A sandwich method-based polystyrene microplastics rapid detection method and kit

Abstract

This invention belongs to the field of environmental monitoring and analytical chemistry technology, and provides a rapid detection method and kit for polystyrene microplastics based on a sandwich method. It includes: a capture component, a magnetic bead-peptide conjugate with a specific peptide covalently coupled to it; and a detection component, a luminescent detection probe formed by covalently coupling bovine serum albumin with a chemiluminescent label and the specific peptide; the specific peptide has an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. The peptide of this invention possesses extremely strong specific binding ability, which can reduce cross-reactions by precisely matching the microplastic target structure, thereby improving detection specificity from the root cause; the use of magnetic beads to load the peptide, with the ultra-large specific surface area of ​​the magnetic beads, can significantly improve the binding efficiency between the peptide and the microplastic; combined with the luminescent group or fluorescent group coupled to the peptide, no additional enzyme-labeled secondary antibody incubation and enzyme-catalyzed colorimetric reaction are required.

Description

Technical Field

[0001] This invention belongs to the field of environmental detection and analytical chemistry technology, and particularly relates to a rapid detection method and kit for polystyrene microplastics based on the sandwich method. Background Technology

[0002] Common types of microplastics include polyethylene (PE), polypropylene (PP), polystyrene (PS), and polyvinyl chloride (PVC). As typical pollutants, they exhibit strong environmental mobility and bioaccumulation, and are widely present in environmental media such as marine water and freshwater, as well as biological samples such as blood, breast milk, organs, and feces. This poses a potential and significant threat to ecosystem homeostasis and the human immune, reproductive, and metabolic systems. Health risk assessment of microplastics has become a research focus in the interdisciplinary field of medicine and environmental science. Therefore, accurate and efficient detection of microplastics in environmental and biological samples is not only crucial for environmental monitoring and pollution control, but also essential for rapid screening and quantitative analysis under clinical conditions. This is a key problem urgently needing to be solved in the fields of environmental health and medical testing.

[0003] Current microplastic detection methods generally suffer from key problems such as insufficient sensitivity, poor specificity, high detection costs, and cumbersome operation procedures. Specific pain points are as follows: 1. Traditional detection techniques (such as microscopic observation and spectral analysis) have high detection limits, making it difficult to capture low concentrations of microplastics, let alone effectively identify nanoscale microplastics or microplastics in biological samples, resulting in a high rate of missed detection for trace contamination; 2. Although immunoassay uses antibodies as recognition elements, antibody preparation is complex, and antibodies have significant steric hindrance. Various proteins in biological samples have strong non-specific adsorption capabilities for microplastics (such as polystyrene), preventing antibodies from reaching unblocked areas and making them unsuitable for complex detection scenarios. Furthermore, antibodies are easily affected by pH changes and protease degradation in complex biological matrices, resulting in poor stability; 3. Existing detection procedures require multiple sample pretreatment steps and rely on high-precision, expensive instruments, resulting in low automation and prolonged detection cycles; 4. Most detection methods lack specificity and are easily interfered with by other coexisting particulate matter in the environment (such as dust and fibers), leading to biased detection results and affecting data reliability. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a rapid detection method and kit for polystyrene microplastics based on the sandwich method, aiming to solve the key problems mentioned in the background art.

[0005] In a first aspect, the present invention provides a rapid detection kit for polystyrene microplastics based on a sandwich method, comprising:

[0006] The capture component is a magnetic bead-peptide conjugate with a specific peptide immobilized by covalent coupling;

[0007] The detection component comprises a luminescent detection probe formed by covalently coupling bovine serum albumin with a chemiluminescent label and the specific polypeptide;

[0008] The specific polypeptide has an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5;

[0009] The specific polypeptides contained in the capture component and the detection component have the same amino acid sequence.

[0010] Further, the preparation method of the magnetic bead-peptide conjugate is as follows: 3 mg of iodinated acetylated silica coated magnetic beads are taken, resuspended in 10 mM phosphate buffer at pH 7.4, 60 μg of the specific peptide is added, activated at 37 °C, and blocked with 10 μL of mercaptoethanol. Finally, 180 μL of 10% bovine serum albumin solution is added, and blocked at 37 °C to obtain the magnetic bead-peptide conjugate.

[0011] Furthermore, the specific method for preparing the luminescent detection probe is as follows:

[0012] Step 1: Dissolve 2 mg of bovine serum albumin in 1 mL of 0.1 M sodium bicarbonate solution with pH 8.6, vortex mix, centrifuge and sonicate, then filter to obtain a clear bovine serum albumin solution.

[0013] Step 2: Add 10 molar amounts of acridine ester to the bovine serum albumin solution and react at room temperature in the dark to allow the acridine ester to initially couple with the bovine serum albumin.

[0014] Step 3: Add 10 μL of 10% glycine stop solution, react at room temperature, desalt using a gravity column, replace the solution with 10 mM triethanolamine at pH 7.4, and centrifuge to wash the column; then centrifuge for 3 minutes to collect the bovine serum albumin-acridone ester conjugate.

