Boronic acid modified gold nanoparticles, method for their macro-scale synthesis and use in immunochromatographic test strips

Abstract

The application provides a phenylboronic acid modified nano-gold, a macro-synthesis method thereof and application of the nano-gold in an immunochromatographic test strip. The synthesis method comprises the following steps: at room temperature, injecting an amino-phenylboronic acid solution into a chloroauric acid solution, and stirring to react, so as to obtain the phenylboronic acid modified nano-gold. The method does not need heating, the reaction time is only 10 minutes, and the single batch yield can reach 5 liters. The obtained nano-gold has a particle size of 20-40 nm, and the surface is rich in phenylboronic acid groups. The immunochromatographic test strip is prepared by using the specific boronic acid affinity interaction between the phenylboronic acid groups on the surface of the nano-gold and the cis- vicinal diol structure of the glycosylation region of the Fc fragment of an antibody, realizing the directional coupling of the Fc fragment of the antibody, preparing an immunolabeling probe, and using the probe to prepare the immunochromatographic test strip for the detection of a target. The application realizes the room temperature macro-preparation of the nano-gold, shortens the antibody coupling time to 5 minutes, has high detection sensitivity, is simple and convenient to operate, is low in cost, and is suitable for large-scale industrialization.

Description

Technical Field

[0001] This invention relates to the fields of nanomaterial preparation and immunoassay technology, and particularly to a phenylboronic acid-modified gold nanoparticle and its large-scale synthesis method at room temperature, as well as the preparation method and usage method of the immunochromatographic test strip based on the gold nanoparticle. Background Technology

[0002] Lateral flow immunoassay (LFIA) strips are widely used in clinical diagnostics, food safety monitoring, and other fields due to their ease of use, low cost, and intuitive results. Among these, LFIA strips using gold nanoparticles as the labeling material are the most common. Currently, the classic synthesis method for gold nanoparticles is the Turkevich method (reduction of chloroauric acid with trisodium citrate). This method requires reflux at high temperatures (approximately 100°C), which is not only energy-intensive and time-consuming but also demands high precision in heating equipment and control, significantly limiting its application in large-scale industrial production. Therefore, developing a method for the rapid, large-scale synthesis of gold nanoparticles at room temperature has significant industrial value.

[0003] Furthermore, the coupling of traditional citrate-reduced gold nanoparticles (Cit-AuNPs) with recognition molecules such as antibodies mainly relies on random physical interactions such as charge adsorption. This non-specific immobilization method results in antibodies exhibiting disordered conformations on the particle surface, such as "lying flat," "lying on their side," or "head down," which partially or completely shields the Fab fragments responsible for antigen recognition, increases steric hindrance, and reduces antigen binding efficiency, thus severely limiting further improvements in detection sensitivity. Summary of the Invention

[0004] This invention addresses the bottlenecks in existing technologies, such as the demanding synthesis conditions and difficulty in large-scale preparation of gold nanoparticles, as well as the randomness and insufficient sensitivity of antibody conjugation. It provides a room-temperature method for the large-scale synthesis of phenylboronic acid-modified gold nanoparticles, along with a method for preparing and using immunochromatographic test strips based on these gold nanoparticles. This method achieves rapid one-pot synthesis of phenylboronic acid-modified gold nanoparticles (PBA-AuNPs) at room temperature, with a single batch yield of up to 5 L. Furthermore, utilizing the phenylboronic acid groups on the surface of these gold nanoparticles, high-performance immunolabeled probes are prepared through boric acid affinity-mediated antibody-directed conjugation technology, which significantly improves detection sensitivity when applied to immunochromatographic test strips.

[0005] To achieve the above technical objectives, the present invention adopts the following technical solution: The first aspect of this invention provides a method for the large-scale synthesis of phenylboronic acid-modified gold nanoparticles, comprising the following steps: adding an aminophenylboronic acid solution to a chloroauric acid solution at room temperature, continuously stirring the reaction until the solution color turns a stable purplish-red, thereby obtaining phenylboronic acid-modified gold nanoparticles (denoted as PBA-AuNPs); wherein, the aminophenylboronic acid in the aminophenylboronic acid solution simultaneously acts as a reducing agent and a surface modifier; the reaction time is 5-20 minutes. The synthesis method provided by this invention can achieve a single-batch yield of 0.1 L to 5 L under laboratory conditions. Compared to traditional techniques that require reflux reactions at approximately 100°C, the reaction conditions of this invention are at room temperature, eliminating the need for additional high-temperature reactors and other specialized equipment, thus facilitating large-scale production and meeting industrial requirements.

[0006] Furthermore, the aminophenylboronic acid is one or more of 4-aminophenylboronic acid, 3-aminophenylboronic acid, and 2-aminophenylboronic acid.

