A three-mode bicolour lateral flow immunochromatographic test paper based on phenylboronic acid gold nanoflower and a detection method

Abstract

This invention discloses a three-modal, two-color lateral flow immunochromatographic test strip based on phenylboronic acid gold nanoflowers and its detection method. This test strip can simultaneously perform qualitative and quantitative tests, with the qualitative mode capable of detecting concentrations as low as 10. 3 Pathogenic microorganisms at CFU / ml; the lowest detectable concentration in colorimetric quantification mode is 10. 3 Pathogenic microorganisms at CFU / ml; the lowest detectable concentration using photothermal and Raman quantitative modes is 10. 2 The invention detects pathogenic microorganisms at cfu / ml levels; the detection can be completed within 15 minutes, and the test results can be observed with the naked eye; the three-modal LFIA improves the sensitivity, accuracy, flexibility and practicality of the detection. The invention has strong specificity for pathogenic microorganisms, high sensitivity, convenient and fast detection process, and safe and reliable test results.

Description

Technical Field

[0001] This invention relates to a lateral flow immunochromatographic test strip, and more particularly to a trimodal dual-color lateral flow immunochromatographic test strip based on gold nanoflowers of phenylboronic acid, and also to a detection method using the above test strip. Background Technology

[0002] Lateral flow immunochromatography (LFIA) is the most important point-of-care testing (POCT) technology, characterized by high stability, speed, portability, and user-friendliness. Traditional LFIA typically uses capture antibodies (CA) combined with nanomaterials (NMs) to synthesize NMs-CA nanoprobes to label bacteria. However, the preparation of NMs-CA probes is often complex and expensive, relying on capture antibodies, which significantly limits the application of LFIA. Patent CN114113592A discloses the application of phenylboronic acid derivatives in LFIA, developing a capture antibody-independent LFIA strategy for rapid and economical detection of pathogenic microorganisms through the strong covalent bond between phenylboronic acid-modified gold nanoparticles and bacteria. However, gold nanoparticle-based LFIA can only achieve qualitative or semi-quantitative detection, has a limited readout method, and a high detection limit, rarely meeting the high sensitivity requirements of certain bioanalytes. Furthermore, capture antibody-independent LFIA strategies cannot achieve quality control lines, potentially leading to false negatives. Summary of the Invention

[0003] Purpose of the invention: The purpose of this invention is to provide a three-modal, two-color lateral flow immunochromatographic test strip based on gold nanoflowers of phenylboronic acid. The second purpose of this invention is to provide a detection method using the above test strip.

[0004] Technical Solution: The present invention relates to a trimodal, dual-color lateral flow immunochromatographic test strip based on phenylboronic acid gold nanoflowers, comprising a polyvinyl chloride (PVC) plate, a sample pad, a nitrocellulose membrane, and an absorbent pad. The sample pad, nitrocellulose membrane, and absorbent pad are sequentially adhered to one side of the PVC plate. The nitrocellulose membrane has a detection line and a control line. Pathogen receptor proteins are linearly arranged to form the detection line, and biotin is linearly arranged to form the control line. The detection line and control line are perpendicular to the direction of liquid diffusion. During detection, pathogen receptor proteins bind to the gold-phenylboronic acid-analyte bacteria conjugate, causing the gold nanoflowers to aggregate on the detection line, which appears blue. Biotin binds to the gold-streptavidin conjugate, causing the gold nanoparticles to aggregate on the control line, which appears red.

[0005] Among them, the gold-phenylboronic acid-bacterial conjugate was prepared by reducing chloroauric acid to obtain gold nanoseeds, then adding chloroauric acid, sodium citrate and phenol to obtain gold nanoflowers. The gold nanoflowers were then mixed with phenylboronic acid, reacted and washed, and resuspended in ultrapure water to obtain phenylboronic acid-functionalized gold nanoflowers. These were then mixed with the bacteria to be tested and bovine serum albumin was added to obtain the gold-phenylboronic acid-bacterial conjugate. The gold-streptavidin conjugate was prepared by adding streptavidin to gold nanoparticles, mixing and then adding bovine serum albumin.

[0006] Preferably, the interface between the sample pad and the nitrocellulose membrane overlaps by 1-2 mm, and the interface between the nitrocellulose membrane and the absorbent pad overlaps by 1-2 mm.

[0007] The lateral flow immunochromatographic detection method based on phenylboronic acid gold nanoflowers of the present invention includes the following steps:

[0008] (1) Preparation of phenylboronic acid-functionalized gold nanoflowers: First, sodium citrate, a reducing agent, was added to chloroauric acid solution to prepare gold nanoseeds. Chloroauric acid, sodium citrate, and phenol were then added to the gold nanoseeds to prepare gold nanoflowers. Phenyboronic acid was added to the gold nanoflowers, and after the reaction, the mixture was washed with ethanol and resuspended in ultrapure water to obtain phenylboronic acid-functionalized gold nanoflowers.

[0009] (2) Preparation of gold-phenylboronic acid-bacterial conjugate: A single colony was added to the culture medium and cultured to obtain bacteria. The bacteria were washed with PBS to obtain bacterial culture. Phenylboronic acid-functionalized gold nanoflowers were added to the bacteria to be tested. After mixing, bovine serum albumin was added to obtain gold-phenylboronic acid-bacterial conjugate.

[0010] (3) Preparation of gold nanoparticles: Prepare chloroauric acid solution, add sodium citrate as reducing agent, cool and let stand to obtain gold nanoparticles;

[0011] (4) Preparation of gold-streptavidin conjugate: gold-streptavidin conjugate was obtained by adding streptavidin to gold nanoparticles, mixing them, and then adding bovine serum albumin.

[0012] (5) Preparation of a mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate: Take the gold-phenylboronic acid-bacterial conjugate solution obtained in step (2) and the gold-streptavidin conjugate solution obtained in step (4) and mix them to obtain a mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate.

