Peptide-modified gold nanoclusters, their preparation methods, and their application in the detection of microplastics

Abstract

This invention relates to biodetection technology, disclosing a peptide-modified gold nanocluster, its preparation method, and its application in the detection of microplastics. The peptide-modified gold nanocluster comprises a gold nanocluster and a peptide LCI modified on the surface of the gold nanocluster, wherein the peptide LCI comprises an amino acid sequence as shown in SEQ ID NO: 1. The preparation method includes the following steps: providing functionalized modified gold nanoclusters; reacting the functionalized gold nanoclusters with the peptide LCI to attach the peptide LCI to the surface of the gold nanoclusters, forming the peptide-modified gold nanocluster. The microplastic detection method includes mixing the peptide-modified gold nanocluster with a test sample that may contain microplastics to obtain a detection solution, and performing colorimetric analysis or absorbance analysis on the detection solution. This gold nanocluster can specifically recognize polypropylene, exhibiting good specificity, low cost, and rapid detection of polypropylene microplastics.

Description

Technical Field

[0001] This invention relates to biodetection technology, specifically to a polypeptide-modified gold nanocluster, its preparation method, and its application in the detection of microplastics. Background Technology

[0002] Microplastics are defined as plastic particles with a diameter of less than 5 mm. Due to their small size, microplastics pose a greater threat to the environment than conventional large-volume plastics, especially in aquatic environments. Under the influence of multiple factors, including physical and biological processes over a long period, plastic fragments form microplastics, which are then washed into rivers, lakes, and oceans by rainwater, posing a significant threat to the survival of aquatic life and the safety of human drinking water.

[0003] With increasing public awareness of ecological environmental protection and growing concern about drinking water safety, the detection of microplastics has attracted growing attention from researchers and the drinking water industry. Currently, methods for detecting microplastics are very limited. Common methods include Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, and Nile red staining. These methods have significant drawbacks. First, the instruments used for FTIR and Raman spectroscopy are expensive and not suitable for widespread use; the detection process is also cumbersome, making them unsuitable for handling large samples. Second, while FTIR and Raman spectroscopy can detect the presence of microplastics, they cannot quantify them in the aquatic environment. Nile red is a solution-induced colorimetric dye that leaves fluorescence when passing through a membrane, masking the fluorescence of substances on the membrane and hindering subsequent observation and counting, leading to errors in the detection results.

[0004] Therefore, providing a simple and rapid new detection method is of great significance for the detection of microplastics. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems of expensive microplastic detection instruments, cumbersome processes, and inability to quantify detection in existing technologies. This invention provides a peptide-modified gold nanocluster, its preparation method, and its application in the detection of microplastics. The gold nanocluster can specifically recognize polypropylene, and has good specificity, low cost, and is simple and fast in detecting polypropylene microplastics.

[0006] To achieve the above objectives, a first aspect of the present invention provides a peptide-modified gold nanocluster, comprising a gold nanocluster and a peptide LCI modified on the surface of the gold nanocluster, wherein the peptide LCI comprises a fragment with an amino acid sequence as shown in SEQ ID NO: 1.

[0007] Preferably, the nucleotide sequence of the gene encoding the polypeptide LCI is shown in SEQ ID NO: 2.

[0008] A second aspect of this invention provides a method for preparing peptide-modified gold nanoclusters, comprising the following steps:

[0009] (1) Provides functionalized modified gold nanoclusters;

[0010] (2) The functionalized gold nanoclusters are contacted with the peptide LCI to connect the peptide LCI to the surface of the gold nanoclusters to form peptide-modified gold nanoclusters.

[0011] The polypeptide LCI includes a fragment with an amino acid sequence as shown in SEQ ID NO: 1.

[0012] Preferably, the preparation method of the functionalized modified gold nanoclusters in step (1) includes: reacting 11-mercaptoundecanoic acid with gold nanoparticles in an alkaline solvent in the dark, then mixing with anhydrous ethanol, and collecting the precipitate by solid-liquid separation.

[0013] Preferably, the alkaline solvent is alkaline ultrapure water.

[0014] Preferably, the molar ratio of 11-mercaptoundecanoic acid to the gold nanoparticles is 3-4:1.

[0015] Preferably, the conditions for the light-protected reaction include: a temperature of 5-40°C and a time of 4-8 hours.

[0016] Preferably, the method for preparing the gold nanoparticles includes: heating a chloroauric acid solution to boiling and then mixing it with a sodium citrate solution, followed by stirring and reaction.

[0017] Preferably, the molar concentration of chloroauric acid in the chloroauric acid solution is 0.15-0.45 mM, and the molar concentration of sodium citrate in the sodium citrate solution is 20-50 mM.

[0018] Preferably, the molar ratio of chloroauric acid in the chloroauric acid solution to sodium citrate in the sodium citrate solution is 1:8-15.

[0019] Preferably, the stirring reaction process includes: first, stirring and refluxing at a temperature of 95-110℃ for 8-12 minutes, and then removing the heat source and continuing the stirring reaction for 10-20 minutes.

[0020] Preferably, the nucleotide sequence of the gene encoding the polypeptide LCI is shown in SEQ ID NO: 2.

[0021] Preferably, the preparation method of the polypeptide LCI includes the following steps:

[0022] S1. The gene encoding the polypeptide LCI is recombined into a vector plasmid using molecular cloning technology to obtain a recombinant plasmid.

[0023] S2. The recombinant plasmid is transformed into competent bacteria, and the bacteria are screened by kanamycin to obtain recombinant bacteria that have been successfully transformed by the recombinant plasmid.

