Peptide fragment for modifying Fe3O4 (at) Au magnetic nanoparticles and preparation method of Fe3O4 (at) Au magnetic nanoparticles

Abstract

The invention provides a peptide fragment for modifying a Fe3O4 (at) Au magnetic nanoparticle and a preparation method of the Fe3O4 (at) Au magnetic nanoparticle, the peptide fragment is an alpha-peptide fragment 1 or / and a beta-peptide fragment 1, and the fragment of the peptide fragment is as shown in SEQ ID NO: 1. The method comprises the following steps: firstly, growing a layer of gold shell on the surface of Fe3O4 in situ to provide more modification sites, and then modifying PS binding peptide (PSBP) by using an Au-S bond to construct the PS NPs selective probe. The ultralow-concentration PS can be quantitatively detected only by monitoring the ultraviolet-visible absorption change of the probe. The peptide modified Fe3O4 (at) Au probe (Fe3O4 (at) Au-PSBP) has good linearity in a range of 0 to 80 pg mL <-1 >. The probe has been successfully applied to actual samples, such as bottled water, edible salt and milk. The adding standard recovery rate is 98.85%-112.10%, and the relative standard deviation (RSD) is 2.53%-13.80%. The provided Fe3O4 (at) Au-PSBP can be easily expanded to other types of NPs by simply replacing peptides, shows huge potential as a universal specific probe, and can sensitively and quickly detect low-concentration NPs in an actual sample.

Description

Technical Field

[0001] The present invention relates to the field of nanoplastics detection, and particularly to a peptide for modifying Fe3O4@Au magnetic nanoparticles and a preparation method of Fe3O4@Au magnetic nanoparticles. Background Art

[0002] Due to the low concentration of MPs / NPs in the environment or actual samples, coupled with their inherent complexity, their separation and detection remain a challenge.

[0003] Currently, the identification and detection of MPs / NPs require the use of multiple devices or instruments. The preliminary identification of MPs is usually carried out by visual inspection, and then appropriate microscopes, including scanning electron microscopes, are used according to the particle size. However, this method has great limitations and can only simply identify larger MPs. Fourier transform infrared spectroscopy (FTIR) is a commonly used technique for the detailed characterization of MPs in environmental samples and can provide more information for component identification. In addition, some rapid determination methods for MPs / NPs based on surface-enhanced Raman spectroscopy (SERS) have also been established. However, there are few rapid, sensitive, and specific detection methods for low-concentration NPs in environmental or actual samples. The reason may be that when measuring low-concentration NPs, it is inevitably interfered by the sample environment and requires ultrasensitive response signals. On the other hand, ligands for the specific detection of NPs are rarely reported.

[0004] Fe3O4 nanoparticles have the characteristics of easy separation, low toxicity, and good biocompatibility, and have great application potential in the biological and medical fields. In addition, Fe3O4 also has the advantage of being easily modified, so it is often combined with other nanoparticles (such as gold nanoparticles or silver nanoparticles) to construct composite probes. PS is one of the most common raw materials in daily plastic products, so PS MPs / NPs are the most common and widely studied objects. Since most MPs / NPs surfaces are hydrophobic, the hydrophobic effect is usually utilized when designing capture and detection methods, but usually its specificity is poor and it cannot target specific types of MPs / NPs. Summary of the Invention

[0005] To solve the above problems, the present invention aims to provide a peptide for modifying Fe3O4@Au magnetic nanoparticles and a preparation method of Fe3O4@Au magnetic nanoparticles, and establish a simple and ultrasensitive detection method for PS NPs. Design and specific process: Fe3O4 was synthesized by hydrothermal method, and a layer of polyethyleneimine (PEI) was coated on its surface through electrostatic interaction to make Fe3O4 positively charged. Then, a gold shell was in-situ grown on the surface of Fe3O4-PEI to provide as many modification sites as possible. PSBP was connected to the surface of Fe3O4@Au through gold-sulfur bonds, thus forming a selective detection probe. PSBP was used as a specific recognition unit to specifically capture PS NPs, while Fe3O4@Au not only played a role in enriching and separating low-concentration NPs in the sample, but also provided a detection response signal. Therefore, it can become a composite probe integrating specific recognition, sample enrichment and signal response.

[0006] The technical solution of the present invention is as follows: A peptide for modifying Fe3O4@Au magnetic nanoparticles, the peptide is α-peptide 1 or / and β-peptide 1, and the peptide 1 fragment is shown in SEQ ID NO:1, Trp-Ser-Pro-Trp-Gly-Met-Trp-Ser-Tyr, abbreviated as WSPWGMWSY; Among them, α and β are modification groups, α is cysteine, and β is mercaptopropionic acid; the α and β are connected to the N-terminus of peptide 1; The Fe3O4@Au magnetic nanoparticles have a core-shell structure, with a Fe3O4 magnetic core and a Au shell layer, and the peptide is connected to the surface of the Au shell layer through gold-sulfur bonds; the Fe3O4@Au magnetic nanoparticles are used to prepare a selective probe for detecting polystyrene nanoplastics.

