WO2023087480A1

WO2023087480A1 - Lateral flow chromatography test paper based on mercaptophenylboronic acid functionalized gold nanoparticles, and detection method therefor

Info

Publication number: WO2023087480A1
Application number: PCT/CN2021/140031
Authority: WO
Inventors: 鞠艳敏; 戴建君; 吴鹏程
Original assignee: 中国药科大学
Priority date: 2021-11-22
Filing date: 2021-12-21
Publication date: 2023-05-25
Also published as: CN114113592A; CN114113592B

Abstract

A lateral flow chromatography test paper based on mercaptophenylboronic acid functionalized gold nanoparticles, and a detection method. The lateral flow chromatography test paper comprises a flat plate (1), a sample pad (2), a nitrocellulose membrane (3) and an absorption pad (4). The lateral flow chromatography detection method can be completed by means of the steps: preparing mercaptophenylboronic acid functionalized gold nanoparticles, preparing a gold-mercaptophenylboronic acid-bacteria conjugate, preparing a lateral flow chromatography test paper strip, and performing lateral flow chromatography detection. Using the strong binding power of phenylboric acid and bacteria overcomes the limitations of a double-antibody sandwich method in conventional lateral flow immunoassays, greatly reducing detection costs, and improving a detection limit; qualitative and quantitative results of pathogenic microorganisms can be obtained by means of the present method, specificity is strong, sensitivity is high, the detection process is convenient and fast, and the detection result is safe and reliable.

Description

A lateral flow chromatography test paper based on mercaptophenylboronic acid functionalized gold nanoparticles and its detection method

technical field

The invention relates to a lateral flow chromatography test paper and a detection method thereof, in particular to a lateral flow chromatography test paper based on mercaptophenylboronic acid functionalized gold nanoparticles and a detection method thereof.

Background technique

Bacteria detection is an important field related to public safety. Lateral flow immunochromatography (LFIA) is an excellent and classic biosensing technology, which is widely used in the field of rapid monitoring of pathogens. Real-time on-site qualitative detection can be achieved by naked eye monitoring detection. The traditional immunological lateral flow chromatography technology for bacterial detection usually uses a double-antibody sandwich method, that is, the "nanomaterial-detection antibody-bacteria-capture antibody" sandwich mode, in which the detection antibody is labeled with a nanomaterial that can generate a readout signal, such as gold Nanoparticles, quantum dots, etc., while the capture antibody is immobilized on the T-line region of the strip. Paired antibodies need to be obtained through a time-consuming screening process, and are limited by factors such as cumbersome preparation processes, chemical instability, or complex cross-linking between antibodies and nanomaterials.

The non-capturing antibody-dependent LFIA method, that is, the "nanomaterial-bacteria-detection antibody" sandwich strategy, usually uses weak binding forces such as electrostatic adsorption, hydrogen bonds, and hydrophobic interactions to capture bacteria, resulting in low reproducibility of results and low detection limits. high. Therefore, it is crucial to capture bacteria using strong binding forces such as covalent bonds to realize the "nanomaterial-bacteria-antibody" sandwich strategy.

The traditional colloidal gold-based lateral flow chromatography detection technology uses a double-antibody sandwich method, which requires the preparation of a "colloidal gold-capture antibody" probe, which is chemically unstable. During the detection process, the probe There may be partial failure of the needle, so the detection limit is high. The existing non-capture antibody-dependent LFIA method uses weak binding forces such as electrostatic adsorption, hydrogen bonding, and hydrophobic interactions to capture bacteria, resulting in a low efficiency of capturing bacteria. Simply put, there are fewer nanoparticles adsorbed on each bacterium , so fewer nanoparticles aggregate on the test strip detection line and thus a higher detection limit.

Contents of the invention

Purpose of the invention: The present invention aims at the problems of high detection limit and insufficient sensitivity of the lateral flow chromatography detection method existing in the prior art, and provides a lateral flow chromatography test paper based on mercaptophenylboronic acid functionalized gold nanoparticles, and also provides the Detection method of lateral flow chromatography.