[0024] S3. After culturing the recombinant bacteria to obtain a culture medium, the culture medium is used to induce the expression of the polypeptide LCI, and then solid-liquid separation is performed to obtain a supernatant, which is then purified.

[0025] Preferably, the nucleotide sequences of the primer pairs encoding the polypeptide LCI gene in step S1 are shown in SEQ ID NO: 3 and SEQ ID NO: 4.

[0026] Preferably, the vector plasmid in step S1 is the pET-28a plasmid.

[0027] Preferably, the competent bacteria in step S2 are competent Escherichia coli.

[0028] Preferably, the molar ratio of the polypeptide LCI to the functionalized gold nanoclusters in step (2) is 50-100:1.

[0029] Preferably, the conditions for the contact reaction include: a temperature of 5-40°C and a time of 20-40 min.

[0030] The third aspect of this invention provides the application of the aforementioned peptide-modified gold nanoclusters or peptide-modified gold nanoclusters prepared by the aforementioned method in the detection of microplastics.

[0031] Preferably, the microplastic is a polypropylene microplastic.

[0032] The fourth aspect of the present invention provides a method for detecting microplastics, the method comprising the following steps: mixing the aforementioned peptide-modified gold nanoclusters or the peptide-modified gold nanoclusters prepared by the aforementioned method with a test sample that may contain microplastics to obtain a test solution, and performing colorimetric analysis or absorbance value detection analysis on the test solution.

[0033] Preferably, the peptide-modified gold nanoclusters are mixed with the sample to be tested in solution form.

[0034] Preferably, the concentration of peptide LCI in the solution of the peptide-modified gold nanoclusters is 5-15 μM, and the concentration of microplastics in the sample to be tested is 0-50 mg / mL.

[0035] Preferably, the volume ratio of the peptide-modified gold nanocluster solution to the sample to be tested is 1:90-100.

[0036] Preferably, the colorimetric analysis process includes: using the solution color of the peptide-modified gold nanoclusters as a control, comparing the color change after the addition of the test sample, which may contain microplastics.

[0037] Preferably, the absorbance detection and analysis process includes: plotting a standard curve of the concentration of the microplastic reference standard versus the absorbance value, and quantifying the microplastics in the sample to be tested based on the standard curve and the absorbance value of the detection solution.

[0038] Preferably, the wavelength of the absorbance value is 550-570 nm.

[0039] The beneficial effects of the present invention through the above technical solution are as follows:

[0040] The peptide-modified gold nanoclusters provided by this invention use peptide LCI as an intermediate. Functionalized gold nanoclusters are used to immobilize peptide LCI, allowing the peptide LCI to bind to the surface of the gold nanoclusters via peptide bonds. Due to its unique structure, the peptide LCI has a specific affinity for polypropylene, thus adsorbing onto the surface of the polypropylene, making the identification of polypropylene more accurate, rapid, and convenient. When the peptide-modified gold nanoclusters provided by this invention are applied to the detection of polypropylene microplastics, under the guidance of peptide LCI, the gold nanoclusters aggregate on the surface of the polypropylene microplastics, causing changes in the solution color and absorbance. This enables the establishment of a polypropylene microplastic detection method with advantages such as high specificity, low cost, simplicity, and speed, achieving qualitative and rapid quantitative analysis of polypropylene microplastics. Attached Figure Description

[0041] Figure 1 This is a schematic diagram illustrating the principle of detecting polypropylene microplastic content in an aquatic environment using peptide-modified gold nanoclusters in this invention.

[0042] Figure 2 This is a graph showing the change in absorbance of the peptide-modified gold nanoclusters as a function of wavelength in Example 1.

[0043] Figure 3 The concentration and absorbance value A of polypropylene microplastics in Example 1 560 Linear relationship graph;

[0044] Figure 4 This is a graph showing the absorbance of the test solutions for each water sample in Example 2 at a wavelength of 560 nm.

[0045] Figure 5 This is a graph showing the content of polypropylene microplastics in each water sample in Example 2. Detailed Implementation

[0046] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0047] The first aspect of the present invention provides a peptide-modified gold nanocluster, comprising a gold nanocluster and a peptide LCI modified on the surface of the gold nanocluster, wherein the peptide LCI comprises a fragment with an amino acid sequence as shown in SEQ ID NO: 1;

[0048] The amino acid sequence of the polypeptide LCI (SEQ ID NO: 1):

[0049] AIKLVQSPNGNFAASFVLDGTKWIFKSKYYDSSKGYWVGIYEVWDRK.

[0050] According to the present invention, preferably, the nucleotide sequence of the gene encoding the polypeptide LCI is as shown in SEQ ID NO: 2;

[0051] The nucleotide sequence of the polypeptide LCI (SEQ ID NO: 2): 5'-GCCATTAAACTGGTTCAGAGCCCGAATGGTAATTTTGCAGCAAGCTTTGTTCTGGATGGCACCAAATGGATCTTCAAAAGCAAATACTATGACAGCAGCAAAGGTTATTGGGTGGGTATTTATGAAGTGTGGGATCGCAAA-3'.

[0052] In this invention, the molar ratio of peptide LCI to gold nanoclusters in peptide-modified gold nanoclusters is preferably 50-100:1.

[0053] A second aspect of this invention provides a method for preparing peptide-modified gold nanoclusters, comprising the following steps:

[0054] (1) Provides functionalized modified gold nanoclusters;

[0055] (2) The functionalized gold nanoclusters are contacted with the peptide LCI to connect the peptide LCI to the surface of the gold nanoclusters to form peptide-modified gold nanoclusters.