[0007] A preparation method of a selective probe for detecting polystyrene nanoplastics by using the peptide to modify Fe3O4@Au magnetic nanoparticles, the method includes the following steps: S1. Synthesize Fe3O4 magnetic nanoparticles by hydrothermal method; S2. Modify Fe3O4@Au with PSBP to obtain a Fe3O4@Au-PSBP composite probe; complete the preparation of the selective probe.

[0008] Preferably, the step S1 specifically includes the following steps: S1.1 0.8-1.2 mg mL -1 Fe3O4 aqueous solution and 0.8-1.2 mg mL -1The PEI (polyethyleneimine) solution is mixed at a volume ratio of (4.5 - 5.5):1, ultrasonicated for 25 - 35 minutes, washed and redissolved to 0.8 - 1.2 mg / mL -1 Fe3O4-PEI solution; S1.2 Add the Fe3O4-PEI solution to AuNPs (gold nanoparticles) at a volume ratio of (4.5 - 5.5):1, ultrasonicate for 0.4 - 0.6 h, wash away the excess AuNPs, and redissolve to 1 - 3 mg per milliliter to obtain Fe3O4-Au seeds; S1.3 Add PVP (polyvinylpyrrolidone) and NH2OH∙HCl to distilled water at a solid-liquid ratio of (0.2 - 0.4):(0.015 - 0.025):100 g / g / mL. The distilled water also adds Fe3O4-Au seeds at a volume ratio of 100:(2 - 4), and ultrasonicate the solution for 12 - 18 minutes; S1.4 Add the HAuCl4 solution containing 1% HAuCl4 to the system obtained in step S1.3 at a volume ratio of (450 - 550):1, continue to ultrasonicate for 14 - 16 minutes to obtain Fe3O4@Au magnetic nanoparticles.

[0009] Further preferably, step S1 specifically includes the following steps: S1.1 1 mg / mL -1 Fe3O4 aqueous solution and 1 mg / mL -1 PEI (polyethyleneimine) solution are mixed at a volume ratio of 5:1, ultrasonicated for 30 minutes, washed and redissolved to 1 mg / mL -1 Fe3O4-PEI solution; S1.2 Add the Fe3O4-PEI solution to AuNPs (gold nanoparticles) at a volume ratio of 5:1, ultrasonicate for 0.5 h, wash away the excess AuNPs, and redissolve to 1 mg per milliliter to obtain Fe3O4-Au seeds; S1.3 Add PVP (polyvinylpyrrolidone) and 0.02 g NH2OH∙HCl to distilled water at a solid-liquid ratio of 0.3:0.02:100 g / g / mL. The distilled water also adds Fe3O4-Au seeds at a volume ratio of 100:3, and ultrasonicate the solution for 15 minutes; S1.4 Add the HAuCl4 solution containing 1% HAuCl4 to the system obtained in step S1.3 at a volume ratio of 500:1, continue to ultrasonicate for 15 minutes to obtain Fe3O4@Au magnetic nanoparticles.

[0010] Preferably, step S2 specifically includes the following steps: S2.1. Dissolve the peptide segment in dimethyl sulfoxide at a concentration of 1.8 - 2.2 mg mL -1 and store it as a PSBP stock solution at 4°C; S2.2. Dilute the PSBP concentration with water to 0.3 - 0.5 mg mL -1 , mix 0.3 - 0.5 mg mL -1 PSBP with 0.8 - 1.2 mg mL -1 Fe3O4@Au at a volume ratio of 1:(0.8 - 1.2). After magnetic separation, discard the unreacted peptide, collect the Fe3O4@Au - PSBP composite probe, wash and redissolve it to 1 mg mL -1 to obtain the Fe3O4@Au - PSBP composite probe.

[0011] More preferably, step S2 specifically includes the following steps: S2.1. Dissolve the peptide segment in dimethyl sulfoxide at a concentration of 2 mg mL -1 and store it as a PSBP stock solution at 4°C; S2.2. Dilute the PSBP concentration with water to 0.4 mg mL -1 , mix 0.4 mg mL -1 PSBP with 1 mg mL -1 Fe3O4@Au at a volume ratio of 1:1. After magnetic separation, discard the unreacted peptide, collect the Fe3O4@Au - PSBP composite probe, wash and redissolve it to 1 mg mL -1 to obtain the Fe3O4@Au - PSBP composite probe.

[0012] More preferably, in step S2.2, PSBP and Fe3O4@Au are mixed and incubated for 6 - 12 h, preferably 6 h.

[0013] The peptide segment or the selective probe obtained by the method is used to detect polystyrene nanoplastics.

