Method for detecting saxitoxin based on transition metal oxide / noble metal particle composite SERS (Surface Enhanced Raman Scattering) substrate

Abstract

The invention belongs to the technical field of toxin detection, and particularly relates to a method for detecting saxitoxin based on a transition metal oxide / noble metal particle composite SERS substrate. The method comprises the following steps: selecting paralytic toxin-saxitoxin in saxitoxin as a detection object; the method comprises the specific steps that firstly, a zinc oxide-silver composite nanoparticle SERS substrate is prepared to be used for enhancing Raman signals of saxitoxin, then SERS spectrums of saxitoxin with different concentrations are collected, the collected spectrums are guided into matlab, and a prediction model is established through a PLS algorithm; treating an actual sample according to a national standard method to obtain a treating fluid, then collecting an SERS spectrum of the treating fluid, and substituting the SERS spectrum into the prediction model to obtain a prediction result; the method is simple and convenient in synthesis step, good in SERS enhancement effect, high in detection speed, high in stability and good in application prospect in the technical field of detection of harmful factors in food and the like.

Description

Technical Field

[0001] The present application relates to the technical field of marine toxin detection, and specifically to a method for detecting shellfish toxins based on a transition metal oxide / precious metal particle composite SERS substrate. Background Art

[0002] Shellfish is an important aquaculture species in my country, with a total annual output of over 15 million tons, mainly including oysters, abalone, snails, mussels, etc. The main shellfish farming areas are Shandong, Fujian, Liaoning and Guangdong. Among them, the annual shellfish production in Fujian Province exceeds 3 million tons, accounting for more than 20% of the national production. Such a large scale of farming mainly depends on the market demand for shellfish food. The amino acid composition of shellfish is very comprehensive. It can provide essential amino acids for humans such as valine and leucine and has a low fat content. It is an important part of the consumer's dietary structure. Shellfish ingest algae in the water by filtering, but the algae filtered by shellfish are prone to toxin-producing algae such as Alexandrium and dinoflagellates. After these toxin-producing algae are ingested by shellfish, they will directly cause algal toxins to accumulate in the shellfish to form shellfish toxins. Toxins that are harmful to the human body include paralytic shellfish toxins, diarrheal shellfish toxins, memory loss shellfish toxins and neurotoxins, which have different degrees of harm to the human body. Therefore, effective monitoring of shellfish toxins is of great significance to protecting consumer health and the healthy development of my country's shellfish farming industry.

[0003] In order to detect shellfish toxins, the state has issued corresponding national standards to strictly limit the toxin content in common shellfish and stipulate specific detection methods. Conventional detection methods for shellfish toxins include mouse bioassay, high performance liquid chromatography, immunoassay, cell assay, etc. These technologies can accurately detect shellfish toxins, but these technologies have high operation requirements and slow detection speed and high detection cost, so they cannot achieve on-site rapid detection. With the integration of information and nanotechnology with spectral technology, spectral technology has been more widely used in the detection of trace pollutants in food. In particular, surface enhanced Raman spectroscopy (SERS) technology has the advantages of high sensitivity and fast detection speed for the detection of trace organic biotoxins due to its signal amplification effect of plasmon resonance (SPR). It has been applied in okadaic acid, carnitine, pesticide residues, and heavy metal residues. The collected SERS spectral variables are usually thousands. By using chemometric models to intelligently screen and regress the collected SERS spectral data, the reliability and scalability of the detection results can be effectively improved. Summary of the invention

[0004] The present invention aims at the problem that after the currently commonly used SERS substrate is complicatedly modified, the larger distance will weaken the signal and the preparation of the substrate is relatively complicated and not conducive to practical application. A transition metal oxide / noble metal particle composite SERS substrate is prepared to collect the spectrum of saxitoxin in shellfish toxins, and the collected SERS spectrum is processed and a quantitative model is established in combination with chemometrics, thereby successfully realizing the rapid detection of saxitoxin in shellfish toxins. The chemometrics adopts the partial least squares (PLS) method.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] A method for detecting shellfish toxins based on a transition metal oxide / noble metal particle composite SERS substrate, the steps are as follows:

[0007] (1) Preparation of zinc oxide nanoparticles (ZnO NPs): ZnONPs particles were prepared using a classic two-step wet chemical reaction method; the operation steps are as follows:

[0008] Dissolve zinc nitrate hexahydrate (Zn(NO3)2·6H20) in ultrapure water to obtain zinc nitrate hexahydrate solution; add ammonia water (NH3·H2O) and continue stirring to react, collect the precipitate by centrifugation after stirring, and then wash it with anhydrous ethanol, collect the precipitate by centrifugation again after washing, and obtain zinc oxide nanoparticles after vacuum drying, which are recorded as ZnO NPs;

[0009] (2) Preparation of zinc oxide nanoparticles / silver nanoparticles (ZnO@Ag): The ZnO NPs prepared in step (1) are used as a carrier to grow and load silver nanoparticles (Ag NPs);

[0010] First, ZnO NPs, polyvinyl pyrrolidone K30 (PVP K30) and glucose were dissolved in ultrapure water, heated to a certain temperature and stirred, and silver nitrate solution (AgNO3) was added under stirring conditions, and then centrifuged, washed and dried to obtain zinc oxide nanoparticles / silver nanoparticles, which were recorded as ZnO@Ag.

[0011] (3) Preparation of saxitoxin standard solution: saxitoxin powder was dissolved in methanol solution to obtain a saxitoxin standard solution with a concentration of 0.001-1000 ng / mL;

[0012] (4) Collection of SERS spectra of saxitoxin in shellfish:

[0013] The ZnO@Ag prepared in step (2) is dissolved in ethanol, and a ZnO@Ag solution is obtained after ultrasonic dispersion, which is evenly dropped on the surface of a silicon wafer. After drying, the saxitoxin standard solution prepared in step (3) is evenly dropped on the surface of a silicon wafer, and then the laser wavelength, spectral integration time, spectral repeated acquisition times, light source power and scanning wavenumber range are set to collect SERS spectra, and SERS spectral data corresponding to saxitoxin standard solutions of different concentrations are obtained;

[0014] (5) Establishment of a quantitative prediction model: Based on the SERS spectral data of the saxitoxin standard solution of corresponding concentration collected in step (4), a quantitative model is established using a PLS algorithm to ultimately obtain a quantitative prediction model;

[0015] (6) Application of actual samples: Take a shellfish sample and obtain a treated liquid after treatment, repeat the operation in step (4), the only difference is that the saxitoxin standard solution is replaced by the treated liquid, and finally obtain a SERS spectrum; then input the obtained SERS spectrum into the quantitative prediction model established in step (5) to obtain the prediction result, thereby realizing the detection of saxitoxin in shellfish samples.

[0016] Preferably, the concentration of the Zn(NO3)2·6H2O solution in step (1) is 0.004-0.01 g / mL, and the concentration of the NH3·H2O is 28 wt %; the volume ratio of the Zn(NO3)2·6H2O solution to NH3·H2O is 125:1.

[0017] Preferably, the stirring reaction temperature in step (1) is 40°C and the rotation speed is 200 rpm; the stirring reaction time is 12-24 h; the centrifugation conditions are 8000 rpm and centrifugation for 15 min; the vacuum drying temperature is 60°C and the time is 12 h.

[0018] Preferably, the concentration range of the silver nitrate solution (AgNO3) in step (2) is 0.1-0.5 mol / L, and the amount of ZnONPs, polyvinyl pyrrolidone K30 (PVP K30) and glucose dissolved in ultrapure water and silver nitrate solution is 0.5 g: 2.5 g: 2 g: 50 mL: 1-5 mL; the stirring time after adding the silver nitrate solution is 0.5-2 h, and the temperature is 80°C.