[0056] The polypeptide LCI includes a fragment with an amino acid sequence as shown in SEQ ID NO: 1.

[0057] According to the present invention, functionalized gold nanoclusters can be commercially available or prepared in-house using methods disclosed in the prior art.

[0058] According to the present invention, preferably, the preparation method of the functionalized modified gold nanoclusters in step (1) includes: reacting 11-mercaptoundecanoic acid with gold nanoparticles in an alkaline solvent in the dark, then mixing with anhydrous ethanol, collecting the precipitate after solid-liquid separation, and obtaining 11-mercaptoundecanoic acid modified gold nanoclusters.

[0059] In this invention, the solution obtained from the light-shielded reaction can be observed using transmission electron microscopy to examine the effect of 11-mercaptoundecanoic acid on the functionalization of gold nanoclusters.

[0060] According to the present invention, preferably, the alkaline solvent is alkaline ultrapure water. Alkaline ultrapure water can be prepared by mixing ultrapure water with a small amount of strong alkali.

[0061] According to the present invention, the amount of 11-mercaptoundecanoic acid and gold nanoparticles used is sufficient to achieve the effect of functionalizing gold nanoclusters with 11-mercaptoundecanoic acid. To further improve the preparation efficiency of functionalized gold nanoclusters, preferably, the molar ratio of 11-mercaptoundecanoic acid to gold nanoparticles is 3-4:1.

[0062] According to the present invention, preferably, the conditions for the light-avoidance reaction include: a temperature of 5-40°C, specifically 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, or any value between the two above; and a time of 4-8 hours, specifically 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, or any value between the two above.

[0063] According to the present invention, gold nanoparticles can be commercially available or prepared by methods disclosed in the prior art. Exemplarily, the preparation method of the gold nanoparticles includes: heating a chloroauric acid solution to boiling and mixing it with a sodium citrate solution, followed by stirring. In this invention, the reaction liquid I obtained by stirring is subjected to solid-liquid separation, and the solid is washed multiple times to remove residual reaction substrates, thereby obtaining gold nanoparticles.

[0064] According to the present invention, preferably, the molar concentration of chloroauric acid in the chloroauric acid solution is 0.15-0.45 mM, and the molar concentration of sodium citrate in the sodium citrate solution is 20-50 mM.

[0065] According to the present invention, preferably, the molar ratio of chloroauric acid in the chloroauric acid solution to sodium citrate in the sodium citrate solution is 1:8-15. The inventors have found that this preferred embodiment is beneficial for increasing the synthesis rate of gold nanoparticles.

[0066] According to the present invention, preferably, the stirring reaction process includes: first, performing a stirring reflux reaction at a temperature of 95-110°C for 8-12 minutes, and then removing the heat source and continuing the stirring reaction for 10-20 minutes. The inventors have found that this preferred embodiment is beneficial for improving the synthesis rate of gold nanoparticles and optimizing the particle size distribution of gold nanoparticles.

[0067] According to the present invention, preferably, the nucleotide sequence of the gene encoding the polypeptide LCI is as shown in SEQ ID NO: 2.

[0068] In this invention, the polypeptide LCI can be obtained through artificial synthesis, or its encoding gene can be synthesized first and then obtained through biological expression.

[0069] The nucleotide sequences provided by this invention can generally be obtained using polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis. For example, those skilled in the art can easily obtain templates and primers based on the nucleotide sequences provided by this invention, and use PCR to amplify the relevant sequences.

[0070] Once the relevant nucleotide sequence is obtained, the relevant amino acid sequence can be obtained in large quantities using recombinant methods. Typically, the obtained nucleotide sequence is cloned into a vector, then transferred into a host cell, and finally isolated from the proliferated host cells using conventional methods.

[0071] In addition, known methods of artificial chemical synthesis can be used to synthesize the relevant nucleotide sequences.

[0072] According to the present invention, preferably, the preparation method of the polypeptide LCI includes the following steps:

[0073] S1. The gene encoding the polypeptide LCI is recombined into a vector plasmid using molecular cloning technology to obtain a recombinant plasmid.

[0074] S2. The recombinant plasmid is transformed into competent bacteria, and the bacteria are screened by kanamycin to obtain recombinant bacteria that have been successfully transformed by the recombinant plasmid.

[0075] S3. After culturing the recombinant bacteria to obtain a culture medium, the culture medium is used to induce the expression of the polypeptide LCI, and then solid-liquid separation is performed to obtain a supernatant, which is then purified.

[0076] According to the present invention, preferably, the nucleotide sequences of the primer pairs encoding the gene of the polypeptide LCI in step S1 are as shown in SEQ ID NO: 3 and SEQ ID NO: 4.

[0077] LCI positive strand primer (SEQ ID NO: 3): TCAGAGCCCGAATGGTAA;

[0078] LCI antisense primer (SEQ ID NO: 4): ACCCACCCAATAACCTTTG.

[0079] According to the present invention, preferably, the vector plasmid in step S1 is pET-28a plasmid.

[0080] According to the present invention, preferably, the competent bacteria in step S2 are competent Escherichia coli.

[0081] According to the present invention, preferably, the culturing process in step S3 includes: inoculating the recombinant bacteria into a culture medium containing kanamycin for at least one culture. The culture medium can be a conventional liquid culture medium in the art, for example, LB liquid medium.