[0014] Preferably, the application operation is as follows: Add 150 μL of the probe Fe3O4@Au - PSBP to 2 mL of PS solutions at different concentrations (0, 10, 20, 40, 80 pg mL -1 ), incubate on a shaker bed for 20 min, then wash and redissolve to 3 mL, measure the absorbance at 755 nm, and construct a standard curve based on the change value of the absorbance. Subsequently, quantify the PS concentration in the sample according to the standard curve.

[0015] Further preferably, the application operation is as follows: the sample treatment method is: add edible salt to distilled water for dissolution according to a solid-liquid ratio of 1:10 g / mL, and take 10 mL of bottled water and milk for standby; after the sample preparation is completed, take 1 mL of bottled water, edible salt solution, and milk respectively, and then add 1 mL of PS solution with different concentrations. The concentration of the PS solution is 0, 40, 100, 160 pg mL -1 ; after mixing evenly, add 150 μL of the probe Fe3O4@Au-PSBP, incubate on an oscillating bed for 20 min, then wash and redissolve to 3 mL, measure the absorbance at 755 nm, and quantify the PS concentration in the sample according to the standard curve and calculate the spike recovery rate.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a PS peptide-modified Fe3O4@Au for simply, selectively and ultrasensitively determining low-concentration PS NPs in food samples. The proposed Fe3O4@Au-PSBP can specifically capture and enrich ultra-low-concentration PS NPs and generate regular response signals. In addition, the proposed Fe3O4@Au-PSBP probe can be easily extended to other types of NPs by simply replacing the polypeptide. As a general probe, it has broad prospects in simply, selectively and highly sensitively detecting harmful NPs in food samples. Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the following-described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0018] Figure 1 TEM images of the prepared AuNPs (a), Fe3O4 (b), and Fe3O4@Au nanocomposites (c); Zeta potential measurement (d).

[0019] Figure 2 (a) UV absorption spectrum of AuNPs; (b) absorption spectra of Fe3O4 and Fe3O4@Au; (c) UV absorption spectra of Fe3O4@Au with gold shells synthesized with different amounts of HAuCl4 (50 - 300 μL). Figure 3. (a) Chemical structural formulas of PSBP-1 and PSBP-2; (b) Changes in absorption intensity (ΔA280) of PSBP-1 and PSBP-2 before and after coupling.

[0020] Figure 4 . (a) Absorption spectra of the supernatant of PSBP-1 blank and PSBP-1 after coupling; (c) Linear relationship between different concentrations of PSBP-1 and ultraviolet absorption values.

[0021] Figure 5 . (a) Changes in absorption intensity (∆A720) of Fe3O4@Au-PSBP under different incubation conditions; (b) Changes in absorption intensity (∆A720) of Fe3O4@Au-PSBP at different PS detection times (10 - 50 min); (c) Absorption spectra of Fe3O4@Au-PSBP with different addition amounts of PS (50 - 250 μL) in Fe3O4@Au-PSBP.

[0022] Figure 6 . (a) Absorption spectra of Fe3O4@Au-PSBP for detecting different concentrations of PS (0 - 80 pg mL -1 ); (b) Linear relationship between the absorption value of Fe3O4@Au-PSBP and the PS concentration in the range of 0 - 80 pg mL -1 .

[0023] Figure 7 The detection method of the present invention is specific to PS nanoplastics.

[0024] Figure 8 The influence of interference from other substances in the food matrix on the detection results. Detailed implementation manners

[0025] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise specified, 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. Although this invention has been described only in terms of preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. In case of conflict with any incorporated literature, the content of this specification shall prevail.

[0028] Various modifications and variations of the specific embodiments of the specification of this invention will be apparent to those skilled in the art without departing from the scope or spirit of this invention. Other embodiments obtained from the specification of this invention will be apparent to those skilled in the art. The specification and examples of this invention are merely exemplary.

[0029] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0030] Materials and Reagents All chemical reagents were of analytical grade and did not require further purification. Ferric chloride hexahydrate (FeCl3∙6H2O, 99%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Hydroxylamine hydrochloride (NH2OH∙HCl) was purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Ethylene glycol (EG, 99.5%), polyethylene glycol-4000 (PEG-4000), sodium acetate trihydrate (NaAc∙3H2O), sodium citrate trihydrate (Na3C6H5O7∙H2O), and dimethyl sulfoxide (DMSO) were purchased from Shanghai Titan Technology Co., Ltd. (Shanghai, China). PSBP-1 (Cys-WSPWGMWSY, Cys-Trp-Ser-Pro-Trp-Gly-Met-Trp-Ser-Tyr, cysteine-tryptophan-serine-proline-tryptophan-glycine-methionine-tryptophan-serine-tyrosine) and PSBP-2 (Mpa-WSPWGMWSY, Mpa-Trp-Ser-Pro-Trp-Gly-Met-Trp-Ser-Tyr, mercaptopropionic acid-tryptophan-serine-proline-tryptophan-glycine-methionine-tryptophan-serine-tyrosine) were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Chloroauric acid (HAuCl4) and polyvinylpyrrolidone (PVP) were purchased from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China), and polyethyleneimine (PEI) was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China).