[0019] Preferably, the drying temperature in step (2) is 60° C. and the drying time is 6 hours.

[0020] Preferably, the laser wavelength in step (4) is 785 nm; the spectrum integration time is 3 s, the spectrum is collected repeatedly 3 times, the light source power is 150 mw, and the scanning beam range is 400 to 3000 cm -1 .

[0021] Preferably, the treatment in step (6) is carried out in accordance with the national standard GB 5009.213-2016 to obtain a treated liquid.

[0022] Compared with the prior art, the advantages of the present invention are:

[0023] (1) The transition metal oxide / noble metal particle composite SERS substrate prepared by the present invention can still provide stable enhancement for the SERS signal of shellfish toxins without complex modification, thanks to the special three-dimensional structure of its composite nanoparticles, which can provide a large number of "Raman hotspots".

[0024] (2) The present invention adopts the PLS algorithm to establish the quantitative model, the model effect is good, and the prediction results are stable.

[0025] (3) The detection method prepared by the present invention has been successfully applied to the rapid detection of saxitoxin among shellfish toxins, and is also applicable to other shellfish toxins. It has fast detection speed, good sensitivity, high stability, and a wide detection range. It has good application prospects in the field of food testing and has the potential for on-site rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 TEM image of ZnO@Ag NPs.

[0027] Figure 2 This is the collected SERS spectrum data diagram.

[0028] Figure 3 This is a diagram of the training and prediction effects of the PLS model obtained in Example 1. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution, and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Embodiment 1:

[0031] (1) Preparation of ZnO NPs: ZnO NPs were prepared using a classic two-step wet chemical reaction method;

[0032] Zinc nitrate hexahydrate (Zn(NO3)2·6H2O) was dissolved in ultrapure water to obtain a zinc nitrate hexahydrate solution with a concentration of 0.005 g / mL; 125 mL of the zinc nitrate hexahydrate solution was added with 1 mL of 28 wt% NH3·H2O solution and the mixture was stirred and reacted at 40°C for 24 hours. After the reaction, a white turbid liquid was obtained, which was cooled to room temperature and then centrifuged at 8000 rpm for 15 minutes. The supernatant was discarded, the white precipitate was collected, and the white precipitate was washed with anhydrous ethanol. After washing, the white precipitate was centrifuged again at 8000 rpm for 15 minutes. The white precipitate obtained after washing with ultrapure water and centrifugation was collected, which was ZnO NPs. The precipitate was dried at 60°C for 12 hours to finally obtain white ZnO NPs powder.

[0033] (2) Preparation of ZnO@Ag NPs:

[0034] Take 0.5g of ZnO NPs, 2.5g of PVP K30 and 2g of glucose and dissolve them together in 50mL of ultrapure water, then quickly inject 5mL of 0.5mol / L AgNO3 solution into the mixed solution, stir continuously at 80℃ for 1h, centrifuge and wash the obtained solution 3 times, and then dry it under vacuum conditions at 60℃ for 6h to obtain ZnO@Ag NPs.

[0035] Figure 1 TEM image of the prepared ZnO@Ag NPs substrate. It can be seen that the composite substrate is in the shape of a nanoflower, and AgNPs are successfully loaded on the surface of ZnO NPs.

[0036] (3) Preparation of saxitoxin standard solution: saxitoxin powder was dissolved in methanol solution to obtain saxitoxin standard solution; the concentrations were 1000, 100, 10, 1, 0.1, 0.01, and 0.001 ng / mL, respectively, for later use;

[0037] (4) SERS spectrum collection: 0.25 g of the prepared ZnO@Ag powder was dissolved in 5 mL of ethanol, and then evenly dropped on the surface of the silicon wafer after ultrasonic dispersion and dried; then the saxitoxin standard solution was evenly dropped on the surface of the silicon wafer, and the laser wavelength was set to 785 nm, the integration time was 3 s, the spectrum was repeatedly collected 3 times, the light source power was 150 mW, and the scanning beam range was 400-3000 cm -1 , collect SERS spectra;

[0038] Seven concentrations, ten replicates per concentration, seven sets of data were measured, and 70 SERS spectra of toxins at different concentrations were obtained. The results are shown in the figure. Figure 2 As shown;

[0039] (5) Establishment of prediction model:

[0040] The SERS spectrum data collected above were imported into Matlab, and then the PLS algorithm was run to screen the variables of the SERS spectrum. The selected variable combination was used to establish the model, and finally a quantitative prediction model was obtained.