[0082] According to the present invention, preferably, the concentration of kanamycin in the culture medium is 0.05-0.15 mg / mL, and the culture conditions include: an inoculum size of 3-5% by volume, specifically 3%, 3.5%, 4%, 4.5%, 5%, or any value between the two values; a temperature of 30-45°C, specifically 30°C, 35°C, 40°C, 45°C, or any value between the two values; and a time of 2-15 h, specifically 2 h, 5 h, 8 h, 11 h, 13 h, 15 h, or any value between the two values.

[0083] According to the present invention, preferably, the process of inducing the expression of the polypeptide LCI includes: mixing the culture medium with isopropyl-β-D-thiogalactoside (IPTG) for induction.

[0084] According to the present invention, preferably, the conditions for the mixed induction include: an IPTG concentration of 80-120 mg / mL, a temperature of 15-25°C, and a time of 30-50 h.

[0085] In the preparation of the above-mentioned polypeptide LCI, the recombinant plasmid, recombinant bacteria, culture medium induction, purification, etc. can all be carried out using conventional methods in this field.

[0086] According to the present invention, preferably, the molar ratio of the polypeptide LCI to the functionalized gold nanoclusters in step (2) is 50-100:1.

[0087] According to the present invention, preferably, the conditions for the contact reaction include: a temperature of 5-40°C, specifically 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, or any value between the two above; and a time of 20-40 min, specifically 20 min, 25 min, 30 min, 35 min, 40 min, or any value between the two above.

[0088] Based on the peptide-modified gold nanoclusters provided above, the inventors discovered that these gold nanoclusters, guided by peptide LCI, can specifically aggregate on the surface of polypropylene microplastics, causing changes in solution color and absorbance, thus enabling rapid qualitative and quantitative analysis of polypropylene microplastics. Based on this, a third aspect of the present invention provides the application of the aforementioned peptide-modified gold nanoclusters or peptide-modified gold nanoclusters prepared by the aforementioned method in the detection of microplastics.

[0089] According to the present invention, preferably, the microplastic is a polypropylene microplastic.

[0090] The fourth aspect of the present invention provides a method for detecting microplastics, the method comprising the following steps: mixing the aforementioned peptide-modified gold nanoclusters or the peptide-modified gold nanoclusters prepared by the aforementioned method with a test sample that may contain microplastics to obtain a test solution, and performing colorimetric analysis or absorbance value detection analysis on the test solution.

[0091] In this invention, the microplastic can be any type of microplastic particle; preferably, the microplastic is polypropylene microplastic. More preferably, the average particle size of the polypropylene microplastic is 80-120 nm.

[0092] According to the present invention, preferably, the peptide-modified gold nanoclusters are mixed with the sample to be tested in solution form. The inventors have found that this preferred embodiment is beneficial for improving the specificity and accuracy of microplastic detection, making detection faster.

[0093] According to the present invention, preferably, the concentration of peptide LCI in the solution of the peptide-modified gold nanoclusters is 5-15 μM, and the concentration of microplastics in the sample to be tested is 0-50 mg / mL.

[0094] According to the present invention, preferably, the volume ratio of the polypeptide-modified gold nanocluster solution to the sample to be tested is 1:90-100.

[0095] According to the present invention, preferably, the colorimetric analysis process includes: comparing the color change after the addition of the test sample, which may contain microplastics, with the solution color of the polypeptide-modified gold nanoclusters as a control.

[0096] According to the present invention, preferably, the absorbance detection and analysis process includes: plotting a standard curve of the concentration of the microplastic reference standard versus the absorbance value; and quantifying the microplastics in the sample to be tested based on the standard curve and the absorbance value of the detection solution. Exemplarily, the process of plotting the standard curve of the concentration of the microplastic reference standard versus the absorbance value includes: preparing microplastic reference standard solutions of different concentrations; mixing the microplastic reference standard solutions of different concentrations with the peptide-modified gold nanoclusters respectively, and then detecting the absorbance; plotting a standard curve based on the concentration and absorbance value of the microplastic reference standard solutions.

[0097] According to the present invention, preferably, the wavelength of the absorbance value is 550-570 nm.

[0098] According to a particularly preferred embodiment of the present invention, see [link to embodiment]. Figure 1 The method for detecting microplastics includes the following steps:

[0099] (1) After heating a chloroauric acid solution with a molar concentration of 0.15-0.45mM to boiling, it is mixed with a sodium citrate solution with a molar concentration of 20-50mM, so that the molar ratio of chloroauric acid to sodium citrate is 1:8-15. Then, the mixture is stirred and refluxed at a temperature of 95-110℃ for 8-12 minutes. After removing the heat source, the mixture is stirred and reacted for another 10-20 minutes to obtain reaction solution I. The reaction solution I is then subjected to solid-liquid separation and washing to obtain gold nanoparticles.

[0100] (2) In alkaline ultrapure water, 11-mercaptoundecanoic acid and gold nanoparticles were mixed at a molar ratio of 3-4:1 and reacted in the dark at a temperature of 5-40℃ for 4-8 hours to obtain reaction solution II. Reaction solution II was mixed with anhydrous ethanol, and the precipitate was collected by solid-liquid separation to obtain gold nanoclusters modified with 11-mercaptoundecanoic acid.

[0101] (3) The 11-mercaptoundecanoic acid-modified gold nanocluster solution was mixed with the peptide LCI solution (the peptide LCI includes an amino acid sequence as shown in SEQ ID NO: 1) so that the molar ratio of gold nanocluster to peptide LCI was 1:50-100. Then, the mixture was subjected to a static contact reaction at a temperature of 5-40℃ for 20-40 min to attach the peptide LCI to the surface of the gold nanocluster, forming a peptide-modified gold nanocluster.

