection signal by catalyzing the oxidation of AEC substrates with H₂O₂ to produce a reddish-brown oxidation product. Therefore:

[0014] The present invention also provides the application of the above-mentioned Cu-TCPP / Au / Pt nanozyme in lateral flow immunochromatographic detection.

[0015] Specifically, a test strip for lateral flow immunochromatography is provided, the lateral flow immunochromatographic test strip comprising a sample pad, a conjugation pad, a nitrocellulose membrane, an absorbent pad, and a PVC liner; wherein a detection line and a control line are sequentially arranged on the nitrocellulose membrane along the chromatography direction; the detection line is coated with anti-AFP mAb2, and the control line is coated with goat anti-mouse IgG antibody.

[0016] The above-mentioned lateral flow immunochromatographic test strips were prepared by the following method:

[0017] (1) Adjust the pH of the Cu-TCPP / Au / Pt nanozyme dispersion to a suitable range with 1~3 mM potassium carbonate, add 2~4 μg anti-AFP mAb1, and incubate at room temperature for 0.5~2 hours to couple mAb1 antibody to the nanozyme surface by electrostatic adsorption; add BSA to block unbound active sites for 0.5~2 hours, centrifuge and wash to obtain Cu-TCPP / Au / Pt-mAb1 probe, and resuspend it in storage buffer containing 5% sucrose, 1.5% BSA, 1% trehalose and 0.5% Tween-20.

[0018] (2) Anti-AFP mAb2 and goat anti-mouse IgG antibodies, both at 0.8~1.1 mg / mL, were sprayed onto a nitrocellulose membrane to form a detection line and a control line, respectively. The spraying volume was 0.8~1.2 μL / cm. The membrane was then dried at 36~40℃ for 1~2 hours. The sample pad was then soaked in a treatment solution, which was a PBS buffer (pH 7.4) containing 1.5% BSA and 0.5% Tetronic 1307, and then dried at 36~40℃ for 10~12 hours.

[0019] (3) Paste the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad onto the PVC backing in sequence, with adjacent parts overlapping by about 2-3 mm. After assembly, cut into test strips with a width of 3-4 mm and dry and store.

[0020] The operation procedure for detecting AFP using the lateral flow immunochromatographic test strip provided by this invention is as follows:

[0021] (1) Under the following colorimetric detection conditions, mix 4~6 μL of Cu-TCPP / Au / Pt-mAb1 probe (1.68 mg / mL) with 94~96 μL of the sample solution to be tested, drop it onto the sample pad of the test strip, perform chromatographic reaction at room temperature for 14~16 minutes, observe the color development of the detection line and the control line, and perform colorimetric detection.

[0022] (2) To further improve sensitivity, after colorimetric detection, under the following catalytic detection conditions, 6-8 μL of catalytic reaction solution is added dropwise to the detection line and control line regions. The catalytic reaction solution is prepared by mixing acetate buffer (pH 3.5-4.5), 0.2-0.4% H2O2, and 20 mM AEC in a volume ratio of 6-8:(0.8-1.2):1. After reacting at room temperature for 2-4 minutes, the color development of the detection line and control line is observed again, and high-sensitivity detection is performed by amplifying the catalytic signal.

[0023] Compared with the prior art, the main technical features and functional advantages of the present invention are as follows:

[0024] This invention utilizes two-dimensional Cu-TCPP MOF nanosheets as a carrier, and in situ loads gold and platinum nanoparticles onto their surface to prepare Cu-TCPP / Au / Pt nanozymes. These nanozymes exhibit excellent peroxidase-like activity. Used as a signal tag in lateral flow immunochromatographic (LFIA) test strips for AFP detection, they significantly amplify the detection signal by catalyzing the oxidation of AEC substrates with H₂O₂ to produce a reddish-brown oxidation product. The test strips of this invention provide both colorimetric and catalytic signal outputs. The visual detection limit under colorimetric LFIA is 5 ng / mL, the visual detection limit under catalytic LFIA is as low as 0.5 ng / mL, and the calculated detection limit under catalytic LFIA is 0.25 ng / mL, with a linear range of 0.5–500 ng / mL. The detection method provided by this invention has the advantages of simple operation, high sensitivity, and low cost, making it suitable for clinical point-of-care diagnosis and primary healthcare testing. Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the preparation of the Cu-TCPP / Au / Pt nanozyme and the principle of LFIA detection in this invention.

