A multi-indicator SERS sensor for detecting spoilage of aquatic products and its application

Abstract

The present invention belongs to the field of chemical testing and analysis, and specifically relates to a multi-indicator SERS sensor for detecting spoilage of aquatic products and its application. Perfluoropolyether is injected into a polytetrafluoroethylene-lined porous membrane by spin coating to form a surface-enhanced Raman scattering film. When droplets containing AuNS@ZIF‑8 evaporate on the substrate, aggregates are produced in the form of SERS sensors. By connecting different SERS probe molecules to AuNSs, the AuNS@ZIF‑8SLIPS substrate can adsorb various gas molecules and respond to various indicators in the system. The established SERS platform successfully implemented the dual indicators of pH and BAs, further improving the accuracy of monitoring the deterioration of aquatic products. These studies have opened up new prospects for the development of new smart packaging.

Description

Technical Field

[0001] The invention belongs to the field of chemical testing and analysis, and particularly relates to a multi-indicator SERS sensor for detecting spoilage of aquatic products and an application thereof. Background Art

[0002] Food safety and quality issues are receiving increasing attention, particularly in the areas of public health and safety control. Due to the susceptibility of food to foodborne illnesses, food spoilage has become a major reason for stringent food safety control requirements. According to 2019 World Food Report, one-third of food is wasted annually, with microbial spoilage being the primary cause. During food spoilage, particularly during the decaying stage of aquatic products, microbial populations increase dramatically, leading to the extensive decomposition of nitrogenous compounds, resulting in the formation of α-keto acids, ammonia, organic acids, sulfides, and various biogenic amines (BAs). These compounds often have an unpleasant odor and high toxicity levels, posing a threat to food safety.

[0003] Surface-enhanced Raman scattering (SERS) is a powerful technique that exploits localized surface plasmon resonance (LSPR) of plasmonic metal nanostructures. It has been widely used in biological and chemical detection due to its advantages such as high sensitivity, significant sample resolution, good multiplexing capability, and non-invasive sampling. Consequently, SERS sensors have been widely used to detect spoilage indicators. However, due to the low adsorption of gas molecules on SERS substrates and the uneven distribution of hotspots, SERS-based sensing substrates exhibit low sensitivity and repeatability in real-world sample applications. Preventing SERS probes from interacting with interfering species in complex environments is difficult and often leads to false positive / negative results.

[0004] Plasmonic metal nanomaterials assembled on metal-organic frameworks (MOFs) have attracted increasing interest in recent years. Notably, MOFs exhibit excellent gas adsorption capabilities due to their extensive, large-surface-area, nanoporous three-dimensional networks, which significantly enhances the detection sensitivity of MOF-functionalized SERS platforms. The porous MOF shell can also function as a molecular sieve, significantly simplifying SERS spectroscopy and preventing corrosion of SERS probe molecules in complex environments. Summary of the Invention

[0005] Currently, a range of fluorescence- and colorimetric-based interfacial sensors have been reported for real-time and simple food spoilage monitoring. However, it is well known that these methods exhibit inherent limitations, including limited sensitivity, low precision, low biocompatibility, and a single monitoring indicator. Therefore, there is an urgent need to establish a suitable sensing platform for effective and real-time monitoring of food spoilage. In particular, the development of a rapid on-site detection method that is universally applicable to the monitoring of aquatic product spoilage is urgently needed.

[0006] Based on this, in order to solve the above-mentioned technical problems, the present invention provides a preparation method and application of a SERS platform for monitoring the corruption of aquatic products. It has the characteristics of simple preparation method, easy portability, short detection time, and high sensitivity. In particular, the technical solution of the present invention can realize the simultaneous monitoring of multiple corruption markers, and has universal applicability for the detection of corruption markers, which is different from the limitation of the prior art that can only detect specific corruption markers. In addition, a well-designed hydrophobic substrate can concentrate the plasma sensitive area and improve the detection sensitivity and repeatability of the SERS substrate.

[0007] The multi-indicator SERS sensor for detecting aquatic product spoilage is obtained by adding mixed liquid A and mixed liquid B to a SERS membrane; the mixed liquid A is obtained by incubating the AuNS@ZIF-8 reinforced material and a probe molecule for monitoring the pH value of the spoilage system for 10-14 hours, and the mixed liquid B is obtained by incubating the AuNS@ZIF-8 reinforced material and a probe molecule for monitoring the BAs content of the spoilage system for 10-14 hours; the AuNS@ZIF-8 reinforced material is obtained by reacting and separating CTAB-coated gold nanostars (AuNSs), methylimidazole and zinc salt.