[0102] (4) Mix the peptide-modified gold nanoclusters (peptide LCI concentration of 5-15 μM) with the test sample (concentration of 0-50 mg / mL) that may contain polypropylene microplastics at a volume ratio of 1:90-100 to obtain the test solution, and perform colorimetric analysis or absorbance analysis on the test solution.

[0103] Using the color of the solution of peptide-modified gold nanoclusters as a control, the color change of the test sample that may contain microplastics after its addition can be compared, or a standard curve of microplastic reference concentration versus absorbance value can be plotted. Based on the standard curve and the absorbance value of the test solution, the microplastics in the test sample can be quantified.

[0104] The present invention will be described in detail below through embodiments.

[0105] In the following examples, absorbance values ​​were measured using a UV spectrophotometer and the UV-professional software; *E. coli* (with the pET-28a plasmid backbone) was purchased from Beijing Qingke Biotechnology Co., Ltd., catalog number DH5a; the AxyPrep™ MPlasmid Miniprep Kit was purchased from Corning Incorporated, catalog number AP-MN-P-250; the AxyPrep™ DNA Gel Extraction Kit was purchased from Corning Incorporated, catalog number AP-GX-50; PrimeSTAR was purchased from TaKaRa, catalog number R045; Exnase... II was purchased from Vazyme, part number C112-02-AB; Buffer was purchased from Vazyme, part number C112-02-AA; Polypropylene microplastics were purchased from Dongguan Zhangmutou Zhongxin Plastics Co., Ltd., model number 100nm; Water samples (ultrapure water, drinking water, groundwater, pond water and seawater) were taken from Qixia District, Nanjing and Nanhui New City Beach Area, Shanghai, respectively; Other reagents and raw materials were all conventional commercial products.

[0106] In the following examples, a 100 mg / mL kanamycin antibiotic solution was prepared as follows: 2 g of kanamycin antibiotic powder was placed in a sterile 50 mL centrifuge tube, then 20 mL of sterile water was added, mixed well, and then filtered through a sterile water filter. The solution was then stored in a 2 mL centrifuge tube at -20°C.

[0107] Preparation of IPTG solution with a concentration of 100 mg / mL: Place 2 g of IPTG powder in a sterile 50 mL centrifuge tube, then add 20 mL of sterile water, mix well, then filter through a sterile water filter, store in a 2 mL centrifuge tube, and store at -20℃.

[0108] Preparation of PBS solution: 8 g / L sodium chloride, 0.2 g / L potassium chloride, 2.9 g / L disodium hydrogen phosphate dodecahydrate, 0.2 g / L potassium dihydrogen phosphate, pH adjusted to 7.4.

[0109] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0110] LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 12 g / L.

[0111] In the following examples, unless otherwise specified, the room temperature is 25±5℃.

[0112] Preparation Example 1: Preparation of Peptide LCI

[0113] S1. The pET-28a plasmid backbone was extracted from E. coli using the AxyPrep™ Plasmid Miniprep Kit. The pET-28a plasmid backbone was cut using double digestion with Nde I and Xho I enzymes. After incubation at 37°C for 1.5 h, the pET-28a digested backbone was obtained by gel extraction using the AxyPrep™ DNA Gel Extraction Kit.

[0114] S2 and the LCI polypeptide gene fragment (nucleotide sequence shown in SEQ ID NO: 2) were synthesized by Beijing Qingke Biotechnology Co., Ltd. Primers for the LCI polypeptide were designed using Primer Premier software, and the designed primer pairs were sent to Beijing Qingke Biotechnology Co., Ltd. for synthesis. Amplification was performed using the PCR reaction system and conditions shown in Table 1. The PCR-amplified samples were run on a gel, and the gel was then recovered to obtain the encoding gene of the LCI polypeptide. The nucleotide sequences of the primer pairs for the LCI polypeptide encoding gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4.

[0115] LCI positive strand primer (SEQ ID NO: 3): TCAGAGCCCGAATGGTAA,

[0116] LCI antisense primer (SEQ ID NO: 4): ACCCACCCAATAACCTTTG;

[0117] Table 1

[0118]

[0119]

[0120] S3. Using the reaction system and conditions shown in Table 2, Gibson assembly was performed to obtain the recombinant plasmid pET-28a-LCI. The assembled recombinant plasmid pET-28a-LCI was transformed into competent Escherichia coli and plated on LB agar plates containing kanamycin. The plates were incubated for 12 hours at 33°C. Eight E. coli colonies were selected from the single colonies that grew on the LB agar plates for colony PCR. The PCR samples were then used for DNA verification. Probable samples were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing to confirm the successful construction of the plasmid.

[0121] Table 2

[0122]

[0123] S4. Inoculate activated Escherichia coli BL21 into LB liquid medium and culture until OD. 600 =0.4, centrifuged at 4000 rpm for 10 min at 4℃, collected the E. coli cells in the precipitate, dissolved the collected E. coli cells in CaCl2 solution (CaCl2 concentration of 0.1M), incubated on ice for 30 min, centrifuged again at 4℃ for 10 min at 4000 rpm, collected the E. coli cells in the precipitate, dissolved the collected E. coli cells in CaCl2 solution containing 10% glycerol (CaCl2 concentration of 0.1M), and obtained competent E. coli;

[0124] S5. The competent E. coli obtained in step S4 were mixed with the recombinant plasmid pET-28a-LCI (concentration of 100 ng / mL) at a volume ratio of 3:100. The mixture was then incubated in a water bath at 42℃ for 90 s to promote bacterial uptake of the recombinant plasmid. After an ice bath for 3 min, the mixture was inoculated into LB liquid medium at an inoculation amount of 0.8% by volume. The mixture was then cultured at 33℃ for 12 h to obtain the transformed competent bacteria.