[0041] (6) Application of actual samples: Take a shellfish sample and pre-treat it according to the national standard method (GB 5009.213-2016) to obtain a treatment solution, repeat the operation in step (4), except that the saxitoxin standard solution is replaced by the treatment solution, and finally obtain a SERS spectrum; then input the obtained SERS spectrum into the quantitative prediction model established in step (5) to obtain the prediction result.

[0042] Figure 3 This is the fitting relationship between the actual value and the predicted value of the toxin concentration in the established prediction model; the results show that the fitting degree is good and the accuracy is high, and it can be used to detect shellfish toxins in real samples.

[0043] Embodiment 2:

[0044] (1) Preparation of ZnO NPs: ZnO NPs were prepared using a classic two-step wet chemical reaction method;

[0045] Zinc nitrate hexahydrate (Zn(NO3)2·6H2O) was dissolved in ultrapure water to obtain a zinc nitrate hexahydrate solution with a concentration of 0.006 g / mL; 125 mL of the zinc nitrate hexahydrate solution was added with 1 mL of 28 wt% NH3·H2O solution and the mixture was stirred and reacted at 40°C for 24 hours. After the reaction, a white turbid liquid was obtained, which was cooled to room temperature and then centrifuged at 8000 rpm for 15 minutes. The supernatant was discarded, the white precipitate was collected, and the white precipitate was washed with anhydrous ethanol. After washing, the white precipitate was centrifuged again at 8000 rpm for 15 minutes. The white precipitate obtained after washing and centrifugation with ultrapure water was collected, which was ZnO NPs. The precipitate was dried at 60°C for 12 hours to finally obtain white ZnO NPs powder.

[0046] (2) Preparation of ZnO@Ag NPs:

[0047] Take 0.5 g of ZnO NPs, 2.5 g of PVP K30 and 2 g of glucose and dissolve them together in 50 mL of ultrapure water. Then quickly inject 5 mL of 0.3 mol / L AgNO3 solution into the mixed solution and stir it continuously at 80 °C for 1 h. The resulting solution is centrifuged and washed 3 times, and then dried under vacuum conditions at 60 °C for 6 h to obtain ZnO@Ag NPs.

[0048] (3) Preparation of saxitoxin standard solution: saxitoxin powder was dissolved in methanol solution to obtain saxitoxin standard solution; the concentrations were 1000, 100, 10, 1, 0.1, 0.01, and 0.001 ng / mL, respectively, for later use;

[0049] (4) SERS spectrum collection: 0.25 g of the prepared ZnO@Ag powder was dissolved in 5 mL of ethanol, and then evenly dropped on the surface of the silicon wafer after ultrasonic dispersion and dried; then the saxitoxin standard solution was evenly dropped on the surface of the silicon wafer, and the laser wavelength was set to 785 nm, the integration time was 3 s, the spectrum was repeatedly collected 3 times, the light source power was 150 mW, and the scanning beam range was 400-3000 cm -1 , collect SERS spectra; seven concentrations, fifteen parallels for each concentration, 7 sets of data were measured, and 105 SERS spectra of toxins at different concentrations were obtained.

[0050] (5) Establishment of prediction model:

[0051] The SERS spectrum data collected above were imported into Matlab, and then the PLS algorithm was run to screen the variables of the SERS spectrum. The selected variable combination was used to establish the model, and finally a quantitative prediction model was obtained.