[0007] Furthermore, the concentration of the aminophenylboronic acid solution is 0.5% to 2.0% (w / v), and the concentration of the chloroauric acid solution is 0.01% to 0.1% (w / v); the mass ratio of the aminophenylboronic acid to the chloroauric acid in the chloroauric acid solution is 5 to 200.

[0008] The second aspect of the present invention provides phenylboronic acid-modified gold nanoparticles prepared by the above-described method for the mass synthesis of phenylboronic acid-modified gold nanoparticles, wherein the surface of the phenylboronic acid-modified gold nanoparticles is modified with phenylboronic acid groups; the average particle size of the phenylboronic acid-modified gold nanoparticles is 20 nm to 40 nm, preferably 23 nm to 25 nm; and the local surface plasmon resonance absorption peak of the phenylboronic acid-modified gold nanoparticles, as determined by ultraviolet-visible absorption spectroscopy, is located at 510 nm to 540 nm.

[0009] A third aspect of the present invention provides an immunolabeling probe comprising the aforementioned phenylboronic acid-modified gold nanoparticles, and a first antibody molecule connected to the surface of the phenylboronic acid-modified gold nanoparticles via a boric acid affinity-mediated directional coupling method; the first antibody molecule comprises an Fc fragment and a Fab fragment disposed opposite to each other; the boric acid affinity-mediated directional coupling method is as follows: the phenylboronic acid groups on the surface of the phenylboronic acid-modified gold nanoparticles specifically covalently bind to the glycosylated cis-vicinal diol structure on the Fc fragment, thereby fixing the Fab fragment in a tail-to-tail configuration away from the surface of the phenylboronic acid-modified gold nanoparticles; the surface of the immunolabeling probe is further coated with a small molecule monosaccharide as a blocking layer.

[0010] Furthermore, the coupling of the first antibody molecule with the phenylboronic acid-modified gold nanoparticles is carried out at pH 8.0 to 9.0 for a coupling time of 3 to 10 minutes; preferably, the coupling time is 5 minutes; optionally, the small molecule monosaccharide is one or more of glucose, fructose, galactose, or mannose and their analogues.

[0011] A fourth aspect of this invention provides an immunochromatographic test strip kit, comprising the aforementioned immunolabeled probe and an immunochromatographic test strip; the immunochromatographic test strip includes a sample pad, a conjugation pad, a nitrocellulose (NC) membrane, and an absorbent pad; the nitrocellulose membrane is provided with a detection line (T line) and a control line (C line), the detection line is coated with a second antibody molecule, and the control line is coated with a universal capture molecule that specifically binds to the immunolabeled probe; wherein the immunolabeled probe is loaded on the conjugation pad or mixed with the sample to be tested before use. The universal capture molecule is a molecule that can capture the antibody.

[0012] Furthermore, the second antibody molecule is used to specifically bind to the target molecule and form a double antibody sandwich complex with the immunolabeled probe; optionally, the target molecule includes antigens, antibodies, proteins, hormones, viruses, or metabolites; the universal capture molecule is an IgG antibody, streptavidin, or protein A / G.

[0013] A fifth aspect of the present invention provides a method for using an immunochromatographic test strip kit, the method being applied to the immunochromatographic test strip kit as described above; comprising the following steps: (1) Pre-treat the sample to be tested to obtain the sample solution; (2) The sample solution to be tested is dropped onto the sample pad of the immunochromatographic test strip, and the solution flows through the conjugation pad, nitrocellulose membrane and absorption pad in sequence for chromatography by capillary action; (3) If the sample to be tested contains the target molecule, a second antibody molecule-target molecule-immunolabeled probe sandwich complex is formed at the detection line, and a universal capture molecule-immunolabeled probe complex is formed at the control line. Both lines show color signals generated by the accumulation of nano-gold modified with phenylboronic acid. (4) Use the naked eye or a reader to read the color signal intensity of the detection line and the control line, and according to the pre-established standard curve, realize the qualitative or quantitative detection of the concentration of the target molecule in the sample to be tested.

[0014] Furthermore, the sample to be tested in step (1) is any one or more of fecal extract, serum, plasma, whole blood, urine, saliva, cerebrospinal fluid, or secretions; the pretreatment is to dilute the sample to be tested with phosphate buffer; the chromatography time in step (2) is 5 to 15 minutes.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects: (1) Simple synthesis method and large-scale production: For the first time, a one-step rapid synthesis of phenylboronic acid-modified gold nanoparticles was achieved at room temperature without any heating equipment. The reaction time is only 10 minutes, and the yield per batch can reach 5 L, which has extremely high production efficiency and batch consistency, fully meeting the needs of industrialization and overcoming the shortcomings of the traditional Turkevich method, such as high temperature, high energy consumption and difficulty in scale-up.