[0125] S6. The transformed competent bacteria from step S5 were inoculated onto LB solid plates containing kanamycin (0.1 mg / mL) for screening culture. Six single bacterial colonies were selected from the LB solid plates containing kanamycin and inoculated onto LB liquid medium containing kanamycin (0.1 mg / mL). The culture was incubated at 37°C for 12 h to obtain seed culture. The seed culture was inoculated into LB liquid medium containing kanamycin (0.1 mg / mL) at an inoculation rate of 4% by volume and incubated at 37°C for 2.5 h to obtain culture medium. IPTG (100 mg / mL) was added to the culture medium and the culture was induced at 20°C for 40 h to produce the peptide LCI. The induced culture medium was centrifuged for 10 min to obtain the supernatant containing the LCI peptide.

[0126] S7. Purify the supernatant containing LCI peptides using a nickel column: Open the cap of the nickel column, discard all the ethanol, immediately add 10 mL of filtered Buffer A until all drops are added, then add 2 mL of the supernatant containing LCI peptides until all drops are added, then add 5 mL of Buffer B until all drops are added, then add 5 mL of Buffer C until 5 drops are added, then immediately use a 2 mL centrifuge tube to collect the target protein until all drops are added, then add another 5 mL of Buffer B until all drops are added, then add another 5 mL of Buffer A until all drops are added, then add 1.5-2 mL of 20% ethanol, plug the top and bottom, and store at 4°C. This allows the LCI protein to bind to the nickel column via its histidine tag, while other proteins flow out. Elute the LCI protein bound to the nickel column with imidazole (20 mM). Imidazole competitively binds to histidine with nickel to elute the LCI protein, yielding a crude LCI protein solution.

[0127] The preparation of Buffer A, Buffer B, and Buffer C with an imidazole concentration of 10M (total volume 500mL) is as follows:

[0128] 10M Imidazole Buffer A: Sodium dihydrogen phosphate (NaH2PO4) 3.9g, sodium chloride 8.7g, imidazole 0.34g.

[0129] 20M Imidazole Buffer B: Sodium dihydrogen phosphate (NaH2PO4) 3.9g, sodium chloride 8.7g, imidazole 0.68g.

[0130] 300M Imidazole Buffer C: Sodium dihydrogen phosphate (NaH2PO4) 3.9g, sodium chloride 8.7g, imidazole 10.2g;

[0131] S8. The crude LCI protein solution was purified a second time through an ultrafiltration tube (3 kDa). The small pores in the ultrafiltration tube were used to concentrate the LCI protein solution by centrifugation at 5000 rpm for 1 h to obtain a peptide LCI solution (the nucleotide sequence of the peptide LCI is shown in SEQ ID NO: 2). The peptide LCI was verified by SDS-PAGE electrophoresis. The separating gel concentration was 5 wt%, and the stacking gel concentration was 12 wt%. SDS-PAGE electrophoresis was performed (stacking gel voltage 80 V, time 30 min; separating gel voltage 120 V, time 90 min). Coomassie brilliant blue was used for staining, and the solution was destained with a mixture of 8 vol% glacial acetic acid and 25 vol% ethanol. The band at 7 kDa was clear.

[0132] Example 1

[0133] (1) Take a 0.3 mM chloroauric acid solution into a 50 mL round bottom flask, place a magnetic flask, attach a spherical condenser, heat to boiling, and while stirring at high speed, quickly add a 40 mM sodium citrate solution so that the molar ratio of chloroauric acid to sodium citrate is 1:12. Reflux for 10 min under vigorous stirring, remove the heat source, continue stirring for 15 min, cool the solution to room temperature to obtain reaction solution I, centrifuge and wash reaction solution I to obtain gold nanoparticles;

[0134] (2) In alkaline ultrapure water, 11-mercaptoundecanoic acid and gold nanoparticles were mixed at a molar ratio of 3.5:1 and reacted at room temperature in the dark for 6 hours to obtain reaction solution II. Reaction solution II was mixed with anhydrous ethanol, and the precipitate was collected by centrifugation to obtain gold nanoclusters modified with 11-mercaptoundecanoic acid. The precipitate was then redissolved in ultrapure water to obtain a solution of gold nanoclusters modified with 11-mercaptoundecanoic acid.

[0135] (3) The 11-mercaptoundecanoic acid-modified gold nanocluster solution was mixed with the peptide LCI solution obtained in Preparation Example 1, such that the molar ratio of gold nanoclusters to peptide LCI was 1:75. The mixture was then allowed to stand at room temperature for 30 min to allow the peptide LCI to attach to the surface of the gold nanoclusters, forming a peptide-modified gold nanocluster solution. (The peptide-modified gold nanocluster solution was added to a cuvette, and the absorbance of the solution at different wavelengths was measured. The results are shown below.) Figure 2 (as shown);

[0136] (4) A solution of peptide-modified gold nanoclusters (with a peptide LCI concentration of approximately 10 μM) was mixed with an aqueous solution containing polypropylene microplastics (concentrations of 0 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, and 20 mg / mL, respectively) at a volume ratio of 1:100 to obtain a detection solution. The absorbance of each detection solution at 560 nm was measured, and the concentration of polypropylene microplastics and absorbance value A were calculated based on the absorbance values.560 The standard curve, the results are as follows Figure 3 As shown, the limit of detection and limit of quantitation for polypropylene microplastics are 100 μg / mL and 1 mg / mL, respectively.