[0008] Preferably, the SERS membrane is obtained by dispersing perfluoropolyether in a polytetrafluoroethylene film and then heating it.

[0009] Specifically, a polytetrafluoroethylene film is adhered to a flat glass sheet, a hydrophobic perfluoropolyether is dispersed on the glass sheet, and after removing excess lubricating liquid, the injected film is heated to form a surface enhanced Raman scattering film, namely the SERS film.

[0010] Specifically, the perfluoropolyether is dispersed by spin coating at a rotation speed of 600-1500 rpm and a time of 30-60 seconds.

[0011] Preferably, the preparation method of the CTAB-coated gold nanostars (AuNSs) is as follows:

[0012] S1: Sodium citrate, citric acid, ethylenediaminetetraacetic acid and tetrachloroauric acid are heated in water for reaction and then cooled to obtain an Au nanoparticle solution;

[0013] S2: mixing the Au nanoparticle solution, tetrachloroauric acid, and hydrochloric acid, adding silver nitrate and ascorbic acid, and reacting for 8-12 minutes to obtain an intermediate product;

[0014] S3: adding cetyltrimethylammonium bromide to the intermediate product, reacting for 1-3 minutes, cooling with ice water, and separating to obtain the CTAB-coated gold nanostars (AuNSs).

[0015] Furthermore, in step S1, the temperature of the heating reaction is 98-100°C.

[0016] Furthermore, in step S1, the heating reaction time is 50-70s.

[0017] Specifically, the preparation method of the AuNS@ZIF-8 reinforced material is as follows: spherical citric acid-stabilized gold nanoparticles (AuNPs) are used as seeds, Ag + CTAB-coated gold nanostars (AuNSs) were synthesized using ions as a blocking agent and ascorbic acid as a reducing agent. A MeIm solution and a Zn(NO3)2·6H2O solution were prepared using the AuNSs solution as a solvent. The MeIm solution and 200 μL of the Zn(NO3)2·6H2O solution were then placed in a glass bottle filled with ultrapure water, magnetically stirred at room temperature for 10-30 minutes, and then allowed to stand. Finally, the particles were collected by centrifugation, washed twice with methanol, and then redispersed in methanol to obtain the AuNS@ZIF-8 reinforced material.

[0018] Preferably, the zinc salt is zinc nitrate and / or zinc acetate.

[0019] Preferably, the molar ratio of the methylimidazole to the zinc salt is 1:1-1.5.

[0020] Preferably, the probe molecule for monitoring the pH value of the corruption system is 4-mercaptobenzaldehyde (4-MBA).

[0021] Preferably, the probe molecule used to monitor the BAs content in the corruption system is 4-mercaptopyridine (4-Mpy).

[0022] The application of the multi-indicator SERS sensor for detecting spoilage of aquatic products comprises the following steps: adding aquatic product samples of different freshness to the multi-indicator SERS sensor for detecting spoilage of aquatic products, performing surface-enhanced Raman scattering, and obtaining the freshness of the aquatic product samples by calculating the relationship between freshness and SERS intensity.

[0023] The present invention provides a novel real-time multi-indicator food spoilage monitoring strategy based on SERS sensors, which consists of AuNS@ZIF-8 and a liquid-infused smooth porous surface (SLIPS) substrate. The platform is simply prepared by concentrating AuNS@ZIF-8 on a SLIP substrate. By attaching different SERS probe molecules to AuNSs, the AuNS@ZIF-8 SLIPS substrate can adsorb various gas molecules and respond to various indicators in the system. Using 4-Mpy and 4-MBA as SERS probe molecules, the pH and BAs spoilage indicators during the shrimp spoilage process were monitored in real time. This further improves the accuracy of aquatic product spoilage monitoring.

[0024] The mechanism of this invention is as follows: Based on glass sheets, nanoparticles, and metal-organic framework composites, this invention proposes a very simple strategy for effective, real-time, and multi-indicator monitoring of aquatic product spoilage. A hydrophobic perfluoropolyether is spin-coated onto a polytetrafluoroethylene-lined porous membrane to form a surface-enhanced Raman scattering (SERS) film. When droplets containing AuNS@ZIF-8 evaporate onto a SLIPS substrate, aggregates form as SERS sensors without the coffee ring effect. Crucially, by attaching different SERS probe molecules to the AuNSs, the AuNS@ZIF-8 SLIPS substrate can adsorb a variety of gas molecules and respond to a variety of indicators in the system. The established SERS platform successfully implements dual indicators of pH and BAs, further improving the accuracy of aquatic product spoilage monitoring. These studies open up new prospects for the development of novel smart packaging.