Technical solution: The lateral flow chromatography test paper based on mercaptophenylboronic acid functionalized gold nanoparticles according to the present invention includes a flat plate, a sample pad, a nitrocellulose membrane and an absorbent pad, and the sample pad, the nitrocellulose membrane and the absorbent pad are pasted in sequence On the surface of one side of the plate, the surface of the nitrocellulose membrane is coated with a detection line, and the detection line is coated with a pathogenic microorganism receptor protein, and the gold-mercaptophenylboronic acid-to-be-tested bacterium conjugate passes through the detection line. It is bound by the receptor protein of pathogenic microorganisms, and the gold-mercaptophenylboronic acid nanoparticles are gathered on the detection line.

Preferably, the sample pad is a polyester film treated with Triton X-100, sodium chloride, Tris-HCl and PB and dried, the junction of the sample pad and the nitrocellulose membrane overlaps by 1-2 mm, and the nitric acid The junction of the cellulose film and the absorbent pad overlaps by 1-2mm.

Preferably, the gold-mercaptophenylboronic acid-bacteria conjugate is prepared by adding mercaptophenylboronic acid to gold nanoparticles to prepare gold nanoparticles functionalized with mercaptophenylboronic acid; adding the gold nanoparticles functionalized with mercaptophenylboronic acid to be tested Bacteria, after mixing, bovine serum albumin was added to obtain a gold-mercaptophenylboronic acid-bacteria conjugate.

The invention also discloses a lateral flow chromatography detection method based on mercaptophenylboronic acid functionalized gold nanoparticles, comprising the following steps:

(1) Preparation of gold nanoparticles functionalized with mercaptophenylboronic acid: prepare chloroauric acid solution, add reducing agent sodium citrate, cool and stand to obtain gold nanoparticles, take gold nanoparticles, add mercaptophenylboronic acid, wash with ethanol after reaction , to obtain gold nanoparticles functionalized with mercaptophenylboronic acid;

(2) Preparation of gold-mercaptophenylboronic acid-bacteria conjugate: take a single colony and add it to the medium for culture, take the bacterial solution and add it to the medium for culture to obtain the bacteria, take the gold nanoparticles functionalized with mercaptophenylboronic acid and add it to the bacteria to be tested, mix Finally, bovine serum albumin was added, and the gold-mercaptophenylboronic acid-tested bacterium conjugate was obtained after mixing;

(3) Preparation of lateral flow chromatography test strips: the sample pad, nitrocellulose membrane and absorbent pad are pasted on the flat plate in order to overlap each other by 1-2mm, and assembled into lateral flow chromatography test paper, the surface of the nitrocellulose membrane A detection line is coated, and the detection line is coated with a pathogenic microorganism receptor protein;

(4) Lateral flow chromatography detection method: get the gold-mercaptophenylboronic acid-to-be-tested bacterium conjugate that step (2) makes, add drop-wise on the sample pad of test strip, sample liquid migrates toward nitrocellulose membrane, wait for The detection bacteria can carry out specific antigen antibody recognition with the pathogenic microorganism receptor protein coated on the detection line on the nitrocellulose membrane, and the gold-mercaptophenylboronic acid nanoparticles gather on the detection line, and T-line bands appear for qualitative judgment; use Image J software analyzes the gray value of the T line and compares it with the standard curve to realize the quantitative determination of the bacterial concentration in the sample.

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 mercaptophenylboronic acid is 0.1-1g/L.

Preferably, in step (2), the bacteria are Gram-positive bacteria or Gram-negative bacteria, the concentration of the bacteria is 0-10 ⁸ CFU/mL, and the mixing time is 0.5-2h.

Preferably, in step (3), the preparation of the sample pad is specifically, immersing the polyester film in the sample pad treatment solution for 10-24 hours, and then drying in an oven at 25-40°C for 2-10 hours to obtain the sample pad, The sample pad treatment solution is composed of 0.1%-1% Triton-100, 1%-5% sodium chloride, Tris-HCl (pH 8.0-10.0).

Preferably, in step (3), the preparation of the nitrocellulose membrane specifically includes distributing the antibody to the bacteria to be detected onto the nitrocellulose membrane at a rate of 0.5-2 μL/cm.

