METHOD FOR PREPARING AGNPS@SASP SUBSTRATE MATERIAL AND APPLICATION THEREOF

Abstract

Disclosed are a method for preparing a AgNPs@SASP substrate material and application thereof. The present disclosure uses cyclohexane as an organic phase and ethanol as an inducer, and uses the interfacial tension of an organic/water interface for self-assembly to arrange the nanoparticles in a single layer. The addition of a low-dielectric solvent as an inducer reduces the charge density on the nanoparticles. As a result, the van der Waals force combined with the reduction of the Coulomb repulsion force cause the nanoparticles to reassemble at the organic/water interface to form a new equilibrium, and a SERS platform of AgNPs in a two-dimensional array is prepared. SERS detection and peak attribution analysis of different food-borne pathogen spores are carried out, and qualitative analysis is carried out by a multivariate statistical method to realize the rapid identification of spore-forming bacteria.

Claims

1. A method for preparing a AgNPs@SASP substrate material, the method comprising: (1) preparation of AgNPs by a seed mediated two-step growth method; (2) preparation of a solid-phase AgNPs@SASP substrate material by self-assembly of AgNPs prepared by a seed mediated two-step growth method, wherein preparing a self-assembled silver nanoparticle metallic film by a one-step method at a water-cyclohexane liquid-liquid interface, which is the AgNPs@SASP substrate material.

2. The method for preparing a AgNPs@SASP substrate material according to claim 1, wherein step (1) preparation of AgNPs by a seed mediated two-step growth method comprises: a. adding ultrapure water to an aqueous trisodium citrate solution, heating, then adding a AgNO.sub.3 solution and a NaBH.sub.4 solution, stirring, thereafter cooling to room temperature, and adding ultrapure water to obtain a AgNPs starting seed solution; b. adding the aqueous trisodium citrate solution to ultrapure water, then adding the AgNO.sub.3 solution and the AgNPs starting seed solution at a constant speed, stirring a resulting mixed solution vigorously for 1 h, cooling to room temperature, then continuing the addition of the aqueous trisodium citrate solution and the AgNO.sub.3 solution, heating and stirring vigorously for 1 h, adding ultrapure water, then cooling to room temperature, and storing at 4° C. away from light.

3. The method for preparing a AgNPs@SASP substrate material according to claim 1, wherein step (2) preparation of a solid-phase AgNPs@SASP substrate material by self-assembly of AgNPs prepared by a seed mediated two-step growth method comprises: enriching the AgNPs prepared by a seed mediated two-step growth method in step (1) by centrifugation, then suspending in absolute ethanol by ultrasonic dispersion, injecting to a water-cyclohexane oil-water interface with a pipette, where silver nanoparticles are drawn to the liquid-liquid interface by surface tension to form a thin film with a monomolecular arrangement floating on a liquid surface after volatilization of cyclohexane, inserting a glass slide or a silicon wafer deep below the liquid surface, making the silver nanoparticle film adhered to the silicon wafer by an inclined inserting and pulling method, and drying with nitrogen.

4. The method for preparing a AgNPs@SASP substrate material according to claim 2, wherein, in step a, a mass fraction of the aqueous trisodium citrate solution is 1%, a mass fraction of the AgNO.sub.3 solution is 1%, a mass fraction of the NaBH.sub.4 solution is 0.1%, and a volume ratio of the aqueous trisodium citrate solution, the ultrapure water, the AgNO.sub.3 solution and the NaBH.sub.4 solution is 20:75:1.7:2.

5. The method for preparing a AgNPs@SASP substrate material according to claim 2, wherein, a heating temperature in step a is 70° C., a heating time is 15 min, and the stirring is conducted by stirring at 70° C. and then cooling to room temperature.

6. The method for preparing a AgNPs@SASP substrate material according to claim 2, wherein in step b, a volume ratio of the ultrapure water, the AgNPs starting seed solution, the AgNO.sub.3 solution added for the first time, and the aqueous trisodium citrate solution added for the first time is 75:10:1.7:2, wherein a mass fraction of the AgNO.sub.3 solution is 1%, and a mass fraction of the aqueous trisodium citrate solution is 1%.