[0031] Instruments Transmission electron microscope (TEM) images were taken using a JEM-2100F (JEOL, Japan) model. X-ray diffraction (XRD) patterns were obtained using an x-ray diffractometer (Malvern Panalytical, Netherlands) moving at a speed of 2° per second. Ultraviolet-visible spectra were measured using a UV 1091PC spectrophotometer (Shanghai AoXi Scientific Instruments, China). Zeta potential was determined using a Zetasizer Nano ZS Zen 3600 (Malvern Panalytical, Netherlands).

[0032] Example 1 1.1 Synthesis of Fe3O4@Au 16 mL of an aqueous solution of Fe3O4 (1 mg mL -1 ), was mixed with 4 mL of a PEI solution (1 mg mL -1 ), and then sonicated for 30 minutes. It was washed and redissolved to 1 mg mL -1 . 20 mL of Fe3O4-PEI was added to 100 mL of AuNPs, sonicated for 0.5 h, the excess AuNPs were washed away, and then redissolved to 1 mg per milliliter to obtain Fe3O4-Au seeds. 0.3 g of PVP and 0.02 g of NH2OH∙HCl were added to 100 mL of distilled water. Subsequently, 3 mL of Fe3O4-Au seeds were added, and the solution was sonicated (30 KHz, for 15 minutes). Then, 200 μL (during the experiment, different addition volumes were studied, specifically 50 μL, 150 μL, 200 μL, 250 μL, 300 μL) of HAuCl4 (the solution contained 1% HAuCl4) was added to the system, and sonication was continued for 15 minutes.

[0033] 1.2 Modification of Fe3O4@Au with PSBP The polypeptides with different thiol groups modified at the ends were PSBP-1 (Cys-WSPWGMWSY, Cysteine-Tryptophan-Serine-Proline-Tryptophan-Glycine-Methionine-Tryptophan-Serine-Tyrosine) and PSBP-2 (Mpa-WSPWGMWSY, Mercaptopropionic acid-Tryptophan-Serine-Proline-Tryptophan-Glycine-Methionine-Tryptophan-Serine-Tyrosine). The peptides were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 2 mg mL -1 and stored as a stock solution at 4 °C. It was diluted with water to 0.4 mg mL -1 . Then, 2 mL of PSBP (0.4 mg mL -1), mix with 2 mL of Fe3O4@Au (1 mg mL -1 ), and conduct specific research on the mixing time corresponding to the incubation time in 2.4 later. After magnetic separation, discard the unreacted peptides, collect the Fe3O4@Au-PSBP composite probe, wash and redissolve it to 1 mg mL -1 .

[0034] 1.3. Detection of PS NPs based on Fe3O4@Au-PSBP probe Add 150 μL of the probe Fe3O4@Au-PSBP to 2 mL of PS solutions with different concentrations (0, 10, 20, 40, 80 pg mL -1 ), incubate for 20 min on an oscillating bed, then wash and redissolve to 3 mL, and then measure the absorbance at 755 nm. Construct a standard curve based on the change value of the absorbance. Subsequently, quantify the PS concentration in the sample according to the standard curve. The sample treatment method is as follows: Dissolve edible salt in distilled water at a solid-liquid ratio of 1:10 g / mL, and take 10 mL of bottled water and milk for standby; after the sample preparation is completed, take 1 mL each of bottled water, edible salt solution, and milk, and then add 1 mL of PS with different concentrations (0, 40, 100, 160 pg mL -1 ) solutions. After mixing evenly, add 150 μL of the probe Fe3O4@Au-PSBP, incubate for 20 min on an oscillating bed, then wash and redissolve to 3 mL, and then measure the absorbance at 755 nm. Quantify the PS concentration in the sample according to the standard curve and calculate the spike recovery rate.

[0035] 2. Results 2.1 Characterization Figure 1 TEM images of the prepared AuNPs (a), Fe3O4 (b), and Fe3O4@Au nanocomposite (c); (d) Zeta potential measurement; The morphology of the prepared Fe3O4@Au was characterized by high-resolution transmission electron microscopy (HRTEM). Figure 1The TEM images of AuNPs, Fe3O4, and Fe3O4@Au are shown in parts a - c respectively. The prepared AuNPs (Figure 1a) and Fe3O4 (Figure 1b) are well - dispersed with uniform particle sizes, and their diameters are approximately 20 nm and 450 nm respectively. As can be clearly seen from Figure 1c, AuNPs are tightly attached to the surface of Fe3O4. The prepared Fe3O4 is first coated with a PEI layer, then AuNPs are adsorbed, and subsequently uniform gold seeds grow anisotropically on Fe3O4. Since Fe3O4 - PEI is negatively charged while AuNPs are positively charged, the gold seeds are loaded through electrostatic binding. Then, through the NH2OH∙HCl reduction method, a gold shell is formed in - situ in the gaps between the gold seeds on the surface of Fe3O4. The results of zeta potential confirm the successful assembly of the PEI layer, the loading of gold seeds, and the formation of the gold shell (Figure 1d).