[0016] (2) Highly efficient antibody-directed conjugation: The PBA-AuNPs synthesized in this invention have phenylboronic acid groups on their surface, eliminating the need for cumbersome post-modification. They can directly utilize the affinity of boric acid to achieve "tail-to-tail" directional conjugation of antibodies. The conjugation time is shortened from more than 60 minutes in the traditional method to 5 minutes, and the Fab fragment is fully exposed, resulting in a significant improvement in antigen capture efficiency.

[0017] (3) Significantly improved detection performance: When the immunolabeled probe prepared in this invention is applied to immunochromatographic test strips, the detection sensitivity is high, and it also has excellent specificity and accuracy.

[0018] (4) High versatility: The PBA-AuNPs preparation method and directional coupling strategy of the present invention are universal. By changing different antibody molecules, it can be used for high-sensitivity detection of a variety of targets, providing an efficient and low-cost universal platform for the field of point detection. Attached Figure Description

[0019] One or more embodiments are illustrated by way of example with reference to the accompanying drawings. These illustrations do not constitute a limitation on the embodiments, and unless otherwise stated, the figures in the drawings are not to be limited by scale.

[0020] Figure 1 This invention provides a method for the room-temperature large-scale synthesis of phenylboronic acid-modified gold nanoparticles and a method for preparing and using its immunochromatographic test strips. Figure 2 Transmission electron microscope image of phenylboronic acid-modified gold nanoparticles provided in an embodiment of the present invention; Figure 3 The standard curve of the phenylboronic acid-modified gold nanoparticle immunochromatographic test strip for detecting alpha-fetoprotein (AFP) in human serum samples provided in this embodiment of the invention; Figure 4 The standard curve of the phenylboronic acid-modified gold nanoparticle immunochromatographic test strip for detecting hemoglobin (Hb) in human fecal samples provided in this embodiment of the invention; Figure 5The standard curve for the immunochromatographic test strip of phenylboronic acid-modified gold nanoparticles used to detect human chorionic gonadotropin (HCG) in human urine samples is provided in an embodiment of the present invention. Detailed Implementation

[0021] The inventors discovered that phenylboronic acid (PBA) derivatives have attracted attention due to their ability to specifically covalently bind to the cis-vicinal diol structure of glycoproteins, addressing the problem of antibody-directed conjugation in the prior art. Since the glycosylation site of an antibody is uniquely located in its Fc fragment, PBA is considered an ideal linker molecule for achieving "tail-to-tail" (Fc fragment facing the particle, Fab fragment facing outward) antibody-directed conjugation. However, existing methods for preparing PBA-functionalized gold nanoparticles often involve post-modification of Cit-AuNPs, which suffers from cumbersome steps, long cycles, and uneven surface modification. Although there are reports of in-situ reduction synthesis of ultrasmall (<10 nm) gold nanoparticles using aminophenylboronic acid (APBA), the resulting particles exhibit extremely weak color signals, failing to meet the needs of clinical colorimetric diagnosis.

[0022] Based on this, the present invention aims to develop a method for preparing gold nanoparticles that can simultaneously achieve room temperature large-scale synthesis, high color intensity, and targeted conjugation of highly active antibodies, and apply it to LFIA, which is of great significance for improving the detection performance of target molecules in clinical, food, or environmental samples.

[0023] The present invention will now be described in detail with reference to specific embodiments.

[0024] In this invention, "room temperature" refers to a typical ambient temperature of 20°C to 30°C. "Large-scale preparation" refers to a single batch yield reaching a scale-up level (≥0.1 L). The "first antibody molecule" and "second antibody molecule" are paired antibodies capable of specifically binding to different epitopes of the same target molecule.

[0025] It should be clarified that, in this invention, the term "sample to be tested" refers to a mixture that may contain or is suspected of containing the target molecule. The types of samples applicable to this invention include bodily fluids (e.g., blood, which may include conventional blood samples after anticoagulation), plasma, serum, urine, semen, saliva, sweat, cell cultures, and tissue extracts. In addition, it may also include solvents, seawater, industrial wastewater, food samples, environmental samples (e.g., soil or water bodies), plant material, eukaryotic cells, bacteria, plasmids, viruses, fungi, and cells derived from prokaryotes. The above-mentioned samples to be tested may be diluted with a diluent before use, as appropriate. For example, to avoid the hook effect, the sample to be tested may be appropriately diluted with a diluent before instrument detection.

[0026] In this invention, the term "target molecule" refers to a substance contained in the sample to be tested during the detection process. The recognition molecule used to detect the target molecule must have the ability to specifically bind to it. The types of target molecules may include, but are not limited to: antigens, peptides, antibodies, amino acids, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), viruses, hormones, drugs, and metabolites.