[0013] (6) Preparation of three-mode dual-color side-flow chromatography test paper of gold nanoflowers of phenylboronic acid: The sample pad, nitrocellulose membrane and absorption pad were sequentially pasted onto the plate to assemble the side-flow chromatography test paper;

[0014] (7) Lateral flow chromatography detection method: Take the mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate prepared in step (5) and drop it onto the sample pad of the test strip. The sample solution migrates to the nitrocellulose membrane. The gold-streptavidin conjugate can recognize the streptavidin-biotin system with the biotin coated on the detection line on the nitrocellulose membrane. The gold nanoparticles aggregate on the quality control line and a C line appears. The C line is red, indicating that the test strip is working normally.

[0015] The bacteria to be detected can undergo specific antigen-antibody recognition with the pathogen receptor protein coated on the detection line on the nitrocellulose membrane. Gold nanoflowers aggregate on the detection line, forming a T-line band that appears blue for qualitative judgment. The RGB values ​​of the T-line are analyzed using ColorPicker software and compared with the standard curve to achieve quantitative judgment of the colorimetric signal of bacterial concentration in the sample.

[0016] Preferably, in step (1), the concentration of the chloroauric acid solution is 0.1%-10%, the concentration of sodium citrate is 0.1%-10%, and the concentration of phenylboronic acid is 0.1-1 g / L; in step (3), the concentration of the chloroauric acid solution is 0.1%-10%, and the concentration of sodium citrate is 0.1%-10%.

[0017] Preferably, in step (2), the bacteria are Gram-positive or Gram-negative bacteria, and the concentration of the bacteria is 0-10. 8 The concentration of bovine serum albumin (BSA) was CFU / mL, the mixing time was 0.1-2 h, and the concentration of BSA was 1%-20%.

[0018] Preferably, in step (4), the concentration of streptavidin is 0.1-10 g / L, the mixing time is 0.1-1 h, and the concentration of bovine serum albumin is 1%-10%.

[0019] Preferably, in step (6), the gold-phenylboronic acid-bacterial conjugate aggregates gold nanoflowers on the detection line by binding with the pathogenic microorganism receptor protein, which appears blue; the gold-streptavidin conjugate aggregates gold nanoparticles on the quality control line by binding with biotin, which appears red.

[0020] Preferably, in step (6), the sample pad is prepared by immersing a polyester film in 0.1%-1% Triton-100, 1%-5% sodium chloride and Tris-HCl in sequence, and then drying it in an oven at 25-40℃ for 2-10 hours.

[0021] Preferably, in step (6), the preparation of the nitrocellulose membrane specifically involves distributing the antibody to be detected onto the nitrocellulose membrane at a rate of 0.5-2 μL / cm to form a detection line, and distributing biotin onto the nitrocellulose membrane at a rate of 0.5-2 μL / cm to form a control line.

[0022] Preferably, the quantitative judgment in step (7) can also be achieved by irradiating the T line with a 635 nm wavelength laser, analyzing the temperature value of the T line with a thermal imager, and comparing it with the standard curve to realize the quantitative judgment of the bacterial concentration in the sample based on the photothermal mode; or by analyzing the Raman intensity of the T line with a Raman spectrometer and comparing it with the standard curve to realize the quantitative judgment of the bacterial concentration in the sample based on the Raman mode.

[0023] This lateral flow immunochromatographic method utilizes gold-phenylboronic acid nanoflowers not only to generate colorimetric, photothermal, and Raman signals, but also to capture bacteria by covalently binding phenylboronic acid to the cis-ortho-dihydroxy structures on the bacterial surface. This avoids the use of traditional capture antibodies, enabling novel, antibody-free, trimodal detection. Users can freely choose different quantitative modes based on available equipment and resources, improving the overall flexibility and sensitivity of the assay. Furthermore, a quality control line is implemented using a streptavidin-biotin system, thus preventing false negatives.

[0024] The obtained gold-phenylboronic acid-bacteria conjugate was added to the sample pad of the test strip. The bacterial concentration was qualitatively determined by colorimetric analysis of the T-line color intensity observed with the naked eye. ColorPicker software was used to analyze the RGB values ​​of the T-line at different bacterial concentrations, and a colorimetric signal standard curve was fitted. A thermal imager was used to analyze the T-line temperature at different bacterial concentrations, and a photothermal signal standard curve was fitted. A Raman spectrometer was used to analyze the Raman intensity of the T-line at different bacterial concentrations, and a Raman signal standard curve was fitted. Based on the RGB values, temperature, and Raman intensity of the T-line of the sample, and according to the standard curves of each signal, a three-modal quantitative determination of the bacterial concentration in the sample was achieved.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0026] (1) The phenylboronic acid-functionalized gold nanoflower three-mode lateral flow chromatography test strip of the present invention is simple to assemble and can quickly and efficiently perform three-mode lateral flow chromatography, and simultaneously complete qualitative and quantitative tests. It has high sensitivity and high specificity; it can realize the on-site instant detection of a broad spectrum of bacteria, greatly save detection costs and improve the detection limit, and has universal applicability in the field of pathogen detection.

[0027] (2) The three-modal lateral flow chromatography detection method based on phenylboronic acid-functionalized gold nanoflowers of the present invention can detect a minimum concentration of 10 in the qualitative mode. 3 Pathogenic microorganisms at CFU / ml; the lowest detectable concentration in colorimetric quantification mode is 10. 3 Pathogenic microorganisms at CFU / ml; the lowest detectable concentration using photothermal and Raman quantitative modes is 10. 2The detection method can detect pathogenic microorganisms at a concentration of cfu / ml; the detection can be completed within 15 minutes, and the test results can be observed with the naked eye; the sensitivity of photothermal and Raman signals is improved by an order of magnitude compared with colorimetric signals; the three-modal LFIA improves the sensitivity, accuracy, flexibility and practicality of the detection method, which is highly specific and sensitive to pathogenic microorganisms, convenient and fast in the detection process, and the test results are safe and reliable. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the side-flow chromatography test strip of the present invention;

[0029] Figure 2 This is a transmission electron microscope image of the gold nanoflowers of the present invention;

[0030] Figure 3 The UV-Vis absorption spectrum of gold nanoflowers modified with phenylboronic acid;

[0031] Figure 4 Raman spectrum of gold-phenylboronic acid nanoflowers;

[0032] Figure 5 Photothermal image of gold-phenylboronic acid nanoflowers;

[0033] Figure 6 Scanning electron microscope (SEM) images of gold-phenylboronic acid nanoflowers co-incubated with *Escherichia coli* and *Staphylococcus aureus*, where a is an SEM image of *Escherichia coli* co-incubated with gold nanoflowers; b is an SEM image of *Escherichia coli* co-incubated with gold-phenylboronic acid nanoflowers; c is an SEM image of *Staphylococcus aureus* co-incubated with gold nanoflowers; and d is an SEM image of *Staphylococcus aureus* co-incubated with gold-phenylboronic acid nanoflowers.