Preferably, in step (4), the software analyzes the T-line gray value under each bacterial concentration, and fits to formulate a standard curve; according to the T-line gray value of the sample to be detected, according to the standard curve, it is possible to quantitatively determine the concentration of the sample in the sample. concentration of bacteria.

Invention principle:

The inventor found in the research center that phenylboronic acid can be reversibly combined with the cis-ortho-dihydroxyl structure contained in the lipopolysaccharide and glycoprotein on the surface of bacteria, and dehydrated to form a cyclic ester, which can be used as a recognition molecule for microorganisms; Applied to lateral flow immunochromatography to realize bacterial detection.

In lateral flow immunochromatography, it is necessary to rely on antibody recognition and capture of specific bacteria, and the combination can be achieved by relying on the structural characteristics of the bacteria themselves to form a "nanomaterial-bacteria" complex. In the present invention, the phenylboronic acid on the surface of the nanomaterial Form a six-membered ring borate diol ester with the cis-ortho-dihydroxy structure on the surface of bacteria, and realize the recognition and capture of bacteria by nanomaterials through covalent bonding. The gold-mercaptophenylboronic acid nanoparticles used in this lateral flow immunochromatography method are not only used to generate signals, but also can use phenylboronic acid to covalently bind to the cis-ortho-dihydroxyl structure contained in lipopolysaccharides and glycoproteins on the bacterial surface. To capture bacteria, only a single antibody is required for better results than traditional sandwich formats.

Different from the weak binding force such as electrostatic attraction, hydrophobic interaction and hydrogen bond used in the prior art, it is the first time to use strong binding force such as covalent bond to capture bacteria, and design a new type of non-capture antibody-dependent lateral flow immunochromatography method; the new LFIA has universal applicability in the field of pathogen detection; the synthesis of gold-mercaptophenylboronic acid nanoparticles and the preparation process of lateral flow chromatography test strips are cost-effective, fast, simple and labor-saving. After adding the prepared gold-mercaptophenylboronic acid-bacteria conjugate dropwise to the sample pad of the test strip, observe the color intensity of the T line of the NC film with the naked eye, and then perform colorimetric qualitative judgment of the bacterial concentration or use Image J software to analyze the gray color of the T line. Quantitatively determine the concentration of bacteria. The real-time on-site detection of broad-spectrum bacteria is realized, which greatly saves the detection cost and improves the detection limit.

Beneficial effect: compared with the prior art, the present invention has the following significant advantages:

The invention provides a fast, highly sensitive, highly specific on-site detectable lateral flow chromatography detection method based on mercaptophenylboronic acid functionalized gold nanoparticles, which can simultaneously complete qualitative and quantitative tests; the lowest detectable concentration is The detection of 10 ³ cfu/ml pathogenic microorganisms can be completed within 10 minutes, and the test results can be observed only with the naked eye. The invention has strong specificity for pathogenic microorganisms, high sensitivity, convenient and quick detection process, and safe and reliable detection results.

Description of drawings

Fig. 1 is the schematic diagram of lateral flow chromatography test strip of the present invention;

Fig. 2 is the naked-eye observation figure of the test result of lateral flow chromatography test strip of the present invention;

Fig. 3 is the Image J software analysis schematic diagram of the test result of lateral flow chromatography test strip of the present invention;

Fig. 4 is the transmission electron micrograph of gold nanoparticle of the present invention;

Fig. 5 is the ultraviolet-visible absorption spectrogram of the gold nanoparticles modified by mercaptophenylboronic acid;

Fig. 6 is the infrared spectrogram of the gold nanoparticles modified by mercaptophenylboronic acid, gold nanoparticles, and mercaptophenylboronic acid;

Figure 7 is a scanning electron microscope image and an infrared spectrum image of co-incubation of gold-mercaptophenylboronic acid nanoparticles with Escherichia coli O157: H7 and Staphylococcus aureus;

Figure 8 is an optimization diagram of five detection parameters of the lateral flow immunochromatography based on gold-mercaptophenylboronic acid;

Figure 9 Under this detection method, taking Escherichia coli O157:H7 as an example, the control group, 10 ⁰ , 10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ cfu/ml E. coli O157: The experimental results of bands corresponding to H7.