[0027] Example 1 The target molecule is alpha-fetoprotein (AFP); the first antibody molecule is anti-AFP monoclonal antibody 1 (mouse-derived); the second antibody molecule is anti-AFP monoclonal antibody 2 (mouse-derived, recognizing different epitopes); the sample to be tested is human serum. The detection principle is as follows: PBA-AuNPs and anti-AFP monoclonal antibody 1 are directionally conjugated via boric acid affinity to prepare an immunolabeled probe. When the sample contains AFP, during chromatography on the test strip, AFP first binds to the PBA-AuNPs-anti-AFP antibody 1 probe on the binding pad, and is subsequently captured by anti-AFP monoclonal antibody 2 immobilized on the NC membrane detection line, forming an "antibody 2—AFP—antibody 1—PBA-AuNPs" sandwich complex. Gold nanoparticles accumulate on the T line to form a red band. Goat anti-mouse IgG immobilized on the C line can capture excess probes to form a red control band. The AFP concentration can be quantitatively detected by the T line signal intensity.

[0028] Specific implementation steps: 1. Large-scale preparation of phenylboronic acid-modified gold nanoparticles (PBA-AuNPs) at room temperature Referring to the preparation method described above, at room temperature (25°C), 135 mL of a 10 mg / mL 4-aminophenylboronic acid solution was rapidly injected into 5 L of a 1 mg / mL chloroauric acid solution, and the mixture was stirred at 500 rpm for 10 minutes. The solution color changed from light yellow to colorless and finally to a stable wine red. Figure 1 PBA-AuNPs were obtained. A small amount was subjected to UV-Vis spectroscopy, and the LSPR absorption peak was found to be at 536 nm; transmission electron microscopy showed that the average particle size was 23.5 nm. Figure 2 The PBA-AuNPs solution exhibits good monodispersity. The resulting PBA-AuNPs solution was stored at 4°C protected from light for later use.

[0029] 2. Preparation of immunolabeled probe (PBA-AuNPs—anti-AFP antibody 1) A boric acid affinity-mediated directional coupling method was employed. 1 mL of the PBA-AuNPs solution prepared in step 1 was centrifuged at 15000 rpm for 10 min, the supernatant was discarded, and the precipitate was reconstituted in 1 mL of ultrapure water. The pH was adjusted to 8.5 with 0.1 M K₂CO₃ solution. 5 μg of anti-AFP monoclonal antibody 1 was added, and the mixture was incubated with gentle shaking at room temperature for 5 min. Then, 100 μL of 1 mg / mL glucose solution was added, and incubation continued for 30 min to block the unreacted phenylboronic acid groups. Finally, the mixture was centrifuged at 15000 rpm for 10 min, and the precipitate was reconstituted in 200 μL of probe preservation buffer (0.01 M PBS containing 1% Tween-20, 1% BSA, and 0.1% ProClin 300, pH 7.4) to obtain the PBA-AuNPs-anti-AFP antibody 1 immunolabeled probe, which was stored at 4°C for later use.

[0030] 3. Assembly of immunochromatographic test strips (1) Coating the NC membrane: Anti-AFP monoclonal antibody 2 was diluted to 3.0 mg / mL with coating buffer (0.01 M PBS, pH 7.4, containing 5% sucrose and 1% NaCl), and sprayed onto the NC membrane at a rate of 1 μL / cm using a dip-coating gold sprayer to form a detection line (T line); goat anti-mouse IgG antibody was diluted to 1.0 mg / mL with the same buffer and sprayed onto the NC membrane to form a control line (C line), with a spacing of 5 mm between the two lines. The coated NC membrane was then dried overnight in a 37°C oven.

[0031] (2) Treatment of conjugate pads: The glass fiber conjugate pads were immersed in the pretreatment solution (20 mM sodium borate buffer, pH 8.0, containing 1% BSA, 0.25% Tween-20 and 0.1% ProClin 300) for 30 minutes, and then dried overnight in an oven at 37°C.

[0032] (3) Spraying with gold: The PBA-AuNPs-anti-AFP antibody 1 probe prepared in step 2 was diluted to 1.5 times the original concentration with probe dilution buffer (0.01 MPBS, pH 7.4, containing 5% sucrose, 1% BSA, 0.5% sodium caseinate, 1% PVP, 0.1% Tween-20), and uniformly sprayed onto the treated conjugate pad with a loading of 3 μL per test strip, and dried at 37°C for 2 hours.

[0033] (4) Assembly and strip cutting: The sample pad, conjugate pad (gold-plated), NC film coated with T-line and C-line, and absorbent pad are stacked sequentially on the PVC backing plate, with each component overlapping by 2 mm to ensure capillary flow continuity. The assembled plate is cut into 3.8 mm wide individual test strips using a strip cutter, and then placed in plastic cartridges, dried and sealed for storage.