[0036] 2.2 UV - Vis Absorption Spectra The UV absorption peak of AuNPs is obvious. The effective coating of Fe3O4 with Fe3O4 and Fe3O4@Au was verified by AuNPs and measured with a UV - Vis spectrometer. Figure 2 a shows the UV absorption spectrum of the synthesized AuNPs, and its UV absorption peak is around 520 nm. As Figure 2 shown in b, the maximum UV absorption peak of the Fe3O4@Au solution has a red - shift phenomenon relative to AuNPs. This observation indicates that the gold shell layer has been effectively coated on the surface of Fe3O4. The results show that gold has a significant influence on the UV absorption signal of Fe3O4@Au. As can be seen from Figure 2 c, the amount of HAuCl4 used in the formation of the gold shell also has a significant influence on the UV absorption signal of the obtained probe. When the amount of HAuCl4 used is less than 200 μL, the UV absorption peak of Fe3O4@Au is relatively flat and unclear, which will lead to a decrease in the sensitivity of subsequent detection. When the addition amount of HAuCl4 is greater than 200 μL, the UV absorption peak of Fe3O4@Au is relatively steeper, showing a more obvious peak. However, this also significantly increases the detection cost. Therefore, in order to ensure that the probe has a sufficiently high sensitivity while controlling the cost of the detection probe, we choose 200 μL as the amount of HAuCl4 used in the subsequent experiment.

[0037] 2.3 Coupling of PSBP and Fe3O4@Au The binding peptides on the surface of Fe3O4@Au - PSBP are very important for the specific recognition of PS NPs. That is to say, the more binding peptides on the surface, the higher the sensitivity of the probe. In the design of the terminal thiol groups, the structural diagrams of two peptide sequences with different terminal modification groups were compared ( Figure 3a). Although the main peptide sequences are the same, the terminal modification groups are different, which can also affect specific interactions due to reasons such as structural hindrance. The changes in absorbance before and after the binding of PSBP-1 and PSBP-2 were compared, and the results showed that the absorbance of PSBP-1 decreased more significantly, indicating a higher binding efficiency ( Figure 3 b). Therefore, PSBP-1 was adopted in the present invention. The binding amount of PSBP-1 was calculated based on the change in ultraviolet absorption rate before and after binding ( Figure 4 ). After incubation with Fe3O4@Au, the absorption rate of PSBP-1 decreased significantly, indicating that the -SH groups provided by cysteine had a high binding efficiency with gold. There was a good linear relationship between the amount of polypeptide and absorbance, and the linear relationship was Y = 2.37465 - 0.00253 (R 2 = 0.9995) (Y: absorbance, X: concentration of PSBP-1). Therefore, the binding amount of PSBP-1 on Fe3O4@Au was 0.041 mg.

[0038] 2.4 Optimization of Fe3O4@Au-PSBP The incubation time was studied (the peptide PSBP-1 was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 2 mg mL -1 and stored as a stock solution at 4 °C. It was diluted with water to 0.4 mg mL -1 . Then, 2 mL of PSBP-1 (0.4 mg mL -1 ) was mixed with 2 mL of Fe3O4@Au (1 mg mL -1 ), and the incubation times were 6 h and 12 h respectively. After incubation, it was washed and redissolved to 1 mg mL -1 , and the absorbance was measured at 755 nm), the detection time (150 μL of the probe Fe3O4@Au-PSBP was added to 2 mL of 5 groups of PS solutions with a concentration of 40 pg mL -1 . They were incubated on an oscillating bed for 10 min, 20 min, 30 min, 40 min, and 50 min respectively, then washed and redissolved to 3 mL, and the absorbance was measured at 755 nm), and the concentration of Fe3O4@Au-PSBP (50, 100, 150, 200, and 250 μL of the probe Fe3O4@Au-PSBP were added to 2 mL of 5 groups of PS solutions with a concentration of 40 pg mL -1 respectively. They were incubated on an oscillating bed for 20 min, then washed and redissolved to 3 mL, and the absorbance was measured at 755 nm) were studied to optimize the best detection conditions. After Fe3O4@Au was incubated with PSBP for 6 h, the probe produced a strong detection signal, indicating that 6 h was sufficient for PSBP to bind to the surface of Fe3O4@Au (Figure 5 a). Figure 5 b shows the influence of the detection time. After adding the Fe3O4@Au-PSBP probe into the detection system, we monitored the signal changes for 10 to 50 min. The results showed that the capture of PSNPs by the Fe3O4@Au-PSBP probe was very rapid, and an obvious detection signal appeared around 10 min. The detection signal reached the maximum value and tended to be stable after 20 min. Therefore, the selected detection time was 20 min. The amount of the Fe3O4@Au-PSBP probe had a great influence on the detection sensitivity. If the amount of the probe was too small, the target PS NPs could not be completely captured; if the amount of the probe was too large, the excess probe in the system would also generate interference signals, affecting the detection sensitivity. Figure 5 c shows that there were obvious differences in the ultraviolet-visible absorption intensities with different amounts of the probe. Among them, 150 μL was the best. At this dosage, the PS NPs in the system could be effectively captured, but the amount of the probe was not too much to cause signal interference.