Fig. 10 is a graph showing the results of detection of Escherichia coli O157:H7 content in four food matrices (drinking water, watermelon juice, milk and beef) by the lateral flow chromatography method.

Detailed ways

The technical solution of the present invention will be further described below in conjunction with the accompanying drawings.

Example 1

The preparation process of gold nanoparticles functionalized with mercaptophenylboronic acid: take 100ml of ultrapure water in a conical flask, add 1ml of 1% chloroauric acid immediately after heating and boiling and keep stirring, add 4ml of 1% sodium citrate, and wait for the solution The color gradually changes from colorless to wine red. After the color is stable, continue to boil for 10 minutes, cool to room temperature, and add pure water to make up to 100ml. Take 5ml of the gold nanoparticles prepared above in an eggplant-shaped bottle, add 1ml of mercaptophenylboronic acid, react at room temperature for 12 hours, and wash with ethanol several times to obtain gold nanoparticles functionalized with phenylboronic acid.

Preparation of gold-mercaptophenylboronic acid-bacteria conjugate: Take a single bacterium and drop it into 3ml LB liquid medium for overnight cultivation at 150rpm at 37°C. Take 60μl of the bacterial liquid and culture it in 3ml LB liquid medium for 3 hours to obtain bacteria with a concentration of 10 ⁸ cfu/ml. Bacteria with different concentrations were obtained by doubling dilution. Take 10 μl of gold-mercaptoboronic acid nanoparticles in an EP tube, add 10 μl of bacteria, and mix for 0.5 hours with a mixer, then add 10 μl of bovine serum albumin (BSA), and mix for 0.5 hours with a mixer to obtain gold-mercaptophenylboronic acid - Bacteria conjugates.

Production of test strips: LFIA test strips include three parts: sample pad, nitrocellulose membrane, and absorbent pad. The absorbent pad was not further processed, and the sample pad was treated with Triton-100, sodium chloride, and Tris-HCl (pH 8.0), and then dried at 25°C for 8 hours; the test line area of the nitrocellulose membrane was detected by the detection antibody ( 0.5 mg/mL) at a rate of 0.5 μL/cm. Then, the three parts were sequentially pasted on the polyvinyl chloride flat plate overlapping each other by 1mm, and assembled into LFIA test strips. Finally, strips were cut into 3 mm widths and stored at 4–8 °C for further use.

Example 2

The preparation process of gold nanoparticles functionalized with mercaptophenylboronic acid: take 100ml of ultrapure water in a conical flask, add 1ml of 1% chloroauric acid immediately after heating and boiling and keep stirring, add 4ml of 10% sodium citrate, the solution color From colorless to wine red gradually, after the color is stable, continue to boil for 10 minutes, cool to room temperature, add pure water to make up to 100ml. Take 5ml of the gold nanoparticles prepared above in an eggplant-shaped bottle, add 1ml of mercaptophenylboronic acid, react at room temperature for 12 hours, and wash with ethanol several times to obtain gold nanoparticles functionalized with phenylboronic acid.

Preparation of gold-mercaptophenylboronic acid-bacteria conjugate: Take a single bacterium and drop it into 3ml LB liquid medium for overnight cultivation at 150rpm at 37°C. Take 60μl of the bacterial solution and culture it in 3ml LB liquid medium for 3 hours to obtain bacteria with a concentration of 10 ⁸ cfu/ml. Bacteria with different concentrations were obtained by doubling dilution. Take 100 μl of gold-mercaptoboronic acid nanoparticles in an EP tube, add 100 μl of bacteria, mix for 2 hours in a mixer, then add 50 μl of bovine serum albumin (BSA), and mix for 0.5 hours in a mixer to obtain gold-mercaptophenylboronic acid- Bacterial Conjugates.

Production of test strips: LFIA test strips include three parts: sample pad, nitrocellulose membrane, and absorbent pad. The absorbent pad was not further processed, and the sample pad was treated with Triton-100, sodium chloride, and Tris-HCl (pH 10.0), and then dried at 37°C for 12 hours; the test line area of the nitrocellulose membrane was detected by the detection antibody ( 2 mg/mL) at a rate of 0.8 μL/cm. Then, the three parts were sequentially pasted on the polyvinyl chloride flat plate overlapping each other by 1mm, and assembled into LFIA test strips. Finally, strips were cut into 3-5 mm widths and stored at 4-8 °C for further use.