[0034] 4. Plotting the standard curve for AFP detection AFP standards were diluted with sample diluent (0.01 M PBS) to different concentrations: 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.3 ng / mL, 15.6 ng / mL, 7.8 ng / mL, 3.9 ng / mL, 1.95 ng / mL, 0.98 ng / mL, 0.49 ng / mL, 0.24 ng / mL, 0.12 ng / mL, 0.06 ng / mL, and 0 ng / mL. 70 μL of each of these AFP standard solutions was vertically added to the sample wells of the test strip. After chromatography at room temperature for 10 minutes, the signal intensities of the T and C lines were read using a test strip reader, and the T / C value (the ratio of the detection line intensity to the control line intensity) was calculated. A standard curve was plotted with T / C values ​​on the ordinate and the logarithm of AFP concentration (ln(AFP concentration)) on the x-axis. The experimental results are as follows: Figure 3 As shown, the equation of the linear standard curve is y = 0.3261 ln(x) + 0.0415, R0 2 = 0.9917. The limit of detection of this method is defined as the average of the T / C values ​​obtained from detecting 20 samples with an AFP concentration of 0, plus three times the standard deviation. The calculated limit of detection is 0.08 ng / mL.

[0035] 5. Detect the AFP content in human serum samples. The steps for detecting AFP levels in human serum samples using the immunochromatographic test strip kit of this invention are as follows: (1) Sample pretreatment: Take the serum sample to be tested and dilute it 10 times with the above sample diluent (e.g., 10 μL serum + 90 μL diluent), and mix thoroughly.

[0036] (2) Detection: Take 70 μL of diluted serum sample and add it to the sample well of the test strip. Analyze at room temperature for 10 minutes.

[0037] (3) Result interpretation: Place the test strip into the reader, read the intensity of the T line and C line and calculate the T / C value. Substitute the obtained T / C value into the standard curve equation in step 4 to calculate the actual concentration of AFP in the serum sample.

[0038] Example 2 The target molecule is human hemoglobin (Hb); the first antibody molecule is anti-human hemoglobin monoclonal antibody 1; the second antibody molecule is anti-human hemoglobin monoclonal antibody 2 (mouse-derived, recognizing different epitopes); the labeling material is phenylboronic acid-modified gold nanoparticles synthesized in Example 1 of this invention; the sample to be tested is a human fecal sample.

[0039] Specific implementation steps: 1. Preparation of immunolabeled probe (PBA-AuNPs-anti-Hb antibody 1) A boric acid affinity-mediated directional coupling method was employed. 1 mL of the PBA-AuNPs solution prepared in Example 1 was centrifuged at 15000 rpm for 10 min, the supernatant was discarded, and the precipitate was reconstituted in 1 mL of ultrapure water. The pH was adjusted to 8.5 with 0.1 M K₂CO₃ solution. 5 μg of anti-human hemoglobin monoclonal antibody 1 (anti-Hb mAb1) was added, and the mixture was gently incubated with shaking at room temperature for 5 min. Then, 100 μL of 1 mg / mL glucose solution was added, and incubation continued for 30 min to block the unreacted phenylboronic acid groups. Finally, the mixture was centrifuged at 15000 rpm for 10 min, and the precipitate was reconstituted in 200 μL of probe preservation solution (0.01 M PBS containing 1% Tween-20, 1% BSA, and 0.1% ProClin 300, pH 7.4) to obtain the PBA-AuNPs-anti-Hb antibody 1 immunolabeled probe, which was stored at 4°C for later use.

[0040] 2. Assembly of immunochromatographic test strips (1) Coating the NC membrane: Anti-human hemoglobin monoclonal antibody 2 (anti-Hb mAb2) was diluted to 3.0 mg / mL with coating buffer (0.01 MPBS, pH 7.4, containing 5% sucrose and 1% NaCl), and sprayed onto the NC membrane at a rate of 1 μL / cm using a gold sprayer to form a detection line (T line); goat anti-mouse IgG antibody was diluted to 1.0 mg / mL with the same buffer and sprayed onto the NC membrane to form a control line (C line), with a spacing of 5 mm between the two lines. The coated NC membrane was placed in a 37°C oven to dry overnight.

[0041] (2) Treatment of conjugate pads: The glass fiber conjugate pads were immersed in the pretreatment solution (20 mM sodium borate buffer, pH 8.0, containing 1% BSA, 0.25% Tween-20 and 0.1% ProClin 300) for 30 minutes, and then dried overnight in an oven at 37°C.