[0025] The technical solution of the present invention has the following advantages over the prior art:

[0026] Combined with the developed procedure for accurate aggregation of nanoparticles using smooth hydrophobic surfaces, the multifunctional sensing platform strategy is believed to be applicable to a wider range of sensing devices, such as fluorescence, Raman, and infrared spectrometers, to achieve economical, simple, rapid, flexible, and portable detection.

[0027] Furthermore, this strategy is opening up new possibilities for a wide range of applications, not only in food spoilage monitoring but also in biosensors and environmental monitoring.

[0028] (1) In the SERS sensing platform described in the present invention, when droplets containing AuNS@ZIF-8 nanoparticles evaporate on the SLIPS substrate, the nanoparticles can be accurately aggregated, achieving strong SERS enhancement.

[0029] (2) The present invention realizes the simultaneous monitoring of multiple corruption markers by connecting different probe molecules, while avoiding mutual interference between different markers and improving the accuracy of monitoring.

[0030] (3) Combined with the developed procedure for accurate aggregation of nanoparticles using smooth hydrophobic surfaces, the multifunctional sensing platform strategy is believed to be applicable to a wider range of sensing devices, such as fluorescence, Raman, and infrared spectrometers, to achieve economical, simple, rapid, flexible, and portable detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is a high-resolution transmission electron microscopy photograph of AuNS and AuNS@ZIF-8 in Example 1 of the present invention;

[0032] Figure 2is the X-ray diffraction pattern of AuNS and AuNS@ZIF-8 in Example 1 of the present invention;

[0033] Figure 3 is the SERS spectra of AuNS@ZIF-8@4-MBA at different concentrations of putrescine in Example 3 of the present invention;

[0034] Figure 4 is the SERS spectra of AuNS@ZIF-8@4-Mpy at different pH values ​​in Example 4 of the present invention;

[0035] Figure 5 This is a diagram of the spoilage process of whole shrimp at 25° C. monitored in real time using the SERS sensing platform described in Example 5 of the present invention. DETAILED DESCRIPTION

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0037] Example 1

[0038] (1) 3.5 mL of 60 mM sodium citrate and 1.5 mM citric acid were added to 144 mL of boiling water (ultrapure water) and heated continuously for 30 min to synthesize AuNPs seeds.

[0039] (2) Add 0.1 mL of 30 mM ethylenediaminetetraacetic acid and 1 mL of 25 mM aqueous chloroauric acid (HAuCl4) to the AuNP seed crystals. After 60 seconds of reaction, the addition is stopped, the temperature is lowered to 95°C, and the reaction flask is immersed in ice water to interrupt the reaction. Au nanoparticles with an average core diameter of approximately 13 nm are obtained.

[0040] (3) 40 mL of 0.25 mM HAuCl4 aqueous solution was mixed with 0.06 mL of 1 M HCl and 3 mL of AuNPs seeds and stirred at room temperature to obtain AuNSs. Subsequently, 0.3 mL of 1 mM silver nitrate and 0.3 mL of 66.67 mM ascorbic acid were injected simultaneously. After 10 minutes, 10 mL of 0.2 M hexadecyltrimethylammonium bromide aqueous solution (CTAB) was added. After 2 minutes, the flask was immersed in an ice water bath to interrupt the reaction. The mixture was centrifuged at 4000 RCF (relative centrifugal force) for 10 minutes and the mixture was washed with 5 × 10 -4 The CTAB solution was washed twice and finally dispersed in 5 mL of 5 × 10 -4 M CTAB solution to ensure the stability of AuNSs during storage.

[0041] (4) A 2 M MeIm solution and a 0.08 M Zn(NO3)2·6H2O solution were prepared using the AuNSs solution as solvent. 800 μL of the MeIm solution and 200 μL of the Zn(NO3)2·6H2O solution were then placed in a glass bottle containing 600 μL of ultrapure water, magnetically stirred at room temperature for 30 min, and allowed to stand for 30 min. Finally, the particles were collected by centrifugation (4000 rpm, 5 min), washed twice with methanol, and then redispersed in 1 mL of methanol.