[0034] Figure 7 The image shows the optimization results of four detection parameters for the gold-phenylboronic acid-based lateral flow immunochromatography method. In the image, a represents the optimized band parameter for the incubation time of bacteria and nanoparticles; b represents the optimized band parameter for the immunoreaction time; c represents the optimized band parameter for the amount of phenylboronic acid modification; and d represents the optimized band parameter for the volume ratio of gold-phenylboronic acid-bacteria and gold-streptavidin probes.

[0035] Figure 8 Using this detection method, taking Escherichia coli as an example, the control group and 10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 Image showing the naked-eye observation results of the bands corresponding to CFU / ml E. coli;

[0036] Figure 9 Using this detection method, taking Escherichia coli as an example, the control group and 10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 Image of colorimetric signal analysis results of bands corresponding to CFU / ml E. coli using ColorPicker software;

[0037] Figure 10 Using this detection method, taking Escherichia coli as an example, the control group and 10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 Image of photothermal signal thermal imaging analysis results of the bands corresponding to CFU / ml E. coli;

[0038] Figure 11 Using this detection method, taking Escherichia coli as an example, the control group and 10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 Raman spectroscopy analysis results of the bands corresponding to CFU / ml E. coli;

[0039] Figure 12 The images show the results of naked-eye observation, colorimetric, Raman, and photothermal signal analysis for detecting Escherichia coli in artificial urine using this lateral flow chromatography method; where a is the naked-eye observation result; b is the colorimetric signal result; c is the Raman signal result; and d is the photothermal signal result.

[0040] Figure 13 The images show the results of qualitative pattern detection for Example 1 and Comparative Example 1; where a is the naked-eye observation result for Example 1 and b is the naked-eye observation result for Comparative Example 1. Detailed Implementation

[0041] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0042] like Figure 1 As shown, the phenylboronic acid gold nanoflower three-modal dual-color lateral flow chromatography test paper of the present invention includes a polyvinyl chloride plate 1, a sample pad 2, a nitrocellulose membrane 3, and an absorbent pad 4. The sample pad 2, the nitrocellulose membrane 3, and the absorbent pad 4 are sequentially pasted on one side of the polyvinyl chloride plate 1. The interface between the sample pad 2 and the nitrocellulose membrane 3 overlaps by 2 mm, and the interface between the nitrocellulose membrane 3 and the absorbent pad 4 overlaps by 2 mm. The nitrocellulose membrane is provided with a detection line 5 and a control line 7. Pathogenic microorganism receptor proteins 6 are arranged linearly to form the detection line 5, and biotin 8 is arranged linearly to form the control line 7. The detection line 5 and the control line 7 are perpendicular to the liquid diffusion direction. The gold-phenylboronic acid-test bacteria conjugate binds to the pathogenic microorganism receptor protein 6, causing the gold nanoflowers to aggregate on the detection line 5, which appears blue; the gold-streptavidin conjugate binds to the biotin 8, causing the gold nanoparticles to aggregate on the control line 7, which appears red.

[0043] Example 1

[0044] This invention relates to a lateral flow immunochromatographic detection method based on phenylboronic acid gold nanoflowers, comprising the following steps:

[0045] (1) Preparation of phenylboronic acid-functionalized gold nanoflowers: First, 1% sodium citrate reducing agent was added to a 1% chloroauric acid solution to prepare gold nanoseeds. Then, 1% chloroauric acid, 1% sodium citrate and 30mM phenol were added to the gold nanoseeds to prepare gold nanoflowers. 0.2g / L phenylboronic acid was added to the gold nanoflowers. After the reaction, the mixture was washed with ethanol and resuspended in ultrapure water to obtain phenylboronic acid-functionalized gold nanoflowers.

[0046] (2) Preparation of gold-phenylboronic acid-bacterial conjugate: A single colony was added to the culture medium and cultured to obtain bacteria. The bacteria were then washed with PBS to obtain a concentration of 10. 7 cfu mL -1 Bacterial culture: Add phenylboronic acid-functionalized gold nanoflowers to the test bacteria, mix for 0.5 h, and then add 10% bovine serum albumin to obtain gold-phenylboronic acid-test bacteria conjugate.

[0047] (3) Preparation of gold nanoparticles: Prepare a 1% chloroauric acid solution, add a 1% reducing agent sodium citrate, and cool and let stand to obtain gold nanoparticles.

[0048] (4) Preparation of gold-streptavidin conjugate: gold-streptavidin conjugate was obtained by adding streptavidin at a concentration of 1 g / L to gold nanoparticles, mixing them, and then adding bovine serum albumin at a concentration of 10%.

[0049] (5) Preparation of a mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate: Take the gold-phenylboronic acid-bacterial conjugate solution obtained in step (2) and the gold-streptavidin conjugate solution obtained in step (4) and mix them to obtain a mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate.

[0050] (6) Preparation of lateral flow chromatography test strips: The LFIA test strip consists of three parts: a sample pad, a nitrocellulose membrane, and an absorbent pad. The absorbent pad is not further treated, while the sample pad is treated with 0.5% Triton-100, 3% sodium chloride, and Tris-HCl (pH 8.0), and then dried at 37°C for 12 hours. The sample pad, nitrocellulose membrane, and absorbent pad are sequentially pasted onto a plate, overlapping each other by 1-2 mm, to assemble the lateral flow chromatography test strip. The surface of the nitrocellulose membrane is coated with a detection line and a control line. The antibody to be detected is dispensed onto the nitrocellulose membrane at a rate of 1 μL / cm to form the detection line, and biotin is dispensed onto the nitrocellulose membrane at a rate of 1 μL / cm to form the control line.