Example 3

The preparation process of gold nanoparticles functionalized with mercaptophenylboronic acid: take 100ml of ultrapure water in a conical flask, add 1ml of 10% chloroauric acid immediately after heating and boiling and keep stirring, add 4ml of 5% sodium citrate, and wait for a few minutes Afterwards, the color of the solution gradually changed from colorless to wine red. After the color became stable, continue to boil for 10 minutes, cool to room temperature, and add pure water to make up to 100ml. Take 5ml of the gold nanoparticles prepared above in an eggplant-shaped bottle, add 1ml of mercaptophenylboronic acid, react at room temperature for 12 hours, and wash with ethanol several times to obtain gold nanoparticles functionalized with phenylboronic acid.

Preparation of gold-mercaptophenylboronic acid-bacteria conjugate: Take a single bacterium and drop it into 3ml LB liquid medium for overnight cultivation at 150rpm at 37°C. Take 60μl of the bacterial solution and culture it in 3ml LB liquid medium for 3 hours to obtain bacteria with a concentration of 10 ⁸ cfu/ml. Bacteria with different concentrations were obtained by doubling dilution. Take 100 μl of gold-mercaptoboronic acid nanoparticles in an EP tube, add 100 μl of bacteria, mix for 1 hour in a mixer, then add 50 μl of bovine serum albumin (BSA), and mix for 0.5 hours in a mixer to obtain gold-mercaptophenylboronic acid- Bacterial Conjugates.

Production of test strips: LFIA test strips include three parts: sample pad, nitrocellulose membrane, and absorbent pad. The absorbent pad was not further processed, and the sample pad was treated with Triton-100, sodium chloride, and Tris-HCl (pH 8.0), and then dried at 37°C for 12 hours; the test line area of the nitrocellulose membrane was detected by the detection antibody ( 2 mg/mL) at a rate of 1 μL/cm. Then, the three parts were sequentially pasted on the polyvinyl chloride flat plate overlapping each other by 2 mm, and assembled into an LFIA test strip. Finally, strips were cut into 3-5 mm widths and stored at 4-8 °C for further use.

Example 4

The preparation process of gold nanoparticles functionalized with mercaptophenylboronic acid: take 100ml ultrapure water in a conical flask, add 1ml 3% chloroauric acid immediately after heating and boiling and keep stirring, add 5% sodium citrate 4ml after 5 minutes After 2 minutes, the color of the solution gradually changed from colorless to wine red. After the color stabilized, continue to boil for 10 minutes, cool to room temperature, and add pure water to make up to 100ml. Take 5ml of the gold nanoparticles prepared above in an eggplant-shaped bottle, add 1ml of mercaptophenylboronic acid, react at room temperature for 12 hours, and wash with ethanol several times to obtain gold nanoparticles functionalized with phenylboronic acid.

Production of test strips: LFIA test strips include three parts: sample pad, nitrocellulose membrane, and absorbent pad. The absorbent pad was not further processed, and the sample pad was treated with Triton-100, sodium chloride, and Tris-HCl (pH 10.0), and then dried at 37°C for 12 hours; the test line area of the nitrocellulose membrane was detected by the detection antibody ( 2 mg/mL) at a rate of 2 μL/cm. Then, stick the three parts on the polyvinyl chloride flat plate in order to overlap each other by 1-2mm, and assemble into the LFIA test strip. Finally, strips were cut into 3-5 mm widths and stored at 4-8 °C for further use.

Comparative example 1

The preparation process of gold nanoparticles functionalized with mercaptophenylboronic acid: take 100ml of ultrapure water in a conical flask, add 1ml of 12% chloroauric acid immediately after heating and boiling and keep stirring, add 4ml of 12% sodium citrate after 5 minutes After 2 minutes, the color of the solution gradually changed from colorless to wine red. After the color stabilized, continue to boil for 20 minutes, cool to room temperature, and add pure water to make up to 100ml. Take 5ml of the gold nanoparticles prepared above in an eggplant-shaped bottle, add 1ml of mercaptophenylboronic acid, react at room temperature for 12 hours, and wash with ethanol several times to obtain gold nanoparticles functionalized with phenylboronic acid.