[0039] 2.5 Detection of PS by Fe3O4@Au-PSBP Under the optimal experimental conditions (using Fe3O4@Au with a 200 μL gold shell, conjugated with PSBP-1, incubated for 6 hours; the optimal detection conditions were: detection time 20 min, probe dosage 150 μL), the analytical performance of the developed Fe3O4@Au-PSBP probe was evaluated ( Figure 6 ). The ultraviolet-visible absorption intensity in the solution increased with the increase in the concentration of PS NPs ( Figure 6 a). In the range of 0 to 80 pg mL -1 , the Δ absorption (the change in the absorption intensity of PS NPs at 755 nm) had a linear relationship with the concentration of PS NPs. The linear relationship was Y = 0.0025X + 0.0223 (R 2 = 0.9021) (Y: Δ absorption value, X: concentration of PS NPs) ( Figure 6 b). This probe had a very high detection sensitivity and could detect low-concentration PS NPs in the system.

[0040] 2.6 Detection of real samples The selectivity of the proposed Fe3O4@Au-PSBP probe for detecting PS NPs was investigated in actual samples. In bottled water samples, the recoveries of spiked PS NPs were 99.10% - 101.96%; in edible salt samples, the recoveries were 98.85 - 102.67%; in milk samples, the recoveries were 104.30 - 112.10% (Table 1). The probe successfully detected spiked PS in bottled water, edible salt, and milk with good recoveries. The results indicate that the probe provides a simple, rapid, and sensitive method for the reliable analysis of ultra-low concentrations of PS NPs in actual samples.

[0041] Table 1. Detection of PS in spiked samples

[0042] Example 2 Under the optimal preparation conditions of Example 1: The polypeptide PSBP-1 was changed to PSBP-2, and the others were the same as in Example 1.

[0043] 1.1 Synthesis of Fe3O4@Au Mix 16 mL of Fe3O4 (1 mg mL -1 ) aqueous solution with 4 mL of PEI solution (1 mg mL -1 ), and then sonicate for 30 minutes. Wash and redissolve to 1 mg mL -1 . Add 20 mL of Fe3O4-PEI to 100 mL of AuNPs, sonicate for 0.5 h, wash away the excess AuNPs, and redissolve to 1 mg per milliliter to obtain Fe3O4-Au seeds. Add 0.3 g of PVP and 0.02 g of NH2OH∙HCl to 100 mL of distilled water. Subsequently, add 3 mL of Fe3O4-Au seeds and sonicate the solution for 15 minutes. Then, add 200 μL of HAuCl4 (containing 1% HAuCl4 in the solution) to the system and continue to sonicate for 15 minutes.

[0044] 1.2. Modification of Fe3O4@Au with PSBP The terminal-modified polypeptides were PSBP-1 (Cys-WSPWGMWSY, Cysteine-Tryptophan-Serine-Proline-Tryptophan-Glycine-Methionine-Tryptophan-Serine-Tyrosine). The peptide was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 2 mg mL -1 and stored as a stock solution at 4 °C. Dilute with water to 0.4 mg mL -1 . Then, add 2 mL of PSBP (0.4 mgmL -1), and incubated with 2 mL of Fe3O4@Au (1 mg mL -1 ) for 6 h. After magnetic separation, the unreacted peptides were discarded, and the Fe3O4@Au-PSBP composite probe was collected, washed, and redissolved to 1 mg mL -1 .

[0045] 1.3. Detection of PS NPs based on the Fe3O4@Au-PSBP probe 150 μL of the probe Fe3O4@Au-PSBP was added to 2 mL of PS solutions at different concentrations (0, 10, 20, 40, 80 pg mL -1 ), incubated on an orbital shaker for 20 min, then washed and redissolved to 3 mL, and the absorbance was measured at 755 nm. A standard curve was constructed based on the change in absorbance value. Subsequently, the PS concentration in the sample was quantified according to the standard curve. The sample treatment method was as follows: table salt was added to distilled water at a solid-liquid ratio of 1:10 g / mL to dissolve, and 10 mL of bottled water and milk were taken for standby; after sample preparation, 1 mL of each of bottled water, table salt solution, and milk was taken and then added to 1 mL of PS at different concentrations (0, 40, 100, 160 pg mL -1 ). After mixing evenly, 150 μL of the probe Fe3O4@Au-PSBP was added, incubated on an orbital shaker for 20 min, then washed and redissolved to 3 mL, and the absorbance was measured at 755 nm. The PS concentration in the sample was quantified according to the standard curve and the spike recovery rate was calculated.