[0042] (3) Spraying with gold: The PBA-AuNPs-anti-Hb antibody 1 probe prepared in step 2 was diluted to 1.5 times the original concentration with probe dilution buffer (0.01 M PBS, pH 7.4, containing 5% sucrose, 1% BSA, 0.5% sodium caseinate, 1% PVP, 0.1% Tween-20), and uniformly sprayed onto the treated conjugate pad with a loading of 3 μL per test strip, and dried at 37°C for 2 hours.

[0043] (4) Assembly and strip cutting: The sample pad, conjugate pad (gold-plated), NC film coated with T-line and C-line, and absorbent pad are stacked sequentially on the PVC backing plate, with each component overlapping by 2 mm to ensure capillary flow continuity. The assembled plate is cut into 3.8 mm wide individual test strips using a strip cutter, and then placed in plastic cartridges, dried and sealed for storage.

[0044] 3. Construction of standard curves for detecting fecal hemoglobin (Hb) and comparison of sensitivity. (1) Preparation of standard: The Hb standard was diluted with sample diluent (0.01 M PBS) to different concentrations: 1000 ng / mL, 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.3 ng / mL, 15.6 ng / mL, 7.8 ng / mL, 3.9 ng / mL, 1.95 ng / mL, 0.98 ng / mL, 0.49 ng / mL, 0.24 ng / mL, 0.12 ng / mL and 0 ng / mL.

[0045] (2) Detection and curve fitting: Take 70 μL of the above-mentioned Hb standard solutions of different concentrations and add them vertically to the sample wells of the PBA-AuNPs test strip and the control Cit-AuNPs test strip of the present invention. After chromatography at room temperature for 10 minutes, use a test strip reader to read the signal intensity of the T line and the C line, and calculate the T / C value (the ratio of the intensity of the detection line to the intensity of the control line). Plot a standard curve with the T / C value on the ordinate and the Hb concentration on the abscissa.

[0046] (3) Experimental results: The linear standard curve equation of the PBA-AuNPs test strip of the present invention is y = 0.0054x +0.0872, R 2 = 0.9968, linear range is 3.9~125 ng / mL ( Figure 4 The limit of detection was defined as the average of the T / C values ​​obtained from detecting 20 samples with a Hb concentration of 0, plus three times the standard deviation, which was calculated to be 1.95 ng / mL.

[0047] 4. Pretreatment methods for fecal samples Because feces are a semi-solid matrix, a two-step pre-dilution treatment is required to match the clinical positive threshold (100 ng / mL) and reduce matrix interference. The specific procedure is as follows: Take 10 mg of the fecal sample to be tested, add it to 1 mL of sample diluent, vortex thoroughly to prepare a homogenate, and let it stand for 30 seconds to allow large particles to settle; take 7 μL of the homogenate supernatant and mix it with 63 μL of sample diluent (1:10 secondary dilution) to obtain the final sample solution to be tested.

[0048] 5. Detection of Hb content in fecal samples and clinical validation Sample detection: Take 70 μL of the fecal sample solution processed in step 4 and add it to the sample well of the test strip of this invention. Perform chromatography at room temperature for 10 minutes. Read the T / C value using a reader, substitute it into the standard curve equation of step 4, and calculate the actual concentration of Hb in the fecal sample.

[0049] Example 3 The target molecule is human human chorionic gonadotropin (HCG); the first antibody molecule is anti-HCG monoclonal antibody 1; the second antibody molecule is anti-HCG monoclonal antibody 2 (mouse-derived, recognizing different epitopes); the labeling material is phenylboronic acid-modified gold nanoparticles synthesized in Example 1 of this invention; the sample to be tested is a human urine sample.

[0050] Specific implementation steps: 1. Preparation of immunolabeled probe (PBA-AuNPs-antiHCG antibody 1) A boric acid affinity-mediated directional coupling method was employed. 1 mL of the PBA-AuNPs solution prepared in Example 1 was centrifuged at 15000 rpm for 10 min, the supernatant was discarded, and the precipitate was reconstituted in 1 mL of ultrapure water. The pH was adjusted to 8.5 with 0.1 M K₂CO₃ solution. 5 μg of anti-HCG monoclonal antibody 1 (anti-Hb mAb1) was added, and the mixture was gently incubated with shaking at room temperature for 5 min. Then, 100 μL of 1 mg / mL glucose solution was added, and incubation continued for 30 min to block the unreacted phenylboronic acid groups. Finally, the mixture was centrifuged at 15000 rpm for 10 min, and the precipitate was reconstituted in 200 μL of probe preservation solution (0.01 M PBS containing 1% Tween-20, 1% BSA, and 0.1% ProClin 300, pH 7.4) to obtain the PBA-AuNPs-anti-HCG antibody 1 immunolabeled probe, which was stored at 4°C for later use.