[0026] Figure 2 The image shows the morphological characterization of Cu-TCPP / Au / Pt prepared in Example 1 of this invention.

[0027] Figure 3 The Michaelis equation curve and double reciprocal plot are shown for the Cu-TCPP / Au / Pt nanozyme of Example 1 of the present invention.

[0028] Figure 4 This invention optimizes the colorimetric system conditions for the LFIA detection platform using Cu-TCPP / Au / Pt nanozyme as a signal tag in Example 2 of the present invention.

[0029] Figure 5 This invention optimizes the catalytic system conditions for the LFIA detection platform using Cu-TCPP / Au / Pt nanozyme as a signal tag in Example 2 of the present invention.

[0030] Figure 6 This is an example of LFIA images and standard curves of colorimetric and catalytic signals used for the detection of AFP concentrations of different target substances in Example 3 of the present invention. Detailed Implementation

[0031] The technical solution of the present invention will be described in detail below with reference to specific embodiments. These embodiments are only for a better understanding of the technical solution of the present invention and should not be construed as limiting the present invention.

[0032] Example 1: Preparation and peroxidase-like performance characterization of Cu-TCPP / Au / Pt nanozymes

[0033] First, 7.2 mg Cu(NO3)2·3H2O, 20 μL 1.0 M trifluoroacetic acid, and 20 mg polyvinylpyrrolidone were dissolved in 24.0 mL of a mixed solvent of ethanol and DMF in a volume ratio of 1:3, and transferred to a 50 mL reaction vessel. Then, 8 mg of 1,000-tetra(4-carboxyphenyl)porphyrin (TCPP) was dissolved in 8.0 mL of a mixed solvent of ethanol and DMF in a volume ratio of 1:3. The mixture was added to the above system with stirring, sonicated for 10 min, and then reacted solvothermically at 80 °C for 4 h. The product was washed twice with ethanol, centrifuged at 8000 rpm for 10 min, collected, and vacuum dried to obtain Cu-TCPP nanosheets. 4 mg of Cu-TCPP nanosheets were dispersed in 2 mL of HAuCl4·3H2O solution (10 mg / mL) in 47.2 mL of ultrapure water. After ultrasonic mixing, the mixture was magnetically stirred for 1.5 h. Then, 0.8 mL of NaBH4 solution (7.6 mg / mL) was added and the mixture was stirred vigorously for 0.5 h. The product was collected by centrifugation at 10000 rpm for 15 min, washed three times with ultrapure water, and vacuum dried to obtain Cu-TCPP / Au. 4 mg of Cu-TCPP / Au was dispersed in 1 mL of 20 mM H2PtCl6·6H2O solution in 20 mL of ultrapure water. The mixture was stirred at room temperature for 1 h. Then, 2 mL of NaBH4 solution (3 mg / mL) was added and the mixture was stirred vigorously for 3 h. The product was centrifuged at 10000 rpm for 15 min and washed three times with ultrapure water to obtain Cu-TCPP / Au / Pt nanozyme.

[0034] Electron microscopy revealed that the nanozyme exhibited a two-dimensional sheet-like structure with fine nanoparticles distributed on its surface. Figure 2 Steady-state kinetic analysis of Cu-TCPP / Au / Pt nanozymes is as follows: Figure 3 As shown, the results indicate that the nanozyme has excellent peroxidase activity.

[0035] Example 2: Preparation and System Optimization of LFIA Test Strip Detection Platform

[0036] (1) Preparation of LFIA test strip detection platform

[0037] Preparation of Cu-TCPP / Au / Pt-mAb probe: Take the above 0.168 mg / mL Cu-TCPP / Au / Pt dispersion, add 20 μL of 0.1 M K2CO3 to adjust the pH, then add 3 μL of 1 mg / mL AFP detection antibody, and incubate at room temperature for 1 hour. Add 100 μL of 10% BSA for blocking for 1 hour. After centrifugation and washing, resuspend the probe in a solution containing 5% sucrose, 1.5% BSA, 1% trehalose and 0.5% Tween-20 to obtain the Cu-TCPP / Au / Pt-mAb probe.