[0015] Step 4: Take bovine serum albumin-acrididine ester conjugate, add SMCC to make the molar ratio of SMCC to bovine serum albumin-acrididine ester conjugate 12:1, react at 37℃ to activate bovine serum albumin-acrididine ester conjugate and SMCC, and then desalt.

[0016] Step 5: Collect the desalted bovine serum albumin-acridone ester conjugate in a tube, add 5 μL of 10% Brij23, and mix well; separately dissolve the specific polypeptide in 100 μL of dimethyl sulfoxide, and add 46.1 μL of the specific polypeptide solution to the tube. The molar ratio of the specific polypeptide to the bovine serum albumin-acridone ester conjugate is 8:1. After mixing well, react at 4°C to obtain the reaction solution.

[0017] Step 6: Desalt and purify the reaction solution using PBST buffer, which is a phosphate buffer containing 0.05% Tween-20.

[0018] Furthermore, an application of a sandwich-based rapid detection kit for polystyrene microplastics in the detection of polystyrene microplastics, wherein the kit rapidly detects the concentration of polystyrene microplastics.

[0019] Secondly, the present invention provides a rapid detection method for polystyrene microplastics based on a sandwich method, wherein the rapid luminescence detection of polystyrene microplastics is performed using the sandwich method based on the sandwich method via homologous peptides.

[0020] The rapid luminescence detection of polystyrene microplastics using the homologous peptide sandwich method specifically involves: mixing the magnetic bead-peptide conjugate, the luminescence detection probe, the polystyrene microplastics, and the analytical buffer solution; washing after the reaction, adding 0.1% hydrogen peroxide solution, and then luminescence under excitation by 0.25 mol / L sodium hydroxide solution; detecting the generated light signal using a chemiluminescence analyzer; and calculating the concentration of polystyrene microplastics based on the light signal.

[0021] Thirdly, the present invention provides a magnetic bead-peptide conjugate with a specific polypeptide immobilized by covalent coupling, wherein the specific polypeptide has an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

[0022] Fourthly, the present invention provides a luminescent detection probe formed by covalently coupling bovine serum albumin with a chemiluminescent label and a specific polypeptide, wherein the specific polypeptide has an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

[0023] Fifthly, the present invention provides a polystyrene microplastic test strip or test card, the test strip or test card comprising a fluorescent detection probe formed of a biotinylated covalently modified specific polypeptide, the fluorescent detection probe working in conjunction with streptavidin-phycoerythrin, the specific polypeptide having an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

[0024] Furthermore, the preparation method of the fluorescent detection probe is specifically as follows:

[0025] Step 1: Dissolve 0.1 mg of peptide in 100 μL of phosphate buffer, add 0.15 mg of maleimide biotin, mix gently, and react overnight at 4 °C to achieve biotinylation modification of the peptide and obtain peptide-biotin conjugate solution.

[0026] Step 2: Transfer the peptide-biotin conjugate solution to a dialysis bag and dialyze overnight at 4°C using PBST buffer, changing the PBST buffer 2-3 times during the process to remove unreacted biotin and small molecule impurities.

[0027] Furthermore, the method of using the test strip or test card is as follows: the fluorescent detection probe, polystyrene microplastic, streptavidin magnetic microspheres and streptavidin-phycoerythrin are mixed and reacted, washed after the reaction is completed, and dropped onto a nitrocellulose membrane for observation using a fluorescence microscope.

[0028] The present invention has the following technical effects:

[0029] (1) Using specific peptides instead of monoclonal antibodies as recognition elements has the following core advantages: peptides have strong specific binding ability, which can reduce cross-reaction by accurately matching the target structure of microplastics and improve the detection specificity from the root of recognition; at the same time, as a small molecule chemical entity, peptides have a chemical structure stability that is far superior to that of biological macromolecules such as antibodies and are not affected by the secondary structure of proteins; and peptides do not need to rely on complex biological culture systems to obtain, and the cost is significantly lower than that of antibodies.

[0030] (2) In terms of reaction system design, a magnetic bead-loaded peptide mode is adopted. The ultra-large specific surface area of ​​the magnetic beads can significantly improve the binding efficiency of peptides and microplastics, and the magnetic separation characteristics can quickly and directionally remove unbound impurities, directly eliminating the multiple washing steps in the existing technology; coupled with the luminescent group or the fluorescent group coupled to the peptide, there is no need for additional enzyme labeling secondary antibody incubation and enzyme-catalyzed colorimetric reaction. Through the simplification of technical steps, the operational efficiency is improved. Furthermore, by using a single biotinylated peptide, combined with commercially available streptavidin magnetic beads and streptavidin phycoerythrin, the purpose of magnetic separation concentration and fluorescent specific labeling can be directly achieved in one step, reducing detection time and improving detection efficiency. Attached Figure Description

[0031] Exemplary embodiments of the present invention can be more fully understood by referring to the following figures:

[0032] Figure 1 This is a graph showing the results of ELISA testing on the binding ability of the bacteriophage clone of Example 1 of the present invention to a polystyrene enzyme-labeled plate.