[0052] (6) Application of actual samples: Take a shellfish sample and pre-treat it according to the national standard method (GB 5009.213-2016) to obtain a treatment solution, repeat the operation in step (4), except that the saxitoxin standard solution is replaced by the treatment solution, and finally obtain a SERS spectrum; then input the obtained SERS spectrum into the quantitative prediction model established in step (5) to obtain the prediction result.

[0053] Embodiment 3:

[0054] (1) Preparation of ZnO NPs: ZnO NPs were prepared using a classic two-step wet chemical reaction method;

[0055] Zinc nitrate hexahydrate (Zn(NO3)2·6H2O) was dissolved in ultrapure water to obtain a zinc nitrate hexahydrate solution with a concentration of 0.01 g / mL; 125 mL of the zinc nitrate hexahydrate solution was added with 1 mL of 28 wt% NH3·H2O solution and the mixture was stirred and reacted at 40°C for 24 hours. After the reaction, a white turbid liquid was obtained, which was cooled to room temperature and then centrifuged at 8000 rpm for 15 minutes. The supernatant was discarded, the white precipitate was collected, and the white precipitate was washed with anhydrous ethanol. After washing, the white precipitate was centrifuged again at 8000 rpm for 15 minutes. The white precipitate obtained after washing with ultrapure water and centrifugation was collected, which was ZnO NPs. The precipitate was dried at 60°C for 12 hours to finally obtain white ZnO NPs powder.

[0056] (2) Preparation of ZnO@Ag NPs:

[0057] Take 0.5 g of ZnO NPs, 2.5 g of PVP K30 and 2 g of glucose and dissolve them together in 50 mL of ultrapure water. Then quickly inject 4 mL of 0.5 mol / L AgNO3 solution into the mixed solution and stir continuously at 80 °C for 1 h. The resulting solution is centrifuged and washed 3 times, and then dried under vacuum conditions at 60 °C for 6 h to obtain ZnO@Ag NPs.

[0058] (3) Preparation of saxitoxin standard solution: saxitoxin powder was dissolved in methanol solution to obtain saxitoxin standard solution; the concentrations were 1000, 100, 10, 1, 0.1, 0.01, and 0.001 ng / mL, respectively, for later use;

[0059] (4) SERS spectrum collection: 0.25 g of the prepared ZnO@Ag powder was dissolved in 5 mL of ethanol, and then evenly dropped on the surface of the silicon wafer after ultrasonic dispersion and dried; then the saxitoxin standard solution was evenly dropped on the surface of the silicon wafer, and the laser wavelength was set to 785 nm, the integration time was 3 s, the spectrum was repeatedly collected 3 times, the light source power was 150 mW, and the scanning beam range was 400-3000 cm -1 , collect SERS spectra; seven concentrations, twenty parallels for each concentration, measure 7 sets of data, and obtain 140 SERS spectra of toxins at different concentrations;

[0060] (5) Establishment of prediction model:

[0061] The SERS spectrum data collected above were imported into Matlab, and then the PLS algorithm was run to screen the variables of the SERS spectrum. The selected variable combination was used to establish the model, and finally a quantitative prediction model was obtained.

[0062] (6) Application of actual samples:

[0063] Take a shellfish sample and pre-treat it according to the national standard method (GB 5009.213-2016) to obtain a treatment solution, repeat the operation in step (4), the only difference is that the saxitoxin standard solution is replaced by the treatment solution, and finally obtain a SERS spectrum; then input the obtained SERS spectrum into the quantitative prediction model established in step (5) to obtain a prediction result.