Preparation of gold-mercaptophenylboronic acid-bacteria conjugate: Take a single bacterium and drop it into 3ml LB liquid medium for overnight cultivation at 150rpm at 37°C. Take 60μl of the bacterial solution and culture it in 3ml LB liquid medium for 3 hours to obtain bacteria with a concentration of 10 ⁸ cfu/ml. Bacteria with different concentrations were obtained by doubling dilution. Take 5 μl of gold-mercaptoboronic acid nanoparticles in an EP tube, add 120 μl of bacteria, mix with the mixer for 0.4 hours, then add 5 μl of bovine serum albumin (BSA), and mix with the mixer for 0.1 hour to obtain gold-mercaptophenylboronic acid - Bacteria conjugates.

Production of test strips: LFIA test strips include three parts: sample pad, nitrocellulose membrane, and absorbent pad. The absorbent pad was not further processed, and the sample pad was treated with Triton-100, sodium chloride, and Tris-HCl (pH 7.0), and then dried at 50°C for 1 h; the test line area of the nitrocellulose membrane was detected by the detection antibody ( 0.2 mg/mL) at a rate of 0.2 μL/cm. Then, stick the three parts on the polyvinyl chloride flat plate in order to overlap each other by 1-2mm, and assemble them into LFIA test strips. Finally, strips were cut into 3-5 mm widths and stored at 4-8 °C for further use.

As shown in Figure 1, it is a schematic diagram of the prepared lateral flow chromatography test strip, 1 is a polyvinyl chloride flat plate, 2 is an absorbent pad, 3 is a nitrocellulose membrane, 4 is an absorbent pad, 5 is a detection line, 6 To detect antibodies on the line.

As shown in Figure 2, it is the result diagram of the LFIA used in the detection of Escherichia coli O157:H7. For samples with a bacterial concentration greater than or equal to 10 ³ cfu mL ^-1 , the T line of the test strip is obviously colored after detection, and the detection limit is 10 ³ cfu mL ^-1 ; for samples with a bacterial concentration less than 10 ³ cfu mL ^-1 , the T-line of the test strip will not show color after testing.

As shown in Figure 3, it is the curve graph of positive samples and negative samples analyzed by Image J software.

As shown in Figure 4, it is a transmission electron microscope image of gold-mercaptophenylboronic acid nanoparticles, which shows that the synthesized gold phenylboronic acid nanoparticles have a uniform appearance, are spherical, and have a particle size of about 20nm, indicating the successful synthesis of gold phenylboronic acid nanoparticles .

As shown in Figure 5, it is the ultraviolet-visible absorption spectrum figure of gold-mercaptophenylboronic acid nanoparticles, showing that the ultraviolet-visible light absorption spectrum figure of gold-mercaptophenylboronic acid nanoparticles has an absorption peak at 525nm, which is red-shifted by 5nm relative to gold nanoparticles, Indicating the successful synthesis of gold phenylboronic acid nanoparticles.

As shown in FIG. 6 , it is an infrared spectrogram of phenylboronic acid, gold nanoparticles, and gold phenylboronic acid nanoparticles. The strong absorption peak at 3452 cm ^-1 of gold phenylboronic acid nanoparticles indicates the OH vibration of boric acid, and the peak around 1647 cm ^-1 is attributed to the C=C vibration of benzene ring. In addition, the absorption peak of phenylboronic acid at ²⁵⁶⁵ cm was SH vibration, while in gold phenylboronic acid nanoparticles, the peak at 2565 ^cm disappeared due to the formation of Au-S bonds, indicating the successful synthesis of gold phenylboronic acid nanoparticles.

As shown in Figure 7, for the research on the binding ability of gold-mercaptophenylboronic acid nanoparticles to bacteria: taking the Gram-negative bacteria Escherichia coli O157:H7 and the Gram-positive bacteria Staphylococcus aureus as examples, the gold-mercaptophenylboronic acid nanoparticles Co-incubated with Escherichia coli O157:H7 and Staphylococcus aureus respectively, the red arrows in the scanning electron microscope image indicate the nanoparticles bound to the surface of Escherichia coli O157:H7 and Staphylococcus aureus, indicating that gold-mercaptophenylboronic acid nanoparticles are compatible with Gram-negative bacteria Or Gram-positive bacteria have a strong binding effect.