[0046] The method for determining the coupling rate was as follows: the peptide was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 2 mg mL -1 and stored as a stock solution at 4 °C. It was diluted with water to 0.4 mg mL -1 . Then, 2 mL of PSBP (0.4 mg mL -1 ) was mixed with 2 mL of distilled water and 2 mL of Fe3O4@Au (1 mg mL -1 ) respectively and incubated for 6 h. The difference in absorbance values between the blank group after 6 h and the supernatant after magnetic separation to remove magnetic beads after coupling with Fe3O4@Au was measured at 280 nm. According to the standard curve and the calculated amount of coupling, the coupling rate was obtained. The results are shown in Table 2.

[0047] Table 2

[0048] As can be seen from Table 2, the coupling efficiency of polypeptide PSBP-1 is higher than that of PSBP-2, and the detection sensitivity of the probe obtained by coupling with PSBP-1 will be higher than that of the probe obtained by coupling with PSBP-2. Example 3 Under the optimal preparation conditions of Example 1: Change the detection materials in Step 1.3 to PS, PP, PE, PMMA, PET, and PVC, and add 150 μL of the probe Fe3O4@Au-PSBP to the PS, PP, PE, PMMA, PET, and PVC solutions with the same concentration of 40 pg / mL, mix well, incubate on an orbital shaker for 20 min, then wash and redissolve to 3 mL, and then measure the absorbance at 755 nm. The results are shown in -1 ... Figure 7 .

[0049] As Figure 7 can be seen, the detection method of the present invention has high specificity for PS nanoplastics, can accurately identify PS nanoplastics, and does not respond to other common nanoplastics such as PP and PE. These high specificities provide a reliable means for the detection of PS nanoplastics, which helps to accurately identify and quantitatively analyze PS nanoplastics in complex samples. Example 4 Under the optimal preparation conditions of Example 1: The detection materials in 1.3 are PS and PS + different ions, and the specific concentrations are K + (45 μM), Na + (45 μM), Cl -1 (45 μM), lactose (Lac, 45 μM), bovine serum albumin (BSA, 4.5 μM), and glucose (Glu, 4.5 μM), and the concentration of PS is 40 pg / mL -1 . Then add 150 μL of the probe Fe3O4@Au-PSBP, mix well, incubate on an orbital shaker for 20 min, then wash and redissolve to 3 mL, and then measure the absorbance at 755 nm. As Figure 8 can be seen, the detection method of the present invention has extremely high specificity for PS nanoplastics. This method can accurately identify and distinguish PS nanoplastics, effectively avoid the interference of other substances in the food matrix, has strong peptide-binding ability, and ensures the accuracy and reliability of the detection results. The technical solution of the present invention is explained by the above embodiments, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above specific embodiments to be implemented. Any improvement made by those skilled in the art based on the present invention, or the equivalent replacement of the materials selected by the present invention, etc., all fall within the protection scope of the patent.

Claims

1. A peptide segment for modifying Fe3O4@Au magnetic nanoparticles, characterized in that: The peptide segment is α-peptide segment 1 or / and β-peptide segment 1, and the peptide segment fragment is shown in SEQ ID NO: 1, Among them, α and β are modification groups, α is cysteine, and β is mercaptopropionic acid; The α and β are connected to the N-terminus of peptide segment 1; The Fe3O4@Au magnetic nanoparticles are of a core-shell structure, having a Fe3O4 magnetic core and an Au shell, and the peptide segment is connected to the surface of the Au shell via a gold-sulfur bond; the Fe3O4@Au magnetic nanoparticles are used to prepare a selective probe for detecting polystyrene nanoplastics.

2. A method for preparing a selective probe for detecting polystyrene nanoplastics using the peptide-modified Fe3O4@Au magnetic nanoparticles according to claim 1, characterized in that: The method comprises the following steps: S1. Synthesis of Fe3O4 magnetic nanoparticles by hydrothermal method; S2. Peptide modified Fe3O4@Au to obtain Fe3O4@Au-PSBP composite probe; completed the preparation of selective probe.

3. The method for preparing a selective probe for detecting polystyrene nanoplastics using peptide-modified Fe3O4@Au magnetic nanoparticles according to claim 2, characterized in that: The step S1 specifically includes the following steps: S1.1 0.8-1.2 mg mL -1 Fe3O4 aqueous solution with 0.8-1.2 mg mL -1 PEI solution was mixed at a volume ratio of (4.5-5.5):1, sonicated for 25-35 minutes, washed and redissolved to 0.8-1.2 mg mL -1 Fe3O4-PEI solution; S1.2 Add Fe3O4-PEI solution to AuNPs at a volume ratio of (4.5-5.5):1, ultrasonicate for 0.4-0.6 h, wash away excess AuNPs, and then dissolve to 1-3 mg / ml to obtain Fe3O4-Au seeds; S1.3 PVP and NH2OH∙HCl were added to distilled water at a solid-liquid ratio of (0.2-0.4):(0.015-0.025):100 g / g / mL, and Fe3O4-Au seeds were added to the distilled water at a volume ratio of 100:(2-4), and the solution was sonicated for 12-18 minutes; S1.4 Add HAuCl4 solution containing 1% HAuCl4 to the system obtained in step S1.3 at a volume ratio of (450-550):1, and continue ultrasonic treatment for 14-16 minutes to obtain Fe3O4@Au magnetic nanoparticles.