[0042] Example 2

[0043] The amount of sodium citrate in step (1) of Example 1 was adjusted to 4 mL, 80 mM.

[0044] Other steps were the same as those in Example 1, and the nanomaterial with SERS enhancement was obtained.

[0045] Example 3

[0046] Steps (1)-(4) are the same as in Example 1.

[0047] (5) A polytetrafluoroethylene film is adhered to a flat glass sheet, a hydrophobic perfluoropolyether is dispersed on the glass sheet, excess lubricating fluid is removed, and the injected film is heated to form a surface enhanced Raman scattering (SERS) film, thereby obtaining the SERS sensor (sensing platform).

[0048] (6) AuNS@ZIF-8 was incubated with 4-MBA (1 mM) for 12 h and then centrifuged and dropped onto the membrane surface.

[0049] (7) AuNS@ZIF-8 was incubated with 4-Mpy (100 μM) for 12 h and then centrifuged and dropped onto the membrane surface.

[0050] (8) Fresh shrimp samples were placed in a closed culture dish containing an integrated AuNS@ZIF-8 slip substrate droplet containing SERS probe molecules. Laser light was transmitted through the glass for SERS measurement.

[0051] Example 4

[0052] The concentration of 4-MBA in step (6) of Example 3 was adjusted to 0.5 mM, and the other parameters were kept consistent with Example 3 to obtain the SERS sensor (sensing platform) with the function of monitoring aquatic product spoilage with multiple indicators.

[0053] Example 5

[0054] The 4-MBA (1 mM) in step (6) of Example 3 was adjusted to 4-Mpy (100 μM), and the other steps remained the same as in Example 3 to obtain the SERS sensor (sensing platform) with the function of monitoring aquatic product spoilage with multiple indicators.

[0055] Performance Testing

[0056] (1) High-resolution transmission electron microscopy

[0057] Figure 1 The high-resolution transmission electron microscopy images of AuNSs and AuNS@ZIF-8 nanoparticles in this example are shown in Figure 2. Figure 1 As can be seen from the A, the seed-mediated method synthesized AuNSs with an average diameter of 58.2±1.9nm, a regular, uniform polyhedral shape, and a uniform size distribution. During the synthesis process, the AuNSs were dispersed in a CTAB solution to ensure the stability of the AuNSs and better promote the formation and growth of the MOF shell. Figure 1 Figure B is a high-resolution transmission electron microscopy image of AuNS@ZIF-8. It can be seen that the prepared single ZIF-8 contains a single AuNS with an average diameter of 292.0±5.2nm, which is a submicron truncated rhombic dodecahedron shape.

[0058] (1) X-ray diffraction pattern

[0059] Figure 2 The X-ray diffraction patterns of AuNSs and AuNS@ZIF-8 in Example 1. The X-ray diffraction peaks of bare AuNS are located at 38.2, 44.4, 64.6 and 77.6° ( Figure 1 The blue spectrum in D). These peaks come from the (111), (200), (220) and (311) faces of Au (in Figure 1 The X-ray diffraction spectrum of ZIF-8 is marked with blue triangles at 7.6, 10.7, 13.0, 15.0, 16.7 and 18.3° ( Figure 1 There are 6 additional peaks at the red spectrum in D of ZIF-8, which are consistent with the (011), (002), (112), (022), (013) and (222) crystal planes of ZIF-8 ( Figure 1 The X-ray diffraction spectrum of AuNS@ZIF-8 composite particles ( Figure 1 The black spectrum in D) shows all the characteristic peaks of the above-mentioned bare AuNSs and ZIF-8, and the X-ray diffraction results support the formation of AuNS@ZIF-8 nanocomposites.

[0060] (3) Feasibility analysis

[0061] Figure 3The SERS spectra of the SERS sensor monitoring putrescine concentration are shown in Figure 2. The AuNS@ZIF-8 incubated with the 4-MBA probe molecule was dropped onto a glass slide. The glass slide substrate aggregated with AuNS@ZIF-8@4-MBA was placed in a closed culture dish filled with putrescine at different volume concentrations to allow them to react completely. The Raman spectra were then collected. As the putrescine concentration increased, the 1638 cm -1 The Raman peak of putrescine increased significantly, while the other peaks remained largely unchanged. This peak was assigned to the C=N stretching of the imine, indicating a reaction between the -NH2 group of putrescine and the -CHO group of 4-MBA. This further validated the feasibility of the SERS sensor for monitoring putrescine.