[0038] LFIA test strip preparation: 1 mg / mL AFP capture antibody and 1 mg / mL goat anti-mouse IgG were sprayed onto a nitrocellulose membrane at a rate of 1 μL / cm to serve as the detection line (T line) and control line (C line), respectively, and dried at 37°C for 2 hours. The sample pad was soaked in PBS buffer containing 1.5% BSA and 0.5% Tetronic 1307 and then dried. The treated sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad were sequentially assembled onto a PVC backing and cut into 3 mm wide test strips.

[0039] (2) Optimization of LFIA test strip detection platform system

[0040] To obtain optimal detection performance, this invention optimizes the LFIA detection platform system. Preferably, the colorimetric system detection conditions are: 1.5% BSA + 0.5% S9 for the test strip sample pad blocking solution, 1.68 mg / mL Cu-TCPP / Au / Pt concentration, 2 mM K2CO3 concentration, 3 μg anti-AFP mAb1 dosage, 5 μL Cu-TCPP / Au / Pt-mAb1 probe dosage, and a detection time of 15 minutes. Figure 4 Preferably, the detection conditions for the catalytic system are: pH 4 acetate buffer, 0.3% H2O2 concentration, 7 μL catalyst volume, and 3 minutes catalytic time. Figure 5 ).

[0041] Example 3: Performance testing of LFIA for alpha-fetoprotein detection

[0042] The target compound, alpha-fetoprotein, was selected for LFIA performance testing.

[0043] Linearity and Sensitivity: A series of AFP standard solutions (0, 0.5, 5, 10, 20, 100, 140, 200, 320, 400, and 500 ng / mL) were prepared. 95 μL of each AFP solution was mixed with 5 μL of Cu-TCPP / Au / Pt-mAb probe and added to the sample pad of the test strip. After 15 minutes, the colorimetric signal was observed and recorded. Subsequently, 7 μL of a catalytic reaction solution (pH 4.0, prepared with acetate buffer) containing 0.3% H2O2 and 20 mM AEC was added to the T and C line regions. After reacting for 3 minutes, the amplified signal was observed and recorded again. Figure 6As shown, under colorimetric LFIA, the visual limit of detection (vLOD) is 5 ng / mL, and the calculated limit of detection (cLOD) is 3.11 ng / mL. With catalytic LFIA, the visual limit of detection decreases to 0.5 ng / mL, the linear range is 0.5–500 ng / mL, and the cLOD is 0.25 ng / mL.

[0044] Recovery rates: For serum samples spiked with AFP at concentrations of 20, 200, and 400 ng / mL from healthy individuals, the colorimetric LFIA recovery rate was 96.54%–110.49%, with a relative standard deviation (RSD) of less than 7.80%; the catalytic LFIA recovery rate was 94.24%–107.64%, with an RSD of less than 4.13%.

[0045] The embodiments described above are merely illustrative of implementation methods of the present invention, used to illustrate the technical solutions of the present invention, and are not intended to limit it. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A method for preparing Cu-TCPP / Au / Pt nanozymes for lateral flow immunochromatography, characterized in that, Cu-TCPP / Au / Pt nanozymes were obtained by sequential in-situ reduction of gold and platinum nanoparticles from two-dimensional Cu-TCPP metal-organic framework (MOF) nanosheets. The specific steps are as follows: (1) A copper source, medium-tetra(4-carboxyphenyl)porphyrin (TCPP) and a surfactant were mixed in a solvent and subjected to a solvothermal reaction to obtain Cu-TCPP nanosheets; (2) On this basis, HAuCl4·3H2O and H2PtCl6·6H2O are added in sequence to carry out in-situ reduction reaction to obtain the Cu-TCPP / Au / Pt nanozyme.

2. The preparation method according to claim 1, characterized in that, In step (1), the copper source is copper nitrate, the surfactant is polyvinylpyrrolidone, and the solvent is a mixture of ethanol and N,N-dimethylformamide; the temperature of the solvothermal reaction is 70~80℃ and the time is 3~5 hours.