[0051] (7) Side flow chromatography detection method: Take the mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate prepared in step (5) and drop it onto the sample pad of the test strip. The sample solution migrates to the nitrocellulose membrane.

[0052] Example 2

[0053] This invention relates to a lateral flow immunochromatographic detection method based on phenylboronic acid gold nanoflowers, comprising the following steps:

[0054] (1) Preparation of phenylboronic acid-functionalized gold nanoflowers: First, gold nanoseeds were prepared by adding 0.1% sodium citrate reducing agent to a 0.1% chloroauric acid solution. Then, 0.1% chloroauric acid, 0.1% sodium citrate, and 10mM phenol were added to the gold nanoseeds to prepare gold nanoflowers. The gold nanoflowers were then added to 0.1g / L phenylboronic acid, and after the reaction, they were washed with ethanol and resuspended in ultrapure water to obtain phenylboronic acid-functionalized gold nanoflowers.

[0055] (2) Preparation of gold-phenylboronic acid-bacterial conjugate: A single colony was added to the culture medium and cultured to obtain bacteria. The bacteria were then washed with PBS to obtain a concentration of 10. 3 cfu mL -1 Bacterial culture: Add phenylboronic acid-functionalized gold nanoflowers to the test bacteria, mix for 0.1 h, and then add 1% bovine serum albumin to obtain gold-phenylboronic acid-test bacteria conjugate.

[0056] (3) Preparation of gold nanoparticles: Prepare a 0.1% chloroauric acid solution, add a 0.1% reducing agent sodium citrate, and cool and let stand to obtain gold nanoparticles;

[0057] (4) Preparation of gold-streptavidin conjugate: gold-streptavidin conjugate was obtained by adding 0.1 g / L streptavidin to gold nanoparticles, mixing them, and then adding 1% bovine serum albumin.

[0058] (5) Preparation of a mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate: Take the gold-phenylboronic acid-bacterial conjugate solution obtained in step (2) and the gold-streptavidin conjugate solution obtained in step (4) and mix them to obtain a mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate.

[0059] (6) Preparation of lateral flow chromatography test strips: The LFIA test strip consists of three parts: a sample pad, a nitrocellulose membrane, and an absorbent pad. The absorbent pad is not further treated, while the sample pad is treated with 0.1% Triton-100, 1% sodium chloride, and Tris-HCl (pH 8.5), and then dried at 25°C for 4 hours. The sample pad, nitrocellulose membrane, and absorbent pad are sequentially pasted onto a plate, overlapping each other by 1-2 mm, to assemble the lateral flow chromatography test strip. The surface of the nitrocellulose membrane is coated with a detection line and a control line. The antibody to be detected is dispensed onto the nitrocellulose membrane at a rate of 0.5 μL / cm to form the detection line, and biotin is dispensed onto the nitrocellulose membrane at a rate of 0.5 μL / cm to form the control line.

[0060] (7) Side flow chromatography detection method: Take the mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate prepared in step (5) and drop it onto the sample pad of the test strip. The sample solution migrates to the nitrocellulose membrane.

[0061] Example 3

[0062] This invention relates to a lateral flow immunochromatographic detection method based on phenylboronic acid gold nanoflowers, comprising the following steps:

[0063] (1) Preparation of phenylboronic acid-functionalized gold nanoflowers: First, 5% sodium citrate reducing agent was added to a 5% chloroauric acid solution to prepare gold nanoseeds. Then, 5% chloroauric acid, 5% sodium citrate and 50mM phenol were added to the gold nanoseeds to prepare gold nanoflowers. 0.5g / L phenylboronic acid was added to the gold nanoflowers. After the reaction, the mixture was washed with ethanol and resuspended in ultrapure water to obtain phenylboronic acid-functionalized gold nanoflowers.

[0064] (2) Preparation of gold-phenylboronic acid-bacterial conjugate: A single colony was added to the culture medium and cultured to obtain bacteria. The bacteria were then washed with PBS to obtain a concentration of 10. 5 cfu mL -1Bacterial culture: Add phenylboronic acid-functionalized gold nanoflowers to the test bacteria, mix for 0.3 h, and then add 5% bovine serum albumin to obtain gold-phenylboronic acid-test bacteria conjugate.

[0065] (3) Preparation of gold nanoparticles: Prepare a 5% chloroauric acid solution, add a 5% sodium citrate reducing agent, and cool and let stand to obtain gold nanoparticles.

[0066] (4) Preparation of gold-streptavidin conjugate: gold-streptavidin conjugate was obtained by adding streptavidin at a concentration of 5 g / L to gold nanoparticles, mixing them, and then adding bovine serum albumin at a concentration of 5%.

[0067] (5) Preparation of a mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate: Take the gold-phenylboronic acid-bacterial conjugate solution obtained in step (2) and the gold-streptavidin conjugate solution obtained in step (4) and mix them to obtain a mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate.

[0068] (6) Preparation of lateral flow chromatography test strips: The LFIA test strip consists of three parts: a sample pad, a nitrocellulose membrane, and an absorbent pad. The absorbent pad is not further treated, while the sample pad is treated with 0.3% Triton-100, 4% sodium chloride, and Tris-HCl (pH 9.0), and then dried at 30°C for 8 hours. The sample pad, nitrocellulose membrane, and absorbent pad are sequentially pasted onto a plate, overlapping each other by 1-2 mm, to assemble the lateral flow chromatography test strip. The surface of the nitrocellulose membrane is coated with a detection line and a control line. The antibody to be detected is dispensed onto the nitrocellulose membrane at a rate of 1.5 μL / cm to form the detection line, and biotin is dispensed onto the nitrocellulose membrane at a rate of 1.5 μL / cm to form the control line.