[0137] Example 2

[0138] A peptide-modified gold nanocluster solution was prepared according to steps (1)-(3) in Example 1. The peptide-modified gold nanocluster solution (with a peptide LCI concentration of approximately 10 μM) was mixed with water samples (ultrapure water, drinking water, groundwater, pond water, and seawater) that may contain polypropylene microplastics at a volume ratio of 1:90 to obtain a detection solution. The absorbance values ​​of each detection solution at 550 nm, 560 nm, and 570 nm were measured. The results are as follows: Figure 4 As shown in Table 3; based on absorbance value A 560 Using the standard curve obtained in Example 1, the content of polypropylene microplastics in each water sample was calculated, and the results are as follows: Figure 5 As shown in Table 3.

[0139] Table 3

[0140]

[0141] Example 3

[0142] (1) Take a 0.15 mM chloroauric acid solution into a 50 mL round bottom flask, place a magnetic flask, attach a spherical condenser, heat to boiling, and while stirring at high speed, quickly add a 20 mM sodium citrate solution so that the molar ratio of chloroauric acid to sodium citrate is 1:8. Reflux for 8 min under vigorous stirring, remove the heat source, continue stirring for 10 min, cool the solution to room temperature to obtain reaction solution I, centrifuge and wash reaction solution I to obtain gold nanoparticles;

[0143] (2) In alkaline ultrapure water, 11-mercaptoundecanoic acid and gold nanoparticles were mixed at a molar ratio of 3:1 and reacted at room temperature in the dark for 4 hours to obtain reaction solution II. Reaction solution II was mixed with anhydrous ethanol, and the precipitate was collected by centrifugation to obtain gold nanoclusters modified with 11-mercaptoundecanoic acid. The precipitate was then redissolved in ultrapure water to obtain a solution of gold nanoclusters modified with 11-mercaptoundecanoic acid.

[0144] (3) The 11-mercaptoundecanoic acid-modified gold nanocluster solution was mixed with the peptide LCI solution obtained in Preparation Example 1, so that the molar ratio of gold nanocluster to peptide LCI was 1:50. Then, the static contact reaction was carried out at room temperature for 20 min to connect the peptide LCI to the surface of the gold nanocluster to form a peptide-modified gold nanocluster solution.

[0145] (4) The peptide-modified gold nanocluster solution (the concentration of peptide LCI is about 5 μM) is mixed with the aqueous solution containing polypropylene microplastics (concentrations of 0 mg / mL, 5 mg / mL, 10 mg / mL, and 20 mg / mL) at a volume ratio of 1:90 to obtain the detection solution. The color change of the detection solution is directly observed by visual observation. The color of the detection solution changes from red to black as the concentration of polypropylene microplastics increases from low to high.

[0146] Example 4

[0147] (1) Take a 0.45 mM chloroauric acid solution into a 50 mL round bottom flask, place a magnetic spool in it, attach a spherical condenser, heat it to boiling, and while stirring at high speed, quickly add a 50 mM sodium citrate solution so that the molar ratio of chloroauric acid to sodium citrate is 1:15. Reflux for 12 min under vigorous stirring, remove the heat source, continue stirring for 20 min, cool the solution to room temperature to obtain reaction solution I, centrifuge and wash reaction solution I to obtain gold nanoparticles;

[0148] (2) In alkaline ultrapure water, 11-mercaptoundecanoic acid and gold nanoparticles were mixed at a molar ratio of 4:1 and reacted at room temperature in the dark for 8 hours to obtain reaction solution II. Reaction solution II was mixed with anhydrous ethanol, and the precipitate was collected by centrifugation to obtain gold nanoclusters modified with 11-mercaptoundecanoic acid. The precipitate was then redissolved in ultrapure water to obtain a solution of gold nanoclusters modified with 11-mercaptoundecanoic acid.

[0149] (3) The 11-mercaptoundecanoic acid-modified gold nanocluster solution was mixed with the peptide LCI solution obtained in Preparation Example 1, so that the molar ratio of gold nanocluster to peptide LCI was 1:100. Then, the static contact reaction was carried out at room temperature for 40 min to connect the peptide LCI to the surface of the gold nanocluster to form a peptide-modified gold nanocluster solution.

[0150] (4) The peptide-modified gold nanocluster solution (the concentration of peptide LCI is about 15 μM) was mixed with the aqueous solution containing polypropylene microplastics (concentrations of 0 mg / mL, 10 mg / mL and 20 mg / mL) at a volume ratio of 1:100 to obtain the detection solution. The absorbance of each detection solution at 560 nm was measured. The specific absorbance values ​​were 0.803, 0.644 and 0.462, respectively.

[0151] Comparative Example 1

[0152] At room temperature, 0.001 g of Nile Red biological staining agent was added to 250 mL of acetone to prepare a Nile Red working solution with a concentration of 4 μg / mL. For staining, 0.5 g of salt was dissolved in 10 mL of water samples (ultrapure water, drinking water, groundwater, pond water, and seawater) that may contain polypropylene microplastics. 0.3 mL of the prepared Nile Red working solution was added to stain the water samples light blue. For filtration, a microporous membrane with a diameter of 1 mm and a pore size of 0.45 μm was prepared. The stained water samples were passed through the membrane using a vacuum filtration device. After the membrane dried, the fluorescent substances on the membrane were observed under a fluorescence microscope at an excitation light of 450-590 nm and under 4X magnification. Ten fields of view were randomly selected for counting to estimate the total amount of fluorescent substances on the membrane. The results are shown in Table 4.