[0062] Figure 4 Figure 2 shows the SERS spectra of the SERS sensing platform monitoring pH. The SERS spectra of AuNS@ZIF-8@4-Mpy aggregated on a glass slide at different pH levels are shown. At different pH levels, the 1013 / 1100 cm -1 and 1610 / 1577cm -1 The intensity ratios of the two pairs of Raman spectra are very different. 1013 / 1100 cm -1 and 1610 / 1577cm -1 The intensity ratio of these two pairs of Raman peaks has a sensitive response in the pH range of 4.0 to 9.0. Therefore, the intensity ratio of these two pairs of Raman peaks was selected to monitor the pH response of spoilage.

[0063] (4) Monitoring of actual samples

[0064] AuNS@ZIF-8@4-Mpy and AuNS@ZIF-8@4-MBA were assembled on the same SLIPS substrate as a composite SERS sensor. Specifically, the composite SERS sensor was placed in a closed culture dish with the whole shrimp. The 1638 / 1074 cm -1 The intensity ratio of 1013 / 1100 cm-1 of the 4-Mpy sensor is -1 and 1610 / 1577cm -1 The intensity ratios of the shrimp meat and shrimp oil have changed significantly, indicating that the freshness of the shrimp meat has changed significantly.

[0065] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A multi-indicator SERS sensor for detecting spoilage of aquatic products, characterized in that: The multi-indicator SERS sensor for detecting spoilage of aquatic products is obtained by adding mixed solution A and mixed solution B into a SERS film; The mixed solution A is obtained by incubating the AuNS@ZIF-8 reinforced material and the probe molecule for monitoring the pH value of the corruption system for 10-14 hours, and the mixed solution B is obtained by incubating the AuNS@ZIF-8 reinforced material and the probe molecule for monitoring the BAs content of the corruption system for 10-14 hours; the AuNS@ZIF-8 reinforced material is obtained by reacting and separating CTAB-wrapped gold nanostars, methylimidazole and zinc salt.

2. The multi-indicator SERS sensor for detecting aquatic product spoilage according to claim 1, wherein: The SERS film is obtained by dispersing perfluoropolyether in a polytetrafluoroethylene film and then heating it.

3. The multi-indicator SERS sensor for detecting aquatic product spoilage according to claim 1, wherein: The preparation method of the CTAB-wrapped gold nanostars is as follows: S1: Sodium citrate, citric acid, ethylenediaminetetraacetic acid and tetrachloroauric acid are heated in water for reaction and then cooled to obtain an Au nanoparticle solution; S2: mixing the Au nanoparticle solution, tetrachloroauric acid, and hydrochloric acid, adding silver nitrate and ascorbic acid, and reacting for 8-12 minutes to obtain an intermediate product; S3: adding cetyltrimethylammonium bromide to the intermediate product, reacting for 1-3 minutes, cooling with ice water, and separating to obtain the CTAB-coated gold nanostars.

4. The multi-indicator SERS sensor for detecting aquatic product spoilage according to claim 3, wherein: In step S1, the temperature of the heating reaction is 98-100°C.

5. The multi-indicator SERS sensor for detecting aquatic product spoilage according to claim 3, wherein: In step S1, the heating reaction time is 50-70 seconds.

6. The multi-indicator SERS sensor for detecting aquatic product spoilage according to claim 1, wherein: The zinc salt is zinc nitrate and / or zinc acetate.

7. The multi-indicator SERS sensor for detecting aquatic product spoilage according to claim 1, wherein: The molar ratio of the methylimidazole to the zinc salt is 1:1-1.

5.

8. The multi-indicator SERS sensor for detecting aquatic product spoilage according to claim 1, wherein: The probe molecule used to monitor the pH value of the corruption system is 4-mercaptobenzaldehyde.

9. The multi-indicator SERS sensor for detecting aquatic product spoilage according to claim 1, wherein: The probe molecule used to monitor the BAs content in the corruption system is 4-mercaptopyridine.

10. Use of the multi-indicator SERS sensor for detecting spoilage of aquatic products according to any one of claims 1 to 9, characterized in that: The method comprises the following steps: adding aquatic product samples of different freshness to the multi-indicator SERS sensor for detecting aquatic product spoilage, performing surface enhanced Raman scattering, and obtaining the freshness of the aquatic product samples by calculating the relationship between freshness and SERS intensity.

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