[0069] (7) Side flow chromatography detection method: Take the mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate prepared in step (5) and drop it onto the sample pad of the test strip. The sample solution migrates to the nitrocellulose membrane.

[0070] Example 4

[0071] This invention relates to a lateral flow immunochromatographic detection method based on phenylboronic acid gold nanoflowers, comprising the following steps:

[0072] (1) Preparation of phenylboronic acid-functionalized gold nanoflowers: First, gold nanoseeds were prepared by adding 10% sodium citrate as a reducing agent to a 10% chloroauric acid solution. Then, 10% chloroauric acid, 10% sodium citrate, and 100mM phenol were added to the gold nanoseeds to prepare gold nanoflowers. The gold nanoflowers were then added to 1g / L phenylboronic acid, and after the reaction, they were washed with ethanol and resuspended in ultrapure water to obtain phenylboronic acid-functionalized gold nanoflowers.

[0073] (2) Preparation of gold-phenylboronic acid-bacterial conjugate: A single colony was added to the culture medium and cultured to obtain bacteria. The bacteria were then washed with PBS to obtain a concentration of 10. 8 cfu mL -1 Bacterial culture: Add phenylboronic acid-functionalized gold nanoflowers to the bacteria to be tested, mix for 1 hour, and then add 8% bovine serum albumin to obtain the gold-phenylboronic acid-bacterial conjugate.

[0074] (3) Preparation of gold nanoparticles: Prepare a 10% chloroauric acid solution, add a 10% sodium citrate reducing agent, and cool and let stand to obtain gold nanoparticles.

[0075] (4) Preparation of gold-streptavidin conjugate: gold-streptavidin conjugate was obtained by adding 10 g / L streptavidin to gold nanoparticles, mixing them, and then adding 8% bovine serum albumin.

[0076] (5) Preparation of a mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate: Take the gold-phenylboronic acid-bacterial conjugate solution obtained in step (2) and the gold-streptavidin conjugate solution obtained in step (4) and mix them to obtain a mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate.

[0077] (6) Preparation of lateral flow chromatography test strips: The LFIA test strip consists of three parts: a sample pad, a nitrocellulose membrane, and an absorbent pad. The absorbent pad is not further treated, while the sample pad is treated with 1% Triton-100, 5% sodium chloride, and Tris-HCl (pH 10.0), and then dried at 35°C for 10 hours. The sample pad, nitrocellulose membrane, and absorbent pad are sequentially pasted onto a plate, overlapping each other by 1-2 mm, to assemble the lateral flow chromatography test strip. The surface of the nitrocellulose membrane is coated with a detection line and a control line. The antibody to be detected is dispensed onto the nitrocellulose membrane at a rate of 2 μL / cm to form the detection line, and biotin is dispensed onto the nitrocellulose membrane at a rate of 2 μL / cm to form the control line.

[0078] (7) Side flow chromatography detection method: Take the mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate prepared in step (5) and drop it onto the sample pad of the test strip. The sample solution migrates to the nitrocellulose membrane.

[0079] Comparative Example 1

[0080] The lateral flow immunochromatographic assay method of Comparative Example 1, compared with Example 1, changed the detection sample solution by replacing the gold-phenylboronic acid-bacterial conjugate solution with a gold-antibody-bacterial conjugate solution, specifically including the following steps:

[0081] (1) Preparation of gold-antibody-bacterial conjugate solution: First, adjust the pH of 10 mL of gold nanoflower solution to 8.0 with 0.2 mol / L K2CO3, then add 40 μg of antibody solution, stir at room temperature for 30 minutes, and then add 10 μg of... 7 cfu mL -1 The bacterial culture was incubated for 0.5 hours, and then 10% bovine serum albumin was added to obtain the gold-antibody-bacterial conjugate.

[0082] (2) Preparation of gold nanoparticles: Prepare a 1% chloroauric acid solution, add a 1% reducing agent sodium citrate, and cool and let stand to obtain gold nanoparticles.

[0083] (3) Preparation of gold-streptavidin conjugate: gold-streptavidin conjugate was obtained by adding streptavidin at a concentration of 1 g / L to gold nanoparticles, mixing them, and then adding bovine serum albumin at a concentration of 10%.

[0084] (4) Preparation of a mixed solution of gold-antibody-bacterial conjugate and gold-streptavidin conjugate: Take the gold-antibody-bacterial conjugate solution obtained in step (1) and the gold-streptavidin conjugate solution obtained in step (3) and mix them to obtain a mixed solution of gold-antibody-bacterial conjugate and gold-streptavidin conjugate.

[0085] (5) Preparation of lateral flow chromatography test strips: The LFIA test strip consists of three parts: a sample pad, a nitrocellulose membrane, and an absorbent pad. The absorbent pad is not further treated, while the sample pad is treated with 0.5% Triton-100, 3% sodium chloride, and Tris-HCl (pH 8.0), and then dried at 37°C for 12 hours. The sample pad, nitrocellulose membrane, and absorbent pad are sequentially pasted onto a plate, overlapping each other by 1-2 mm, to assemble the lateral flow chromatography test strip. The surface of the nitrocellulose membrane is coated with a detection line and a control line. The antibody to be detected is dispensed onto the nitrocellulose membrane at a rate of 1 μL / cm to form the detection line, and biotin is dispensed onto the nitrocellulose membrane at a rate of 1 μL / cm to form the control line.

[0086] (6) Side flow chromatography detection method: Take the mixed solution of gold-antibody-bacterial conjugate and gold-streptavidin conjugate prepared in step (4) and drop it onto the sample pad of the test strip. The sample solution migrates to the nitrocellulose membrane.

[0087] like Figure 13 As shown in the comparison example 1, the lowest concentration that can be detected in the qualitative mode is 10. 6 Pathogenic microorganisms at CFU / ml; Example 1 performed qualitative analysis with a minimum detectable concentration of 10. 3 For pathogenic microorganisms at cfu / ml, the test results can be observed with the naked eye, significantly improving the detection effect and increasing the sensitivity by three orders of magnitude.