[0051] 2. Assembly of immunochromatographic test strips (1) Coating the NC membrane: Anti-HCG monoclonal antibody 2 (anti-Hb mAb2) was diluted to 3.0 mg / mL with coating buffer (0.01 M PBS, pH 7.4, containing 5% sucrose and 1% NaCl), and sprayed onto the NC membrane at a rate of 1 μL / cm using a gold sprayer to form a detection line (T line); goat anti-mouse IgG antibody was diluted to 1.0 mg / mL with the same buffer and sprayed onto the NC membrane to form a control line (C line), with a spacing of 5 mm between the two lines. The coated NC membrane was placed in a 37°C oven to dry overnight.

[0052] (2) Treatment of conjugate pads: The glass fiber conjugate pads were immersed in the pretreatment solution (20 mM sodium borate buffer, pH 8.0, containing 1% BSA, 0.25% Tween-20 and 0.1% ProClin 300) for 30 minutes, and then dried overnight in an oven at 37°C.

[0053] (3) Spraying with gold: The PBA-AuNPs-antiHCG antibody 1 probe prepared in step 2 was diluted to 1.5 times the original concentration with probe dilution buffer (0.01 M PBS, pH 7.4, containing 5% sucrose, 1% BSA, 0.5% sodium caseinate, 1% PVP, 0.1% Tween-20), and uniformly sprayed onto the treated conjugate pad with a loading of 3 μL per test strip, and dried at 37°C for 2 hours.

[0054] (4) Assembly and strip cutting: The sample pad, conjugate pad (gold-plated), NC film coated with T-line and C-line, and absorbent pad are stacked sequentially on the PVC backing plate, with each component overlapping by 2 mm to ensure capillary flow continuity. The assembled plate is cut into 3.8 mm wide individual test strips using a strip cutter, and then placed in plastic cartridges, dried and sealed for storage.

[0055] 3. Construction of standard curves for detecting urine HCG and comparison of sensitivity. (1) Preparation of standard: The HCG standard was diluted with sample diluent (0.01 M PBS) to different concentrations: 1000 mIU / mL, 500 mIU / mL, 200 mIU / mL, 100 mIU / mL, 50 mIU / mL, 25 mIU / mL, 12.5 mIU / mL, 6.25 mIU / mL, 3.125 mIU / mL, 1.56 mIU / mL and 0 mIU / mL.

[0056] (2) Detection and curve fitting: Take 70 μL of the above-mentioned HCG standard solutions of different concentrations and add them vertically to the sample wells of the PBA-AuNPs test strip of the present invention. After chromatography at room temperature for 10 minutes, use a test strip reader to read the signal intensity of the T line and C line, and calculate the T / C value (the ratio of the intensity of the detection line to the intensity of the control line). Plot a standard curve with the T / C value on the ordinate and the HCG concentration on the abscissa.

[0057] (3) Experimental results: The linear standard curve equation of the PBA-AuNPs test strip of the present invention is y = 0.4018 ln(x) - 0.8033, R 2 = 0.9879 ( Figure 5The limit of detection for this method is defined as the average of the T / C values ​​obtained from detecting 20 samples with an HCG concentration of 0, plus three times the standard deviation. The calculated limit of detection is 3.13 mIU / mL.

[0058] 4. Urine sample pretreatment methods Urine samples can be tested directly without complex pretreatment, but simple processing is recommended to reduce interference from pH and salt content. The procedure is as follows: Collect fresh morning urine or random urine, centrifuge at 5000 rpm for 3 minutes to remove cell debris and precipitate, and dilute the supernatant by 2-fold with sample diluent (e.g., 50 μL urine supernatant + 50 μL diluent) to obtain the test sample solution. For samples with extremely high HCG concentrations (>1000 mIU / mL) (e.g., high concentrations in early pregnancy may cause the HOOK effect), the dilution factor can be appropriately increased (e.g., 5-fold or 10-fold).

[0059] 5. Detection of HCG levels in urine samples and clinical validation Sample detection: Take 70 μL of the urine sample solution processed in step 4 and add it to the sample well of the test strip of this invention. Perform chromatography at room temperature for 10 minutes. Read the T / C value using a reader, substitute it into the standard curve equation of step 4, and calculate the actual concentration of HCG in the urine sample.

[0060] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail can be made without departing from the spirit and scope of the present invention. Any person skilled in the art can make their own modifications and alterations without departing from the spirit and scope of the present invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A method for the large-scale synthesis of phenylboronic acid-modified gold nanoparticles, characterized in that, The process includes the following steps: At room temperature, an aminophenylboronic acid solution is added to a chloroauric acid solution, and the reaction is continuously stirred until the solution color turns a stable purple-red, thus obtaining phenylboronic acid-modified gold nanoparticles. In this process, the aminophenylboronic acid in the aminophenylboronic acid solution serves as both a reducing agent and a surface modifier; the reaction time is 5-20 minutes.