[0064] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; therefore, although this specification has described the present invention in detail with reference to the above embodiments, a person skilled in the art should understand that the present invention can still be modified or replaced by equivalents; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for detecting shellfish toxins based on a transition metal oxide / noble metal particle composite SERS substrate, characterized in that: Here are the steps: (1) Preparation of zinc oxide nanoparticles: Dissolving zinc nitrate hexahydrate in ultrapure water to obtain a zinc nitrate hexahydrate solution; then adding ammonia water and continuously stirring the reaction, collecting the precipitate by centrifugation after stirring the reaction, and then washing it with anhydrous ethanol, and then collecting the precipitate by centrifugation again after washing, and obtaining zinc oxide nanoparticles after vacuum drying, which are recorded as ZnO NPs; (2) firstly dissolving the ZnO NPs, polyvinyl pyrrolidone K30 and glucose obtained in step (1) in ultrapure water, heating to a certain temperature and stirring, adding silver nitrate solution under stirring conditions, continuing to stir, centrifuging, washing and drying to obtain zinc oxide nanoparticles / silver nanoparticles, recorded as ZnO@Ag; (3) Preparation of saxitoxin standard solution: saxitoxin powder was dissolved in methanol solution to obtain a saxitoxin standard solution with a concentration of 0.001-1000 ng / mL; (4) Collection of SERS spectra of saxitoxin in shellfish: The ZnO@Ag prepared in step (2) is dissolved in ethanol, and a ZnO@Ag solution is obtained after ultrasonic dispersion, which is evenly dropped on the surface of a silicon wafer. After drying, the saxitoxin standard solution prepared in step (3) is evenly dropped on the surface of a silicon wafer, and then the laser wavelength, spectral integration time, spectral repeated acquisition times, light source power and scanning wavenumber range are set to collect SERS spectra, and SERS spectral data corresponding to saxitoxin standard solutions of different concentrations are obtained; (5) Establishment of a quantitative prediction model: Based on the SERS spectral data of the saxitoxin standard solution of corresponding concentration collected in step (4), a quantitative model is established using a PLS algorithm to ultimately obtain a quantitative prediction model; (6) Application of actual samples: Take a shellfish sample and obtain a treated liquid after treatment, repeat the operation in step (4), the only difference is that the saxitoxin standard solution is replaced by the treated liquid, and finally obtain a SERS spectrum; then input the obtained SERS spectrum into the quantitative prediction model established in step (5) to obtain the prediction result, thereby realizing the detection of saxitoxin in shellfish samples.

2. The method for detecting shellfish toxins based on a transition metal oxide / noble metal particle composite SERS substrate according to claim 1, characterized in that: The concentration of the Zn(NO3)2·6H2O solution in step (1) is 0.004-0.01 g / mL, and the concentration of the NH3·H2O is 28 wt %; the volume ratio of the Zn(NO3)2·6H2O solution to NH3·H2O is 125:

1.

3. The method for detecting shellfish toxins based on a transition metal oxide / noble metal particle composite SERS substrate according to claim 1, characterized in that: The stirring reaction temperature in step (1) is 40°C and the rotation speed is 200 rpm; the stirring reaction time is 12-24 hours; the centrifugation conditions are 8000 rpm and centrifugation for 15 minutes; the vacuum drying temperature is 60°C and the time is 12 hours.

4. The method for detecting shellfish toxins based on a transition metal oxide / noble metal particle composite SERS substrate according to claim 1, characterized in that: The concentration range of the silver nitrate solution in step (2) is 0.1-0.5 mol / L, and the amount of ZnONPs, polyvinyl pyrrolidone K30 and glucose dissolved in ultrapure water and silver nitrate solution is 0.5 g: 2.5 g: 2 g: 50 mL: 1-5 mL; the stirring time after adding the silver nitrate solution is 0.5-2 h, and the temperature is 80°C.

5. The method for detecting shellfish toxins based on a transition metal oxide / noble metal particle composite SERS substrate according to claim 1, characterized in that: The drying temperature in step (2) is 60° C. and the drying time is 6 h.

6. The method for detecting shellfish toxins based on a transition metal oxide / noble metal particle composite SERS substrate according to claim 1, characterized in that: The laser wavelength in step (4) is 785 nm; the spectrum integration time is 3 s, the spectrum is collected 3 times, the light source power is 150 mw, and the scanning beam range is 400 to 3000 cm -1 .

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