[0033] Figure 2 This is a graph showing the detection specificity analysis results of the polypeptide (SEQ ID NO:1)-acrididine ester conjugate in Example 2 of the present invention;

[0034] Figure 3 This is the fluorescence characterization result of Example 5 of the present invention;

[0035] Figure 4 This is the dose-response curve of the polypeptide with the amino acid sequence SEQ ID NO:1 detected by 2.2 μm polystyrene microspheres in Example 6 of the present invention;

[0036] Figure 5 This is the dose-response curve of a polypeptide with the amino acid sequence SEQ ID NO:1 detected by 0.5 μm polystyrene microspheres in Example 6 of the present invention. Detailed Implementation

[0037] To make the technical problems, solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0039] This invention provides a rapid detection kit for polystyrene microplastics based on a sandwich method, comprising:

[0040] The capture component is a magnetic bead-peptide conjugate with a specific peptide immobilized by covalent coupling;

[0041] The detection component consists of a luminescent detection probe formed by covalently coupling bovine serum albumin with a chemiluminescent label and a specific polypeptide.

[0042] The specific polypeptide has an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5;

[0043] The specific peptides contained in the capture and detection components have the same amino acid sequence.

[0044] In some embodiments, the preparation method of the magnetic bead-peptide conjugate is as follows: 3 mg of iodinated silica coated magnetic beads are taken, resuspended in 10 mM phosphate buffer at pH 7.4, 60 μg of specific peptide is added, activated at 37 °C, and blocked with 10 μL of mercaptoethanol. Finally, 180 μL of 10% bovine serum albumin solution is added and blocked at 37 °C to obtain the magnetic bead-peptide conjugate.

[0045] In some embodiments, the method for preparing the luminescent detection probe is as follows:

[0046] Step 1: Dissolve 2 mg of bovine serum albumin in 1 mL of 0.1 M sodium bicarbonate solution with pH 8.6, vortex mix, centrifuge and sonicate, then filter to obtain a clear bovine serum albumin solution.

[0047] Step 2: Add 10 molar amounts of acridine ester to the bovine serum albumin solution and react at room temperature in the dark to allow the acridine ester to initially couple with the bovine serum albumin.

[0048] Step 3: Add 10 μL of 10% glycine stop solution, react at room temperature, desalt using a gravity column, replace the solution with 10 mM triethanolamine at pH 7.4, and centrifuge to wash the column; then centrifuge for 3 minutes to collect the bovine serum albumin-acridone ester conjugate.

[0049] Step 4: Take bovine serum albumin-acrididine ester conjugate, add SMCC to make the molar ratio of SMCC to bovine serum albumin-acrididine ester conjugate 12:1, react at 37℃ to activate bovine serum albumin-acrididine ester conjugate and SMCC, and then desalt.

[0050] Step 5: Collect the desalted bovine serum albumin-acridone ester conjugate in a tube, add 5 μL of 10% Brij23, and mix well; separately dissolve the specific peptide in 100 μL of dimethyl sulfoxide, and add 46.1 μL of the specific peptide solution to the tube. The molar ratio of the specific peptide to the bovine serum albumin-acridone ester conjugate is 8:1. After mixing well, react at 4°C to obtain the reaction solution.

[0051] Step 6: Desalt and purify the reaction solution using PBST buffer, which is a phosphate buffer containing 0.05% Tween-20.

[0052] In some embodiments, a sandwich-based rapid detection kit for polystyrene microplastics is used to detect polystyrene microplastics, and the kit rapidly detects the concentration of polystyrene microplastics.

[0053] In some embodiments, the present invention provides a rapid detection method for polystyrene microplastics based on a sandwich method, using a rapid detection kit for polystyrene microplastics based on a sandwich method, and performing rapid luminescence detection of polystyrene microplastics by homologous peptide sandwich method;

[0054] As can be understood, the homologous peptide sandwich method refers to using the same specific peptide, one part of which is covalently coupled to a solid support as a capture unit, and the other part is covalently coupled to a signal molecule as a detection unit, to achieve sandwich detection of the target analyte.

[0055] In some embodiments, the rapid luminescence detection of polystyrene microplastics using the homologous peptide sandwich method specifically involves: mixing and reacting magnetic bead-peptide conjugates, luminescent detection probes, polystyrene microplastics, and analytical buffer; washing after the reaction is complete, adding 0.1% hydrogen peroxide solution, and then luminescence under excitation by 0.25 mol / L sodium hydroxide solution; detecting the generated light signal using a chemiluminescence analyzer; and calculating the concentration of polystyrene microplastics based on the light signal.