2. The method for large-scale synthesis of phenylboronic acid-modified gold nanoparticles according to claim 1, characterized in that, The aminophenylboronic acid is one or more of 4-aminophenylboronic acid, 3-aminophenylboronic acid, and 2-aminophenylboronic acid.

3. The synthesis method according to claim 1, characterized in that, The concentration of the aminophenylboronic acid solution is 5-20 mg / mL, and the concentration of the chloroauric acid solution is 1-10 mg / mL; the mass ratio of the aminophenylboronic acid to the chloroauric acid in the chloroauric acid solution is 5-200.

4. A phenylboronic acid-modified gold nanoparticle prepared by a large-scale synthesis method of phenylboronic acid-modified gold nanoparticles according to any one of claims 1 to 3, characterized in that, The surface of the phenylboronic acid-modified gold nanoparticles is modified with phenylboronic acid groups; The average particle size of the phenylboronic acid-modified gold nanoparticles is 20 nm to 40 nm, preferably 23 nm to 25 nm; The localized surface plasmon resonance absorption peak of the phenylboronic acid-modified gold nanoparticles, as determined by UV-Vis absorption spectroscopy, is located between 510 nm and 540 nm.

5. An immunolabeled probe, characterized in that, The invention includes the phenylboronic acid-modified gold nanoparticles as described in claim 4, and a first antibody molecule connected to the surface of the phenylboronic acid-modified gold nanoparticles via a directional coupling mediated by boric acid affinity; the first antibody molecule includes an Fc fragment and a Fab fragment arranged opposite to each other. The boric acid affinity-mediated directional coupling method is as follows: the phenylboronic acid groups on the surface of the phenylboronic acid-modified gold nanoparticles are specifically covalently bound to the glycosylated cis-vicinal diol structure on the Fc fragment, so that the Fab fragment is fixed in a tail-to-tail configuration away from the surface of the phenylboronic acid-modified gold nanoparticles. The surface of the immunolabeled probe is also coated with a small molecule monosaccharide as a blocking layer.

6. The immunolabeled probe according to claim 5, characterized in that, The first antibody molecule was coupled to the phenylboronic acid-modified gold nanoparticles at a pH of 8.0 to 9.0 for 3 to 10 minutes; preferably, the coupling time was 5 minutes. Optionally, the small molecule monosaccharide is one or more of glucose, fructose, galactose, or mannose and their analogues.

7. An immunochromatographic test strip kit, characterized in that, Includes the immunolabeled probe as described in claim 5, and the immunochromatographic test strip; The immunochromatographic test strip includes a sample pad, a conjugation pad, a nitrocellulose (NC) membrane, and an absorbent pad; the nitrocellulose membrane is provided with a detection line (T line) and a control line (C line), the detection line is coated with a second antibody molecule, and the control line is coated with a universal capture molecule that specifically binds to the immunolabeled probe; The immunolabeled probe is loaded onto the binding pad or mixed with the sample to be tested before use.

8. The immunochromatographic test strip kit according to claim 7, characterized in that, The second antibody molecule is used to specifically bind to the target molecule and form a double antibody sandwich complex with the immunolabeled probe; optionally, the target molecule includes antigens, antibodies, proteins, hormones, viruses, or metabolites. The universal capture molecule is an IgG antibody, streptavidin, or protein A / G.

9. A method of using an immunochromatographic test strip kit, characterized in that, The method is applied to the immunochromatographic test strip kit as described in any one of claims 8 to 10; and includes the following steps: (1) Pre-treat the sample to be tested to obtain the sample solution; (2) The sample solution to be tested is dropped onto the sample pad of the immunochromatographic test strip, and the solution flows through the conjugation pad, nitrocellulose membrane and absorption pad in sequence for chromatography by capillary action; (3) If the sample to be tested contains the target molecule, a second antibody molecule-target molecule-immunolabeled probe sandwich complex is formed at the detection line, and a universal capture molecule-immunolabeled probe complex is formed at the control line. Both lines show color signals generated by the accumulation of nano-gold modified with phenylboronic acid. (4) Use the naked eye or a reader to read the color signal intensity of the detection line and the control line, and according to the pre-established standard curve, realize the qualitative or quantitative detection of the concentration of the target molecule in the sample to be tested.

10. The method of use according to claim 9, characterized in that, The sample to be tested in step (1) is any one or more of the following: fecal extract, serum, plasma, whole blood, urine, saliva, cerebrospinal fluid, or secretions. The pretreatment involves diluting the sample to be tested using phosphate buffer. The chromatography time in step (2) is 5 to 15 minutes.

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