As shown in Figure 8, 10 ⁸ cfu mL ^-1 of Escherichia coli O157:H7 was used as the standard solution for parameter optimization, the average gray value of the T line was used as the judgment standard, and the parameter value corresponding to the highest average gray value of the T line was used as Optimal experimental parameters. Bacteria and nanoparticles were incubated for 100 min, 100 μg/ml mercaptophenylboronic acid modification amount, pH 7.0 system environment, 10 min immune reaction time, and 80 μl 5 mg/ml probe amount were used as the best experimental parameters for the entire detection process.

As shown in Figure 9, it is the standard curve for the detection of Escherichia coli O157:H7 by the LFIA platform, which shows that the detection limit of the LFIA detection Escherichia coli O157:H7 is 10 ³ cfu·mL ^-1 , and the linear range is 10 ³ to 10 ⁷ cfu·mL ^-1 , R ² =0.9989.

As shown in Figure 10, it shows the detection results of Escherichia coli O157:H7 in artificially polluted drinking water, watermelon juice, milk, and beef samples, and the detection limits are 10 ⁴ cfu·mL ^-1 , 10 ⁵ respectively cfu·mL ^-1 .10 ⁶ cfu·mL ^-1 , 10 ⁶ cfu·mL ^-1 .

Optimization of test strip detection parameters: This patent discusses the detection conditions (the amount of modification of mercaptophenylboronic acid, the pH value of the bacterial suspension, the volume of gold-mercaptophenylboronic acid dropped on the sample pad, the relationship between gold-mercaptophenylboronic acid and bacteria) Incubation time, immune reaction time) on the experimental results, in order to obtain fast and accurate results. All these parameters were optimized using Escherichia coli O157:H7 standard solution with a concentration of 10 ⁸ cfu mL ^-1 , and the gray value of the detection line on the test strip was used as the judgment standard.

Detection procedure: Taking Escherichia coli O157:H7 as an example, take 30-40 μL of gold-mercaptophenylboronic acid-bacteria conjugate dropwise on the sample pad of the test strip, and the gold-mercaptophenylboronic acid-bacteria conjugate migrates to the NC membrane for 10 minutes The red band (T line) on the NC film can be observed with the naked eye, and qualitative judgment can be made; the gray value of the T line can be analyzed by Image J software and linear fitting can be used to make a standard curve, which can be used for quantitative judgment of E. coli O157:H7 in the sample .

Application in real samples: Four food samples (drinking water, watermelon juice, milk and beef) were selected to demonstrate the wide applicability of the lateral flow chromatography in the field of rapid detection. Specifically, the four food matrices were pretreated first, and then a series of concentrations (0-10 ⁸ cfu/mL) of Escherichia coli O157:H7 were added to obtain the sample solution to be tested. When the concentration of the standard decreases, the gray value on the T line gradually decreases, which is consistent with the standard curve. The detection limits of bacteria in the four food matrices were 10 ⁴ cfu/mL, 10 ⁵ cfu/mL, 10 ⁶ cfu/mL and 10 ⁶ cfu/mL ^-1 , respectively.