[0056] In some embodiments, the present invention provides a magnetic bead-peptide conjugate with a specific polypeptide immobilized by covalent coupling, the specific polypeptide having an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

[0057] In some embodiments, the present invention provides a luminescent detection probe formed by covalently coupling bovine serum albumin with a chemiluminescent label and a specific polypeptide, the specific polypeptide having an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

[0058] In some embodiments, the present invention provides a polystyrene microplastic test strip or test card, the test strip or test card comprising a fluorescent detection probe formed of a biotinylated covalently modified specific polypeptide, the fluorescent detection probe working in conjunction with streptavidin-phycoerythrin, the specific polypeptide having an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

[0059] In some embodiments, the preparation method of the fluorescent detection probe is specifically as follows:

[0060] Step 1: Dissolve 0.1 mg of peptide in 100 μL of phosphate buffer, add 0.15 mg of maleimide biotin, mix gently, and react overnight at 4 °C to achieve biotinylation modification of the peptide and obtain peptide-biotin conjugate solution.

[0061] Step 2: Transfer the peptide-biotin conjugate solution to a dialysis bag and dialyze overnight at 4°C using PBST buffer, changing the PBST buffer 2-3 times during the process to remove unreacted biotin and small molecule impurities.

[0062] In some embodiments, the method of using the test strip or test card is as follows: a fluorescent detection probe, polystyrene microplastics, streptavidin magnetic microspheres and streptavidin-phycoerythrin are mixed and reacted, washed after the reaction is completed, and dropped onto a nitrocellulose membrane for observation using a fluorescence microscope.

[0063] Example 1:

[0064] The target peptides that can specifically recognize polystyrene (PS) were obtained by screening using phage display technology. The specific steps are as follows:

[0065] (1) Solid phase coating: The highly adsorbed polystyrene ELISA plate is used as the screening target to form a solid phase screening interface for affinity screening with random peptide libraries.

[0066] (2) Blocking: The coated wells were blocked with phosphate buffer containing 1% bovine serum albumin to eliminate non-specific binding sites.

[0067] (3) Incubation and panning of phage libraries: The NEB random 12-peptide library was incubated at 1x10⁻¹⁰. 9 The phages were added into the wells coated with polystyrene microspheres and incubated at room temperature for 1 hour, which enabled the polystyrene-specific phages to be captured.

[0068] (4) Washing: Discard unbound phages and wash with PBST buffer (phosphate buffer containing 0.1% Tween-20, v / v). Perform three rounds of panning, increasing the Tween-20 content in the PBST buffer in each round (from 0.1% to 0.5%). Replace the blocking protein in each round with bovine serum albumin, human serum albumin, and whey protein to apply selective pressure and eliminate non-specific interference from proteins, thereby gradually enriching phage clones with high specificity, high affinity, and high stability to the surface of polystyrene microspheres.

[0069] (5) Elution and amplification: Specifically bound phages were eluted with glycine-hydrochloric acid buffer (pH=2.2) and neutralized with Tris-HCl buffer (pH=9.0). The eluted phages were then used to infect Escherichia coli host bacteria for amplification. The amplified phages were used for the next round of screening.

[0070] (6) Clone identification and sequencing: After three rounds of panning, phage clones from the final round were randomly selected, and their binding activity with polystyrene enzyme labels blocked by different proteins was preliminarily verified by ELISA, such as... Figure 1 As shown, taking a single positive phage clone as an example, regardless of whether whey protein or bovine serum albumin was used to block polystyrene ELISA plates, the phage clone ELISA results all showed that the screened single positive phage clones had extremely strong affinity for polystyrene. DNA sequencing was performed on the single positive phage clones with high binding signals, and the displayed polypeptide amino acid sequence was obtained after translation.

[0071] The amino acid sequences of five peptides with excellent binding ability to polystyrene were finally determined, namely MFWPFYPSKSNHGGGS (SEQ ID NO:1), WIINPRATVYALGGGS (SEQ ID NO:2), WDFWPYSWEARGGGS (SEQ ID NO:3), MFWWPYPSKYALGGGS (SEQ ID NO:4), and APLTGSSWETHGGGS (SEQ ID NO:5).

[0072] (7) Chemical synthesis and modification of polypeptides: For detection purposes, polypeptides with amino acid sequences as shown in SEQ ID NO:1-SEQ ID NO:5 were chemically synthesized by solid-phase synthesis.

[0073] Example 2:

[0074] The specific experimental steps for identifying the specificity and affinity of peptides with polystyrene are as follows:

[0075] (1) Solution preparation: Horseradish peroxidase (HRP) working solution containing 1% (w / v) skim milk powder was used as the analysis buffer; a peptide-acridin ester (AE) conjugate working solution was prepared to a final concentration of 50 μg / mL; a polystyrene (PS) microsphere suspension with a concentration of 2.5 mg / mL was prepared; a silica-coated magnetic microsphere (Fe3O4@SiO2) particle suspension (concentration 1.429 mg / mL) was taken.

[0076] (2) Sample addition and incubation reaction: The absorbance of the prepared 2.5 mg / mL polystyrene microsphere suspension and Fe3O4@SiO2 suspension were measured, and the absorbance ratio between the two was calculated. Based on this ratio, the required dilution factor of Fe3O4@SiO2 suspension was calculated to ensure that the number of effective adsorption sites on the surfaces of the two was approximately equal. 50 μL of analytical buffer (blank group), 50 μL of polystyrene microsphere suspension (polystyrene microsphere competition group), and 50 μL of diluted Fe3O4@SiO2 particle suspension (magnetic microsphere group) were added to each well of the polystyrene microplate, respectively; then 100 μL of peptide-acrididine ester conjugate working solution was added to each well; after sealing the polystyrene microplate, it was placed in a constant temperature shaker or incubator at 37℃ and reacted at a suitable speed for 1 hour to allow the peptide to fully bind to the solid phase surface.