[0088] Comparative Example 2

[0089] The lateral flow immunochromatographic assay method of Comparative Example 2 was modified by changing the preparation of the agar-streptavidin conjugate in step (4), specifically including the following steps:

[0090] (1) Preparation of phenylboronic acid-functionalized gold nanoflowers: First, gold nanoseeds were prepared by adding sodium citrate at a concentration of 12% to a chloroauric acid solution. Then, chloroauric acid at a concentration of 12%, sodium citrate at a concentration of 12%, and phenol at a concentration of 8mM were added to the gold nanoseeds to prepare gold nanoflowers. The gold nanoflowers were then taken and phenylboronic acid at a concentration of 1.2g / L was added. After the reaction, the mixture was washed with ethanol and resuspended in ultrapure water to obtain phenylboronic acid-functionalized gold nanoflowers.

[0091] (2) Preparation of gold-phenylboronic acid-bacterial conjugate: A single colony was added to the culture medium and cultured to obtain bacteria. The bacteria were then washed with PBS to obtain a concentration of 10. 7 cfu mL -1 Bacterial culture: Add phenylboronic acid-functionalized gold nanoflowers to the test bacteria, mix for 1.5 h, and then add 12% bovine serum albumin to obtain gold-phenylboronic acid-test bacteria conjugate.

[0092] (3) Preparation of gold nanoparticles: Prepare a 12% chloroauric acid solution, add a 12% sodium citrate reducing agent, and cool and let stand to obtain gold nanoparticles.

[0093] (4) Preparation of gold-streptavidin conjugate: gold-streptavidin conjugate was obtained by adding streptavidin at a concentration of 0.05 g / L to gold nanoparticles, mixing them, and then adding bovine serum albumin at a concentration of 0.5%.

[0094] (5) Preparation of a mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate: Take the gold-phenylboronic acid-bacterial conjugate solution obtained in step (2) and the gold-streptavidin conjugate solution obtained in step (4) and mix them to obtain a mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate.

[0095] (6) Preparation of lateral flow chromatography test strips: The LFIA test strip consists of three parts: a sample pad, a nitrocellulose membrane, and an absorbent pad. The absorbent pad is not further treated, while the sample pad is treated with 0.5% Triton-100, 3% sodium chloride, and Tris-HCl (pH 8.0), and then dried at 45°C for 1 hour. The sample pad, nitrocellulose membrane, and absorbent pad are sequentially pasted onto a plate, overlapping each other by 1-2 mm, to assemble the lateral flow chromatography test strip. The surface of the nitrocellulose membrane is coated with a detection line and a control line. The antibody to be detected is dispensed onto the nitrocellulose membrane at a rate of 1 μL / cm to form the detection line, and biotin is dispensed onto the nitrocellulose membrane at a rate of 1 μL / cm to form the control line.

[0096] (7) Side flow chromatography detection method: Take the mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate prepared in step (5) and drop it onto the sample pad of the test strip. The sample solution migrates to the nitrocellulose membrane.

[0097] In Comparative Example 2, the amount of streptavidin added was too small, resulting in a very faint C-line or that could not be observed. At the same time, the amount of bovine serum albumin combined was too small, which could lead to false positives.

[0098] Comparative Example 3

[0099] The lateral flow immunochromatographic assay method of Comparative Example 3 was modified by changing the rate distribution in step (6), specifically including the following steps:

[0100] (1) Preparation of phenylboronic acid-functionalized gold nanoflowers: First, gold nanoseeds were prepared by adding sodium citrate at a concentration of 12% to a chloroauric acid solution. Then, chloroauric acid at a concentration of 12%, sodium citrate at a concentration of 12%, and phenol at a concentration of 8mM were added to the gold nanoseeds to prepare gold nanoflowers. The gold nanoflowers were then taken and phenylboronic acid at a concentration of 1.2g / L was added. After the reaction, the mixture was washed with ethanol and resuspended in ultrapure water to obtain phenylboronic acid-functionalized gold nanoflowers.

[0101] (2) Preparation of gold-phenylboronic acid-bacterial conjugate: A single colony was added to the culture medium and cultured to obtain bacteria. The bacteria were then washed with PBS to obtain a concentration of 10. 7 cfu mL -1 Bacterial culture: Add phenylboronic acid-functionalized gold nanoflowers to the test bacteria, mix for 1.5 h, and then add 12% bovine serum albumin to obtain gold-phenylboronic acid-test bacteria conjugate.

[0102] (3) Preparation of gold nanoparticles: Prepare a 12% chloroauric acid solution, add a 12% sodium citrate reducing agent, and cool and let stand to obtain gold nanoparticles.

[0103] (4) Preparation of gold-streptavidin conjugate: gold-streptavidin conjugate was obtained by adding streptavidin at a concentration of 1 g / L to gold nanoparticles, mixing them, and then adding bovine serum albumin at a concentration of 10%.

[0104] (5) Preparation of a mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate: Take the gold-phenylboronic acid-bacterial conjugate solution obtained in step (2) and the gold-streptavidin conjugate solution obtained in step (4) and mix them to obtain a mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate.

[0105] (6) Preparation of lateral flow chromatography test strips: The LFIA test strip consists of three parts: a sample pad, a nitrocellulose membrane, and an absorbent pad. The absorbent pad is not further treated, while the sample pad is treated with 0.5% Triton-100, 3% sodium chloride, and Tris-HCl (pH 8.0), and then dried at 45°C for 1 hour. The sample pad, nitrocellulose membrane, and absorbent pad are sequentially pasted onto a plate, overlapping each other by 1-2 mm, to assemble the lateral flow chromatography test strip. The surface of the nitrocellulose membrane is coated with a detection line and a control line. The antibody to be detected is dispensed onto the nitrocellulose membrane at a rate of 0.2 μL / cm to form the detection line, and biotin is dispensed onto the nitrocellulose membrane at a rate of 0.2 μL / cm to form the control line.

[0106] (7) Side flow chromatography detection method: Take the mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate prepared in step (5) and drop it onto the sample pad of the test strip. The sample solution migrates to the nitrocellulose membrane.