[0153] Table 4

[0154]

[0155] As can be seen from the results in Tables 3 and 4, the peptide-modified gold nanoclusters and detection method provided by this invention can achieve rapid qualitative and quantitative analysis of polypropylene microplastics in an aqueous environment, with advantages such as high specificity, low cost, simplicity and speed.

[0156] Comparative Example 2

[0157] A solution of gold nanoclusters modified with thioaniline (with a concentration of approximately 15 μM) was used instead of the peptide-modified gold nanoclusters solution obtained in Example 1. The thioaniline-modified gold nanoclusters solution was mixed with an aqueous solution containing polypropylene microplastics (concentrations of 0 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, and 20 mg / mL) at a volume ratio of 1:110 to obtain a detection solution. The detection solution was unstable and greatly affected by pH, making it impossible to determine the relationship between the concentration of polypropylene microplastics and absorbance.

[0158] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A peptide-modified gold nanocluster, characterized in that, The invention comprises gold nanoclusters and a polypeptide LCI modified on the surface of the gold nanoclusters, wherein the polypeptide LCI comprises an amino acid sequence as shown in SEQ ID NO: 1; and the nucleotide sequence of the gene encoding the polypeptide LCI is shown in SEQ ID NO:

2. The preparation method of the peptide-modified gold nanoclusters includes the following steps: (1) Provide functionalized gold nanoclusters; the preparation method of the functionalized gold nanoclusters includes: reacting 11-mercaptoundecanoic acid with gold nanoparticles in an alkaline solvent in the dark, then mixing with anhydrous ethanol, and collecting the precipitate by solid-liquid separation; (2) The functionalized gold nanoclusters are contacted with the peptide LCI to connect the peptide LCI to the surface of the gold nanoclusters to form peptide-modified gold nanoclusters.

2. The polypeptide-modified gold nanoclusters according to claim 1, characterized in that, In step (1), the alkaline solvent is alkaline ultrapure water; The molar ratio of 11-mercaptoundecanoic acid to the gold nanoparticles is 3-4:1; The conditions for the light-protected reaction include: a temperature of 5-40℃ and a time of 4-8h.

3. The polypeptide-modified gold nanoclusters according to claim 2, characterized in that, The method for preparing the gold nanoparticles includes: heating a chloroauric acid solution to boiling and then mixing it with a sodium citrate solution, followed by stirring and reaction. The molar concentration of chloroauric acid in the chloroauric acid solution is 0.15-0.45 mM, and the molar concentration of sodium citrate in the sodium citrate solution is 20-50 mM. The molar ratio of chloroauric acid in the chloroauric acid solution to sodium citrate in the sodium citrate solution is 1:8-15; The stirring reaction process includes: first, stirring and refluxing at a temperature of 95-110℃ for 8-12 minutes, then removing the heat source and continuing the stirring reaction for 10-20 minutes.

4. The polypeptide-modified gold nanoclusters according to claim 1, characterized in that, The preparation method of the polypeptide LCI includes the following steps: S1. The gene encoding the polypeptide LCI is recombined into a vector plasmid using molecular cloning technology to obtain a recombinant plasmid. S2. The recombinant plasmid is transformed into competent bacteria, and the bacteria are screened by kanamycin to obtain recombinant bacteria that have been successfully transformed by the recombinant plasmid. S3. After culturing the recombinant bacteria to obtain a culture medium, the culture medium is used to induce the expression of the polypeptide LCI, and then solid-liquid separation is performed to obtain a supernatant, which is then purified. The nucleotide sequences of the primer pairs encoding the gene of the polypeptide LCI described in step S1 are shown in SEQ ID NO: 3 and SEQ ID NO: 4; The vector plasmid mentioned in step S1 is pET-28a plasmid; The competent bacteria mentioned in step S2 are competent Escherichia coli.

5. The polypeptide-modified gold nanoclusters according to claim 1, characterized in that, In step (2), the molar ratio of the polypeptide LCI to the functionalized gold nanoclusters is 50-100:1; The conditions for the contact reaction include: a temperature of 5-40°C and a time of 20-40 min.

6. The application of the polypeptide-modified gold nanoclusters according to any one of claims 1 to 5 in the detection of microplastics; The microplastic is a polypropylene microplastic.

7. A method for detecting microplastics, characterized in that, The method includes the following steps: mixing the peptide-modified gold nanoclusters according to any one of claims 1 to 5 with a test sample that may contain microplastics to obtain a detection solution, and performing colorimetric analysis or absorbance detection analysis on the detection solution.

8. The detection method according to claim 7, characterized in that, The peptide-modified gold nanoclusters are mixed with the sample to be tested in solution. The concentration of peptide LCI in the solution of the peptide-modified gold nanoclusters is 5-15 μM, and the concentration of microplastics in the sample to be tested is 0-50 mg / mL. The volume ratio of the peptide-modified gold nanocluster solution to the sample to be tested is 1:90-100. The colorimetric analysis process includes: using the solution color of the peptide-modified gold nanoclusters as a control, comparing the color change after the addition of the test sample that may contain microplastics; The absorbance detection and analysis process includes: plotting a standard curve of the concentration of the microplastic reference standard versus the absorbance value; and quantifying the microplastics in the sample to be tested based on the standard curve and the absorbance value of the test solution. The wavelength of the absorbance value is 550-570nm.

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