[0077] (3) Washing: After the reaction is complete, discard the reaction solution in each well of the microplate. Add 300 μL of PBST washing buffer (containing 0.05% Tween-20 phosphate buffer, v / v) to each well, let stand for a while, then discard the PBST washing buffer. Repeat this washing process 7 to 8 times to completely remove unbound peptide-acridone ester conjugates.

[0078] (4) Detection: After cleaning, 0.1% hydrogen peroxide solution was added to each well, and then luminescence was emitted under the excitation of 0.25 mol / L sodium hydroxide solution; the signal values ​​of five different sequence peptides binding to polystyrene are shown in Table 1.

[0079] Table 1. Signal values ​​of five different sequence peptides binding to polystyrene.

[0080]

[0081] The detection specificity analysis results of the polypeptide (SEQ ID NO:1)-acrididine ester conjugate are as follows: Figure 2 As shown, this experiment aims to evaluate the specific binding ability of five different peptide sequences to polystyrene materials. Using polystyrene ELISA plates as solid substrates, the binding level of the peptides to the substrates was characterized by the luminescence signal intensity of the peptide (SEQ ID NO:1)-acrididine ester conjugate. At the same time, polystyrene microspheres were introduced as competitive binders to verify the targeted binding characteristics of the peptides to polystyrene materials.

[0082] The results showed that the signal values ​​of the peptides with amino acid sequences SEQ ID NO:1-SEQ ID NO:5 reacting with Fe3O4@SiO2 microspheres were at the same level as those in the polystyrene background wells, both exhibiting high signal values. However, after competitive elution with polystyrene microspheres, the signal values ​​decreased significantly. This indicates that the peptides hardly bind to Fe3O4@SiO2 microspheres, suggesting that this could be used as a separation and enrichment system. The significant signal value of the peptides binding to the polystyrene microplate, influenced by competitive elution with polystyrene microspheres, indicates that the peptides strongly and specifically bind to the polystyrene surface.

[0083] Example 3:

[0084] The luminescent detection probe, formed by covalently conjugating bovine serum albumin with a chemiluminescent label and a specific polypeptide, is developed using the following steps:

[0085] (1) Pretreatment of bovine serum albumin: Dissolve 2 mg of bovine serum albumin in 1 mL of 0.1 M sodium bicarbonate solution with pH=8.6, vortex mix, and after brief centrifugation and sonication for 3-5 minutes, filter through a filter membrane to obtain a clear bovine serum albumin solution.

[0086] (2) Acridinium ester coupling reaction: Add 10 molar amounts of acridinium ester to the bovine serum albumin solution and react at room temperature in the dark for 30 minutes to allow acridinium ester to be initially coupled with bovine serum albumin.

[0087] (3) Termination of reaction and desalting: Add 10 μL of 10% glycine stop solution and react at room temperature for 30 minutes. Desalting was performed using a gravity column. The solution was replaced with 10 mM triethanolamine at pH 7.4. The column was washed 5 times at 1000 g centrifugation for 2 minutes each time. Then the sample was centrifuged at 1000 g for 3 minutes and the bovine serum albumin-acridone ester conjugate was collected.

[0088] (4) SMCC crosslinking reaction: Take bovine serum albumin-acridone ester conjugate, add SMCC crosslinking agent, so that the molar ratio of SMCC to bovine serum albumin-acridone ester conjugate is 12:1, react at 37℃ for 30 minutes to realize the activation of bovine serum albumin-acridone ester conjugate and SMCC; desalt to remove unreacted SMCC.

[0089] (5) Peptide coupling reaction: The desalted bovine serum albumin-acridone ester conjugate was collected in a 5 mL tube, and 5 μL of 10% Brij23 was added and vortexed to mix. In addition, 100 μL of dimethyl sulfoxide was used to dissolve the peptides with amino acid sequences of SEQ ID NO:1-SEQ ID NO:5. 46.1 μL of each peptide (SEQ ID NO:1-SEQ ID NO:5) solution was added to the tube. The molar ratio of peptide to bovine serum albumin-acridone ester conjugate was 8:1. After mixing evenly, the reaction was carried out overnight at 4°C to obtain the reaction solution, thus realizing the coupling of peptide with bovine serum albumin-acridone ester.

[0090] (6) Post-processing and purification: The reaction solution was desalted and purified using PBST buffer (phosphate buffer containing 0.05% Tween-20) to obtain the peptide-bovine serum albumin-acridone ester conjugate.