3. The preparation method according to claim 1 or 2, characterized in that, In step (1), the mass ratio of the copper source to medium-tetra(4-carboxyphenyl)porphyrin (TCPP) is 0.8:1 to 1:1, the mass ratio of the copper source to polyvinylpyrrolidone is 1:2.6 to 1:2.9, and the mass ratio of TCPP to polyvinylpyrrolidone is 1:2.4 to 1:2.

6.

4. The preparation method according to claim 1, characterized in that, In step (2), the reducing agent used in the in-situ reduction reaction is sodium borohydride.

5. The preparation method according to claim 1 or 4, characterized in that, In step (2), the molar ratio of HAuCl4·3H2O to the copper source is 1.6:1 to 1.8:1, and the molar ratio of H2PtCl6·6H2O to the copper source is 0.6:1 to 0.8:

1.

6. A Cu-TCPP / Au / Pt nanozyme prepared by any one of claims 1-5, wherein gold nanoparticles and platinum nanoparticles are loaded in situ on its surface; the nanozyme has excellent peroxidase-like activity and is used as a signal tag for the detection of alpha-fetoprotein (AFP) on a lateral flow immunochromatographic test strip. It significantly amplifies the detection line signal by catalyzing the oxidation of AEC substrate by H2O2 to produce a reddish-brown oxidation product.

7. The application of the Cu-TCPP / Au / Pt nanozyme as described in claim 6 in lateral flow immunochromatography.

8. The application according to claim 7, characterized in that, The invention includes the preparation of a lateral flow immunochromatographic test strip based on Cu-TCPP / Au / Pt nanozyme. The test strip includes a sample pad, a conjugation pad, a nitrocellulose membrane, an absorbent pad, and a PVC liner. The nitrocellulose membrane has a detection line and a control line arranged sequentially along the chromatography direction. The detection line is coated with anti-AFP mAb2, and the control line is coated with goat anti-mouse IgG antibody.

9. The application according to claim 8, characterized in that, The lateral flow immunochromatographic test strip is prepared by the following method: (1) Adjust the pH of the Cu-TCPP / Au / Pt nanozyme dispersion to a suitable range with 1~3 mM potassium carbonate, add 2~4 μg anti-AFP mAb1, and incubate at room temperature for 0.5~2 hours to couple mAb1 antibody to the nanozyme surface by electrostatic adsorption; add BSA to block unbound active sites for 0.5~2 hours, centrifuge and wash to obtain Cu-TCPP / Au / Pt-mAb1 probe, and resuspend it in storage buffer containing 5% sucrose, 1.5% BSA, 1% trehalose and 0.5% Tween-20; (2) Anti-AFP mAb2 and goat anti-mouse IgG antibodies, both at 0.8~1.1 mg / mL, were sprayed onto a nitrocellulose membrane to form a detection line and a control line, respectively. The spraying volume was 0.8~1.2 μL / cm. The membrane was dried at 36~40℃ for 1~2 hours. The sample pad was soaked in the treatment solution and then dried at 36~40℃ for 10~12 hours. The treatment solution was a PBS buffer containing 1.5% BSA and 0.5% Tetronic 1307. (3) Paste the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad onto the PVC backing in sequence, with adjacent parts overlapping by about 2-3 mm. After assembly, cut into test strips with a width of 3-4 mm and dry and store.

10. The application according to claim 9, characterized in that, The procedure for detecting AFP using the lateral flow immunochromatographic test strip is as follows: (1) Under the following colorimetric detection conditions, mix 4~6 μL of Cu-TCPP / Au / Pt-mAb1 probe with a concentration of 1.68 mg / mL with 94~96 μL of the sample solution to be tested, drop it onto the sample pad of the test strip, perform chromatographic reaction at room temperature for 14~16 minutes, observe the color development of the detection line and the control line, and perform colorimetric detection; (2) After colorimetric detection, under the following catalytic detection conditions, 6-8 μL of catalytic reaction solution is added to the detection line and control line area. The catalytic reaction solution is prepared by mixing acetate buffer at pH 3.5-4.5, 0.2-0.4% H2O2 and 20 mM AEC in a volume ratio of (6-8):(0.8-1.2):

1. After reacting at room temperature for 2-4 minutes, the color development of the detection line and control line is observed again, and high-sensitivity detection is performed by amplifying the catalytic signal.

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