4. The method for preparing a selective probe for detecting polystyrene nanoplastics using peptide-modified Fe3O4@Au magnetic nanoparticles according to claim 3, characterized in that: The step S1 specifically includes the following steps: S1.1 1 mg mL -1 Fe3O4 aqueous solution and 1 mg mL -1 PEI solution was mixed at a volume ratio of 5:1, sonicated for 30 min, washed and redissolved to 1 mg mL -1 Fe3O4-PEI solution; S1.2 Add Fe3O4-PEI solution to AuNPs at a volume ratio of 5:1, ultrasonicate for 0.5 h, wash away excess AuNPs, and then dissolve to 1 mg / ml to obtain Fe3O4-Au seeds; S1.3 PVP and 0.02 g NH2OH∙HCl were added to distilled water at a solid-liquid ratio of 0.3:0.02:100 g / g / mL. Fe3O4-Au seeds were also added to the distilled water at a volume ratio of 100:3, and the solution was sonicated for 15 min. S1.4 Add HAuCl4 solution containing 1% HAuCl4 to the system obtained in step S1.3 at a volume ratio of 500:1, and continue ultrasonic treatment for 15 minutes to obtain Fe3O4@Au magnetic nanoparticles.

5. The method for preparing a selective probe for detecting polystyrene nanoplastics using peptide-modified Fe3O4@Au magnetic nanoparticles according to claim 2, characterized in that: The step S2 specifically includes the following steps: S2.

1. The peptide was diluted to 1.8-2.2 mg mL -1 The concentration was dissolved in dimethyl sulfoxide and stored at 4 °C as PSBP stock solution; S2.

2. Dilute PSBP with water to a concentration of 0.3-0.5 mg mL -1 , 0.3-0.5 mg mL -1 PSBP with 0.8-1.2 mgmL -1 Fe3O4@Au was mixed in a volume ratio of 1:(0.8-1.2). After magnetic separation, the unreacted peptides were discarded, and the Fe3O4@Au-PSBP composite probe was collected, washed and redissolved to 1 mg mL -1 , and obtained the Fe3O4@Au-PSBP composite probe.

6. The method for preparing a selective probe for detecting polystyrene nanoplastics using peptide-modified Fe3O4@Au magnetic nanoparticles according to claim 5, characterized in that: The step S2 specifically includes the following steps: S2.

1. The peptide was concentrated to 2 mg mL -1 The concentration was dissolved in dimethyl sulfoxide and stored at 4 °C as PSBP stock solution; S2.

2. Dilute PSBP with water to a concentration of 0.4 mg mL -1 , 0.4 mg mL -1 PSBP with 1 mg mL -1 Fe3O4@Au was mixed in a volume ratio of 1:

1. After magnetic separation, the unreacted peptide was discarded, and the Fe3O4@Au-PSBP composite probe was collected, washed, and redissolved to 1 mg mL -1 , and obtained the Fe3O4@Au-PSBP composite probe.

7. The method for preparing a selective probe for detecting polystyrene nanoplastics using peptide-modified Fe3O4@Au magnetic nanoparticles according to claim 5, characterized in that: The step S2.

2. PSBP and Fe3O4@Au are mixed and incubated for 6-12 h, preferably 6 h.

8. The peptide segment of claim 1 or the selective probe obtained by the method of any one of claims 2 to 7 is used to detect polystyrene nanoplastics.

9. The use according to claim 8, characterized in that: The application operation is as follows: in 2 mL PS solution of different concentrations (0, 10, 20, 40, 80 pg mL -1 150 μL of Fe3O4@Au-PSBP probe was added to the sample, incubated on a shaking bed for 20 min, then washed and redissolved to 3 mL, the absorbance was measured at 755 nm, and a standard curve was constructed based on the change in absorbance. Subsequently, the PS concentration in the sample was quantified based on the standard curve.

10. The use according to claim 8, characterized in that: The application operation is as follows: the sample processing method is: edible salt is added to distilled water at a solid-liquid ratio of 1:10 g / mL to dissolve, and 10 mL of bottled water and milk are taken for standby; after the sample preparation is completed, 1 mL of bottled water, edible salt solution, and milk are taken respectively and then added to 1 mL of PS solution with different concentrations, and the concentration of PS solution is 0, 40, 100, and 160 pg mL -1 After mixing evenly, add 150 μL of probe Fe3O4@Au-PSBP, incubate on a shaking bed for 20 min, then wash and redissolve to 3 mL, measure the absorbance at 755 nm, quantify the PS concentration in the sample according to the standard curve and calculate the spike recovery rate.

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