[0091] Example 4:

[0092] The specific steps for immobilizing magnetic beads-peptide conjugates with specific peptides via covalent coupling are as follows:

[0093] (1) Activation of magnetic beads: Take 3 mg of iodine acetylated silica coated magnetic beads, resuspend them in 10 mM phosphate buffer at pH=7.4, add 60 μg of polypeptide with amino acid sequences of SEQ ID NO:1-SEQ ID NO:5, activate at 37℃ for 30 minutes, block with 10 μL mercaptoethanol, and finally add 180 μL of 10% (w / v) bovine serum albumin solution to the system and block at 37℃ for 1 hour to block the remaining reaction sites on the surface of the magnetic beads and reduce non-specific adsorption, thus obtaining magnetic bead-peptide conjugate;

[0094] Among them, silica-coated magnetic beads refer to core-shell structured magnetic microspheres with iron oxide (Fe3O4) as the core and silica (SiO2) as the outer layer. This structure not only maintains superparamagnetism, but also provides a surface rich in silanol groups (-SiOH) with good biocompatibility, which is convenient for silanization modification, thereby introducing active functional groups such as carboxyl groups.

[0095] Iodoacetylated silica-coated magnetic beads: Iodoacetylated groups are covalently modified onto the surface of silica magnetic microspheres using silanizing agents, achieving mild and efficient coupling with thiolized peptides.

[0096] (2) Preparation of reserve suspension: After sealing, remove the supernatant and resuspend the obtained magnetic bead-peptide conjugate in the final buffer (1×PBS, pH=7.4; 0.5% Brij23; 1.5% BSA) to a final volume of 30 mL to form a homogeneous reserve suspension of magnetic bead-peptide conjugate; the reserve suspension can be stably stored at 4°C for several months.

[0097] Example 5:

[0098] The specific steps of the biotinylated peptide-mediated fluorescent labeling experiment for testing strips are as follows:

[0099] (1) Coupling of peptide with biotin: 0.1 mg of peptide (SEQ ID NO:1) was dissolved in 100 μL of phosphate buffer, 0.15 mg of maleimide biotin was added, and after gentle mixing, the mixture was reacted overnight at 4 °C to obtain a peptide-biotin conjugate (pep-Biotin) solution, thereby achieving biotinylation modification of the peptide.

[0100] (2) Dialysis purification: Transfer the peptide-biotin conjugate solution to a dialysis bag and dialyze overnight at 4°C using PBST buffer. Change the PBST buffer 2-3 times during the process to remove unreacted biotin and small molecule impurities.

[0101] (3) Concentration determination: Take the dialysis peptide-biotin conjugate solution and measure the OD280 value using a UV spectrophotometer; calculate its concentration based on the extinction coefficient of the peptide and dilute it to an appropriate working concentration using phosphate buffer.

[0102] Among them, OD280 value: refers to the absorbance value of the sample at a wavelength of 280nm. Since amino acids such as tryptophan and tyrosine contained in peptides have characteristic absorption at 280nm, the peptide concentration can be calculated by combining this value with the extinction coefficient.

[0103] The extinction coefficient of a peptide refers to the absorbance of a peptide at a specific wavelength (280 nm in this case) under unit concentration and unit optical path length. It is a key parameter for calculating peptide concentration by ultraviolet spectrophotometry.

[0104] (4) Preparation of SA-PE (streptavidin-phycoerythrin) working solution: Dilute SA-PE to a concentration of 1 μg / mL using PBST buffer (phosphate buffer containing 0.05% Tween-20), and prepare a volume of 1 mL for later use;

[0105] SA-PE refers to the conjugate of streptavidin (SA) and phycoerythrin (PE). Streptavidin can bind specifically and strongly to biotin, while phycoerythrin provides a fluorescent signal as a fluorescent probe. It is often used for the fluorescent labeling and detection of biomolecules.

[0106] (5) The sample containing microplastics (polystyrene) was pre-soaked in phosphate buffer to fully wet its surface, which facilitated the subsequent coupling reaction;

[0107] (6) Incubation and binding reaction: Two groups of experiments were set up. SA-PE, SA-MB (streptavidin-coated magnetic beads, purchased from Dana magnetic beads) and the polypeptide-biotin conjugate solution in step (3) were mixed in equal volumes as the experimental group and a solution containing microplastics (polystyrene) was added. SA-PE and SA-MB were added to the microplastic (polystyrene) powder as the control group. Both groups of samples were incubated at 37°C for 30 minutes to promote the binding of SA-PE, SA-MB and biotinylated polypeptides on the surface of microplastics. SA-PE and SA-MB played the role of assisting magnetic separation and fluorescent labeling.

[0108] (7) Cleaning and observation: After incubation, the microplastic samples were magnetically separated and cleaned using PBST buffer (2 minutes, repeated 3 times) to remove unbound microplastics and SA-PE. The concentrated solution after magnetic adsorption was dropped onto a glass slide or nitrocellulose membrane and examined under a fluorescence microscope to compare the fluorescence signal intensity.

[0109] The fluorescence characterization results of the peptide-biotin conjugate-mediated binding of SA-PE to polystyrene microplastics are as follows: Figure 3 As shown, this experiment aims to verify the binding ability of peptide-biotin conjugates to polystyrene. By leveraging the specific and strong binding of SA-PE to biotin, the binding level of biotinylated peptides on the microplastic surface is characterized by fluorescence signal intensity. At the same time, a control group without peptide-biotin conjugates is set up to clarify the mediating role of peptides in the binding process between SA-PE and microplastics.