[0107] The slow partitioning rate of Comparative Example 3 resulted in low antibody levels, leading to insufficient antigen-antibody binding and insufficient streptomycin-antibiotic binding, resulting in insufficient sensitivity and faint T-line development or failure to be observed.

[0108] like Figure 1 The diagram shows a side-flow chromatography test strip. 1 is a polyvinyl chloride plate, 2 is a sample pad, 3 is a nitrocellulose membrane, 4 is an absorption pad, 5 is a detection line, 6 is a pathogen receptor protein, 7 is a control line, and 8 is biotin.

[0109] like Figure 2 The image shown is a transmission electron microscope (TEM) image of gold-phenylboronic acid nanoflowers, which shows that the synthesized gold-phenylboronic acid nanoflowers have a uniform morphology, are flower-like, and have a particle size of about 100 nm, indicating the successful synthesis of gold-phenylboronic acid nanoflowers.

[0110] like Figure 3The image shows the UV-Vis absorption spectrum of gold-phenylboronic acid nanoflowers. It shows that the 20nm gold seeds have an absorption peak at 520nm, the 60nm gold nanoparticles have an absorption peak at 554nm, the 100nm gold nanoflowers have an absorption peak at 642nm, and the gold-phenylboronic acid nanoflowers have an absorption peak at 662nm. The absorption peak is 20nm redshifted relative to the gold nanoflowers, indicating the successful synthesis of gold-phenylboronic acid nanoflowers.

[0111] like Figure 4 The image shown is the Raman spectrum of gold-phenylboronic acid nanoflowers, displayed at 1066 cm⁻¹. -1 The presence of a strong Raman absorption peak, which is stronger than that of gold-phenylboronic acid nanospheres, indicates that gold-phenylboronic acid nanoflowers can generate excellent Raman signals.

[0112] like Figure 5 As shown, the photothermal image of gold-phenylboronic acid nanoflowers shows that the temperature of the nanoflowers increases with time under 635nm laser irradiation, indicating that gold-phenylboronic acid nanoflowers can generate excellent photothermal signals.

[0113] like Figure 6 As shown, the study on the binding ability of gold-phenylboronic acid nanoflowers with bacteria: taking Gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus as examples, gold-phenylboronic acid nanoflowers were co-incubated with Escherichia coli and Staphylococcus aureus, respectively. The red arrows in the scanning electron microscope images indicate the nanoflowers bound to the surface of Escherichia coli and Staphylococcus aureus, indicating that gold-phenylboronic acid nanoflowers have a strong binding effect with both Gram-negative and Gram-positive bacteria.

[0114] like Figure 7 As shown, with 10 7 cfu mL -1 Escherichia coli was used as a standard solution for parameter optimization. The T-line color development effect was used as the judgment criterion, and the parameter value corresponding to the most obvious T-line color development was taken as the optimal experimental parameter. The bacterial incubation time with nanoparticles was 30 min, the immunoreaction time was 15 min, the phenylboronic acid modification concentration was 200 μg / ml, and the probe volume ratio of gold-phenylboronic acid nanoflowers to gold-streptavidin was 1:1 as the optimal experimental parameters for the entire detection process.

[0115] like Figure 8 The image shown is a naked-eye observation of the LFIA for E. coli detection. All control lines on the test strips are red, indicating that the test strips are functioning correctly. For bacterial concentrations greater than or equal to 10... 3 cfu mL -1 For the sample, the T line on the test strip showed clear color development after testing, and the detection limit for the naked eye was 10. 3 cfu mL -1 For bacterial concentrations less than 10 3 cfu mL -1For the sample, the T line on the test strip did not develop color after testing.

[0116] like Figure 9 The figure shows the colorimetric signal standard curve for Escherichia coli detection using this LFIA platform, indicating that the detection limit for the colorimetric signal of E. coli using this LFIA platform is 10. 3 cfu·mL -1 The linear range is 10. 4 ~10 8 cfu·mL -1 R 2 =0.9870.

[0117] like Figure 10 The figure shows the standard curve of photothermal signal for Escherichia coli detected by this LFIA platform, indicating that the detection limit of the photothermal signal for Escherichia coli by this LFIA platform is 10. 2 cfu·mL -1 The linear range is 10. 2 ~10 7 cfu·mL -1 R 2 =0.9818.

[0118] like Figure 11 The figure shows the standard curve of Raman signal for E. coli detected by this LFIA platform, indicating that the detection limit of the Raman signal for E. coli by this LFIA is 10. 2 cfu·mL -1 The linear range is 10. 2 ~10 7 cfu·mL -1 R 2 =0.9904.

[0119] like Figure 12 The image shows the naked-eye observation results, colorimetric, Raman, and photothermal signal results of detecting E. coli content in artificial urine using the LFIA platform. The detection limit for naked-eye observation is 10. 4 cfu·mL -1 ; Colorimetric mode detection limit 10 4 cfu·mL -1 The linear range is 10. 4 ~10 8 cfu·mL -1 R 2 =0.9830; Raman mode detection limit is 10. 3 cfu·mL -1 The linear range is 10. 3 ~10 7 cfu·mL -1 R 2=0.9878; detection limit for photothermal mode is 10. 3 cfu·mL -1 The linear range is 10. 3 ~10 7 cfu·mL -1 R 2 =0.9987.