Claims

A lateral flow chromatography test paper based on mercaptophenylboronic acid functionalized gold nanoparticles, characterized in that the lateral flow chromatography test paper comprises a flat plate (1), a sample pad (2), a nitrocellulose membrane (3) and an absorbent Pad (4), the sample pad (2), nitrocellulose membrane (3) and absorbent pad (4) are pasted on the surface of one side of the flat plate (1) in sequence, and the surface of the nitrocellulose membrane (4) is covered with The detected line (5), the pathogenic microorganism receptor protein (6) is coated on the detection line (5), and the gold-mercaptophenylboronic acid-to-be-tested bacteria conjugate passes through the detection line (5) and is coated with pathogenic microorganisms. The gold-mercaptophenylboronic acid nanoparticles are bound to the detection line by binding to the body protein (6).
Lateral flow chromatography test paper according to claim 1, is characterized in that, the intersection of described sample pad (2) and nitrocellulose membrane (3) overlaps 1-2mm, nitrocellulose membrane (3) and absorbent pad ( 4) The junction overlaps by 1-2mm.
The lateral flow chromatography test paper according to claim 1, characterized in that, the gold-mercaptophenylboronic acid-bacteria conjugate is obtained by adding mercaptophenylboronic acid to gold nanoparticles to prepare gold nanoparticles functionalized with mercaptophenylboronic acid ; The gold nanoparticles functionalized with mercaptophenylboronic acid are added to the bacteria to be tested, and bovine serum albumin is added after mixing to obtain a gold-mercaptophenylboronic acid-bacteria conjugate.
A lateral flow chromatography detection method based on mercaptophenylboronic acid functionalized gold nanoparticles, characterized in that it comprises the following steps:

(1) Preparation of gold nanoparticles functionalized with mercaptophenylboronic acid: prepare chloroauric acid solution, add reducing agent, cool and stand to obtain gold nanoparticles, take gold nanoparticles, add mercaptophenylboronic acid, wash with ethanol after reaction, and obtain mercapto Gold nanoparticles functionalized with phenylboronic acid;

(2) Preparation of gold-mercaptophenylboronic acid-bacteria conjugate: take a single colony and add it to the medium for culture, take the bacterial solution and add it to the medium for culture to obtain the bacteria, take the gold nanoparticles functionalized with mercaptophenylboronic acid and add it to the bacteria to be tested, mix Finally, bovine serum albumin was added, and the gold-mercaptophenylboronic acid-tested bacterium conjugate was obtained after mixing;

(3) Preparation of lateral flow chromatography test strips: the sample pad, nitrocellulose membrane and absorbent pad are pasted on the flat plate in order to overlap each other by 1-2mm, and assembled into lateral flow chromatography test paper, the surface of the nitrocellulose membrane A detection line is coated, and the detection line is coated with a pathogenic microorganism receptor protein;

(4) Lateral flow chromatography detection method: get the gold-mercaptophenylboronic acid-to-be-tested bacterium conjugate that step (2) makes, add drop-wise on the sample pad of test strip, sample liquid migrates toward nitrocellulose membrane, wait for The detection bacteria can carry out specific antigen antibody recognition with the pathogenic microorganism receptor protein coated on the detection line on the nitrocellulose membrane, and the gold-mercaptophenylboronic acid nanoparticles gather on the detection line, and T-line bands appear for qualitative judgment; use Image The J software analyzes the gray value of the T line and compares it with the standard curve to realize the quantitative determination of the bacterial concentration in the sample.
The lateral flow chromatography detection method according to claim 4, characterized in that, in step (1), the reducing agent is sodium citrate, and the concentration of the chloroauric acid solution is 0.1%-10%, and sodium citrate The concentration is 0.1%-10%, and the concentration of mercaptophenylboronic acid is 0.1-1g/L.
The lateral flow chromatography detection method according to claim 4, characterized in that, in step (2), the bacteria are Gram-positive bacteria or Gram-negative bacteria, and the concentration of the bacteria is 0-10 8 CFU/ mL.
The lateral flow chromatography detection method according to claim 4, characterized in that, in step (2), the mixing time is 0.5-2h.
The lateral flow chromatography detection method according to claim 4, characterized in that, in step (3), the preparation of the sample pad is specifically, immersing the polyester film in the sample pad treatment solution for 10-24h, and then Dry at 25-40°C for 2-10 hours to obtain a sample pad.
The lateral flow chromatography detection method according to claim 4, characterized in that, in step (3), the preparation of the nitrocellulose membrane is specifically, distributing the bacterial antibody to be detected at a rate of 0.5-2 μL/cm to on nitrocellulose membrane.
lateral flow chromatography detection method according to claim 4, is characterized in that, in step (4), described software analyzes the T line gray value under each bacterial concentration, fits and formulates standard curve; The T-line gray value, according to the standard curve, realizes the quantitative determination of the bacterial concentration in the sample.