[0110] The results showed that the experimental group exhibited significant red fluorescence in the fluorescence channel, and the fluorescence signal in the bright-field + fluorescence channel highly overlapped with the microplastic region; while the control group showed only weak fluorescence in the fluorescence channel, and almost no fluorescence signal in the microplastic region in the bright-field + fluorescence channel. This indicates that the peptide-biotin conjugate can effectively mediate the binding of SA-PE to the polystyrene microplastic surface, thereby generating a significant fluorescence signal; without the peptide-biotin conjugate, SA-PE is difficult to effectively bind to the microplastic surface, verifying the targeted binding ability of this peptide to polystyrene microplastics.

[0111] Example 6:

[0112] A rapid detection kit for polystyrene microplastics based on a sandwich method (the amino acid sequences of the peptides are SEQ ID NO:1-SEQ ID NO:5) was used to rapidly detect polystyrene microplastics using a homologous peptide sandwich method. In the homologous peptide sandwich method, the magnetic bead-peptide conjugate and the peptide in the conjugate have the same amino acid sequence as the peptide in the bovine serum albumin-acridone ester conjugate. That is, the same specific peptide is used. One part is covalently coupled to a solid support as a capture unit, and the other part is covalently coupled to a signal molecule as a detection unit, thereby realizing the sandwich detection of the target analyte.

[0113] The specific steps are as follows:

[0114] Take 400 μL of the stock suspension of magnetic bead-peptide conjugate prepared in Example 4, wash it once with 0.05% Brij23 prepared with 1×PBS, and then re-vortex it in 40 μL of 0.05% Brij23. Then mix it with 50 μL of polystyrene microspheres of different concentrations and particle sizes (0.5 μm and 2.2 μm) (prepared with 0.05% Brij23), 150 μL of buffer (1×PBS, pH=7.4; 0.5% Brij23; 1.5% BSA), 50 μL of the 5 μg / mL peptide and bovine serum albumin-acridone ester conjugate prepared in Example 3 (prepared with 0.05% Brij23), and react at 37°C and 800 rpm for 30 minutes. After the reaction, wash 4-5 times with washing solution (0.05% Brij23). Finally, 0.1% hydrogen peroxide solution was added to each well, and then luminescence was generated under excitation with 0.25 mol / L sodium hydroxide solution. The generated light signal was detected using a chemiluminescence analyzer, and the signal intensity was positively correlated with the concentration of polystyrene microplastics, thus a dose-response curve was plotted.

[0115] The reactivity results of polystyrene microspheres with different particle sizes and concentrations are shown in Tables 2 and 3; the dose-response curves of polystyrene microspheres with different particle sizes were detected using a peptide with the amino acid sequence SEQ ID NO:1. Figure 4 and Figure 5 As shown.

[0116] Table 2. Reactivity of 2.2 μm polystyrene microspheres at different concentrations with five peptides

[0117]

[0118] Table 3. Reactivity of 0.5 μm polystyrene microspheres at different concentrations with five peptides

[0119]

[0120] The results showed that both polystyrene microspheres of different sizes (0.5 μm and 2.2 μm) could specifically bind to the detection kit (containing peptides with amino acid sequences of SEQ ID NO:1-SEQ ID NO:5) and generate background signals. Moreover, as the concentration of polystyrene microspheres increased, the signal value of the binding between the detection kit and the polystyrene microspheres gradually increased.

[0121] In summary, this invention (1) addresses the core pain points of existing technologies, such as high cost, poor stability, and large steric hindrance, by directly achieving the goal of providing a low-cost, high-stability detection method through the design of peptide-substituted antibodies. (2) Addresses the problems of cumbersome operation and long detection cycle by combining magnetic separation of magnetic beads with direct coupling of luminescent groups, effectively reducing incubation and washing steps and shortening time, precisely meeting the goal of simplifying the operation process. (3) Addresses the defects of limited sensitivity and insufficient specificity by providing a low-cost, high-stability detection method through the substitution of antibodies with specific peptides. On the one hand, peptides themselves possess extremely strong specific binding capabilities. Through precise amino acid sequence design, they can form a highly matched binding mode with polystyrene microplastic targets. Combined with the synergistic effect of the double sandwich structure, cross-reactions with non-target plastic particles or organic impurities can be significantly reduced, fundamentally solving the core defects of existing technologies, such as the tendency of antibodies to cross-react and insufficient specificity. On the other hand, the enrichment effect of magnetic beads on polystyrene microplastics and the signal amplification effect of luminescent groups synergistically enhance detection capabilities, making up for the deficiency of antibody-mediated detection sensitivity. This comprehensively achieves the goal of high sensitivity and high specificity detection, systematically solving the key defects of existing technologies.