Claims

1. A three-modal two-color side-flow chromatography device based on gold nanoflowers of phenylboronic acid, characterized in that, The lateral flow chromatography device includes lateral flow chromatography test paper and reagents. The reagents include gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate. The lateral flow chromatography test paper includes a polyvinyl chloride plate (1), a sample pad (2), a nitrocellulose membrane (3), and an absorbent pad (4). The sample pad (2), the nitrocellulose membrane (3), and the absorbent pad (4) are sequentially attached to one side of the polyvinyl chloride plate (1). The nitrocellulose membrane is provided with a detection line (5) and a control line (7). The biological receptor protein (6) is arranged linearly to form the detection line (5), and the biotin (8) is arranged linearly to form the control line (7). The extension direction of the detection line (5) and the control line (7) is perpendicular to the direction of liquid diffusion. During the detection process, the pathogenic microorganism receptor protein (6) binds to the gold-phenylboronic acid-test bacteria conjugate, which aggregates the gold nanoflowers on the detection line (5), which appears blue. The biotin (8) binds to the gold-streptavidin conjugate, which aggregates the gold nanoparticles on the control line (7), which appears red. The gold-phenylboronic acid-bacterial conjugate was prepared by reducing chloroauric acid to obtain gold nanoseeds, then adding chloroauric acid, sodium citrate and phenol to obtain gold nanoflowers. The gold nanoflowers were then taken, phenylboronic acid was added, and after the reaction, they were washed and resuspended in ultrapure water to obtain phenylboronic acid-functionalized gold nanoflowers. These were then mixed with the bacteria to be tested, and bovine serum albumin was added to obtain the gold-phenylboronic acid-bacterial conjugate. The gold-streptavidin conjugate is obtained by adding streptavidin to gold nanoparticles, mixing them, and then adding bovine serum albumin.

2. The phenylboronic acid gold nanoflower three-mode two-color side-flow chromatography apparatus according to claim 1, characterized in that, The interface between the sample pad (2) and the nitrocellulose membrane (3) overlaps by 1-2 mm, and the interface between the nitrocellulose membrane (3) and the absorbent pad (4) overlaps by 1-2 mm.

3. The lateral flow immunoassay method using the lateral flow chromatography apparatus of claim 1, characterized in that, Includes the following steps: (1) Preparation of phenylboronic acid-functionalized gold nanoflowers: First, sodium citrate, a reducing agent, was added to chloroauric acid solution to prepare gold nanoseeds. Chloroauric acid, sodium citrate and phenol were added to the gold nanoseeds to prepare gold nanoflowers. The gold nanoflowers were then added to phenylboronic acid, and after the reaction, they were washed with ethanol and resuspended in ultrapure water to obtain phenylboronic acid-functionalized gold nanoflowers. (2) Preparation of gold-phenylboronic acid-bacterial conjugate: A single colony was added to the culture medium and cultured to obtain bacteria. The bacteria were washed with PBS to obtain bacterial culture. Phenylboronic acid-functionalized gold nanoflowers were added to the bacteria to be tested. After mixing, bovine serum albumin was added to obtain gold-phenylboronic acid-bacterial conjugate. (3) Preparation of gold nanoparticles: Prepare chloroauric acid solution, add sodium citrate as reducing agent, cool and let stand to obtain gold nanoparticles; (4) Preparation of gold-streptavidin conjugate: gold-streptavidin conjugate is obtained by adding streptavidin to gold nanoparticles, mixing them and then adding bovine serum albumin. (5) Preparation of a mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate: Take the gold-phenylboronic acid-bacterial conjugate solution obtained in step (2) and the gold-streptavidin conjugate solution obtained in step (4) and mix them to obtain a mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate. (6) Preparation of three-mode dual-color side-flow chromatography test paper of gold nanoflowers of phenylboronic acid: The sample pad, nitrocellulose membrane and absorption pad were sequentially pasted onto the plate to assemble the side-flow chromatography test paper; (7) Lateral flow chromatography detection method: Take the mixed solution of gold-phenylboronic acid-bacterial conjugate and gold-streptavidin conjugate prepared in step (5) and drop it onto the sample pad of the test strip. The sample solution migrates to the nitrocellulose membrane. The gold-streptavidin conjugate and biotin forming the quality control line are recognized by the streptavidin-biotin system. The gold nanoparticles aggregate on the quality control line and a C line appears. The C line is red, indicating that the test strip is working normally. The bacteria to be detected and the pathogenic microorganism receptor protein that forms the detection line are specifically identified by antigen and antibody. Gold nanoflowers aggregate on the detection line, forming a T-line band that appears blue for qualitative judgment. The RGB values ​​of the T-line are analyzed using ColorPicker software and compared with a standard curve to achieve quantitative judgment of the colorimetric signal of bacterial concentration in the sample.

4. The detection method according to claim 3, characterized in that, In step (1), the concentration of the chloroauric acid solution is 0.1%-10%, the concentration of sodium citrate is 0.1%-10%, and the concentration of phenylboronic acid is 0.1-1 g / L. In step (3), the concentration of the chloroauric acid solution is 0.1%-10%, and the concentration of sodium citrate is 0.1%-10%.

5. The detection method according to claim 3, characterized in that, In step (2), the bacteria are Gram-positive or Gram-negative bacteria, and the concentration of the bacteria is 0-10. 8 The concentration of bovine serum albumin was CFU / mL, the mixing time was 0.1-2 h, and the concentration of bovine serum albumin was 1%-20%.

6. The detection method according to claim 4, characterized in that, In step (4), the concentration of streptavidin is 0.1-10 g / L, the mixing time is 0.1-1 h, and the concentration of bovine serum albumin is 1%-10%.

7. The detection method according to claim 3, characterized in that, The quantitative judgment in step (7) is replaced by the following method: T line is irradiated with a laser with a wavelength of 635 nm, the temperature value of T line is analyzed by a thermal imager, and compared with the standard curve to realize the quantitative judgment of bacterial concentration in the sample based on photothermal mode; or Raman spectrometer is used to analyze the Raman intensity of T line and compare with the standard curve to realize the quantitative judgment of bacterial concentration in the sample based on Raman mode.

8. The detection method according to claim 3, characterized in that, In step (6), the sample pad is prepared by immersing a polyester film in Triton-100, sodium chloride and Tris-HCl in sequence, and then drying it in an oven at 25-40°C for 2-10 hours.

9. The detection method according to claim 3, characterized in that, In step (6), the nitrocellulose membrane is prepared by dispensing the antibody to be detected onto the nitrocellulose membrane at a rate of 0.5-2 μL / cm to form a detection line, and dispensing biotin onto the nitrocellulose membrane at a rate of 0.5-2 μL / cm to form a control line.

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