[0122] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rapid detection kit for polystyrene microplastics based on a sandwich method, characterized in that, include: The capture component is a magnetic bead-peptide conjugate with a specific peptide immobilized by covalent coupling; The detection component comprises a luminescent detection probe formed by covalently coupling bovine serum albumin with a chemiluminescent label and the specific polypeptide; The amino acid sequence of the specific polypeptide is shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5; The specific polypeptides contained in the capture component and the detection component have the same amino acid sequence.

2. The rapid detection kit for polystyrene microplastics based on the sandwich method as described in claim 1, characterized in that, The specific method for preparing the magnetic bead-peptide conjugate is as follows: 3 mg of iodinated acetylated silica-coated magnetic beads are taken, resuspended in 10 mM phosphate buffer at pH 7.4, 60 μg of the specific peptide is added, activated at 37 °C, and blocked with 10 μL of mercaptoethanol. Finally, 180 μL of 10% bovine serum albumin solution is added, and blocked at 37 °C to obtain the magnetic bead-peptide conjugate.

3. The rapid detection kit for polystyrene microplastics based on the sandwich method as described in claim 2, characterized in that, The specific method for preparing the luminescent detection probe is as follows: Step 1: Dissolve 2 mg of bovine serum albumin in 1 mL of 0.1 M sodium bicarbonate solution with pH 8.6, vortex mix, centrifuge and sonicate, then filter to obtain a clear bovine serum albumin solution. Step 2: Add 10 molar amounts of acridine ester to the bovine serum albumin solution and react at room temperature in the dark to allow the acridine ester to initially couple with the bovine serum albumin. Step 3: Add 10 μL of 10% glycine stop solution, react at room temperature, desalt using a gravity column, replace the solution with 10 mM triethanolamine at pH 7.4, and centrifuge to wash the column; then centrifuge for 3 minutes to collect the bovine serum albumin-acridone ester conjugate. Step 4: Take bovine serum albumin-acrididine ester conjugate, add SMCC to make the molar ratio of SMCC to bovine serum albumin-acrididine ester conjugate 12:1, react at 37℃ to activate bovine serum albumin-acrididine ester conjugate and SMCC, and then desalt. Step 5: Collect the desalted bovine serum albumin-acridone ester conjugate in a tube, add 5 μL of 10% Brij23, and mix well; separately dissolve the specific polypeptide in 100 μL of dimethyl sulfoxide, and add 46.1 μL of the specific polypeptide solution to the tube. The molar ratio of the specific polypeptide to the bovine serum albumin-acridone ester conjugate is 8:

1. After mixing well, react at 4°C to obtain the reaction solution. Step 6: Desalt and purify the reaction solution using PBST buffer, which is a phosphate buffer containing 0.05% Tween-20.

4. The application of the sandwich-based rapid detection kit for polystyrene microplastics as described in claim 1 in the detection of polystyrene microplastics, characterized in that, The kit is for the rapid detection of polystyrene microplastic concentrations and is intended for non-disease diagnostic purposes.

5. A rapid detection method for polystyrene microplastics based on the sandwich method, characterized in that, Using the rapid detection kit for polystyrene microplastics based on the sandwich method as described in claim 1, rapid luminescence detection of polystyrene microplastics was performed by the homologous peptide sandwich method. The rapid detection of polystyrene microplastics via the homologous peptide sandwich method specifically involves: mixing and reacting the magnetic bead-peptide conjugate as described in claim 2, the luminescent detection probe as described in claim 3, polystyrene microplastics, and analytical buffer; washing after the reaction is complete, adding 0.1% hydrogen peroxide solution, and then emitting light under the excitation of 0.25 mol / L sodium hydroxide solution; detecting the generated light signal using a chemiluminescence analyzer; and calculating the concentration of polystyrene microplastics based on the light signal. The detection method described is not for disease diagnosis purposes.

6. A magnetic bead-peptide conjugate with a specific polypeptide immobilized by covalent coupling, characterized in that: The amino acid sequence of the specific polypeptide is shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:

5.

7. A luminescent detection probe, characterized in that: It is formed by covalently coupling bovine serum albumin with a chemiluminescent label and a specific polypeptide, wherein the amino acid sequence of the specific polypeptide is shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:

5.

8. A polystyrene microplastic test paper, characterized in that, The test strip includes a fluorescent detection probe formed from a biotinylated covalently modified specific polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:

5.

9. The polystyrene microplastic test paper as described in claim 8, characterized in that, The specific method for preparing the fluorescent detection probe is as follows: Step 1: Dissolve 0.1 mg of the polypeptide in 100 μL of phosphate buffer, add 0.15 mg of maleimide biotin, mix gently, and react overnight at 4 °C to obtain a polypeptide-biotin conjugate solution. Step 2: Transfer the peptide-biotin conjugate solution to a dialysis bag and dialyze overnight at 4°C using PBST buffer.

10. The polystyrene microplastic test paper as described in claim 9, characterized in that, The specific method for using the test strip is as follows: mix and react the fluorescent detection probe, polystyrene microplastics, streptavidin magnetic microspheres and streptavidin-phycoerythrin, wash after the reaction is complete, drop onto a nitrocellulose membrane, and observe using a fluorescence microscope.

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