7. The method for preparing a AgNPs@SASP substrate material according to claim 2, wherein in step b, an amount of the AgNO.sub.3 solution and an amount of the aqueous trisodium citrate solution added for the second time are the same as those of the first time, and a heating temperature is 70° C.

8. An application of the AgNPs@SASP substrate material prepared by the method according to claim 1 as a SERS substrate.

9. The application according to claim 8, wherein SERS detection is performed on spores of different food-borne pathogen to realize rapid identification of spore-forming bacteria.

10. The application according to claim 9, wherein the spores of food-borne pathogens include at least one of C. perfringens spores, B. subtilis spores, and B. cereus spores.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 is a schematic diagram of the preparation of AgNPs@SASP substrate material and the process of the SERS detection.

[0029] FIG. 2 shows the characterization results of AgNPs prepared by a seed mediated two-step growth method. Panel (A-1) shows a UV-Vis spectrum of the AgNPs seed solution prepared in step (1) a of Example 1, panel (A-2) shows the particle size distribution of the AgNPs seed solution prepared in step (1) a of Example 1, panel (A-3) shows the SEM image of the AgNPs seed solution prepared in step (1) a of Example 1; panel (B-1) shows a UV-Vis spectrum of the AgNPs seed solution in step (1) a treated with a seed mediated two-step growth method, panel (B-2) shows the particle size distribution of AgNPs seed solution in step (1) a treated with a seed mediated two-step growth method, panel (B-3) shows the SEM image of the AgNPs seed solution in step (1) a treated with a seed mediated two-step growth method.

[0030] FIG. 3 is the SEM image of the AgNPs@SASP substrate material prepared in Example 1.

[0031] FIG. 4 shows the comparison results of the enhancement effect of the AgNPs@SASP substrate material prepared in Example 1.

[0032] FIG. 5 shows the reproducibility results of SERS detection for the three spores based on AgNPs@SASP substrate material.

[0033] FIG. 6 shows an average SERS spectra of the spores of three different food-borne pathogens.

[0034] FIG. 7 shows the comparison results of the SERS peak intensity of Ca.sup.2+-DPA of three different spores.

[0035] FIG. 8 is a HCA dendrogram of the SERS spectra of spores of the three food-borne pathogens. A1-A10, C. perfringens spores; B1-B10, B. subtilis spores, C1-C10, B. cereus spores.

[0036] FIG. 9 is a scatter diagram of the SERS spectra of spores of the three food-borne pathogens analyzed by LDA.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] The present disclosure will be further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present disclosure and not to limit the scope of the present disclosure, and those skilled in the art can make some non-essential improvements and adjustments based on the content of the above disclosure.

Example 1

[0038] A method for preparing the AgNPs@SASP substrate material in this example comprises:

[0039] (1) Preparation of AgNPs by a Seed Mediated Two-Step Growth Method;

[0040] a. 75 ml of ultrapure water was added to 20 ml of trisodium citrate with a mass fraction of 1% and heated in a round bottom flask at 70° C. for 15 min, 1.7 ml of AgNO.sub.3 solution with a mass fraction of 1% was added to the resulting mixture, then 2 ml of NaBH.sub.4 solution with a mass fraction of 0.1% was quickly added thereto, the reaction solution was stirred at 70° C., then cooled to room temperature, and then water was added to a volume of 100 ml to obtain a AgNPs starting seed solution.

[0041] b. 75 ml of water and 2 ml of aqueous trisodium citrate solution with a mass fraction of 1% were added in a 100 ml round bottom flask, then 10 ml of the prepared AgNPs starting seed solution and 1.7 mL of AgNO.sub.3 solution with a mass fraction of 1% were added thereto at a constant speed, the mixed solution was vigorously stirred for 1 h, this step was repeated twice (i.e., after cooling to room temperature, 2 ml of 1% aqueous trisodium citrate solution and 1.7 ml of 1% AgNO.sub.3 solution were added, heated at 70° C. and stirred vigorously for 1 h), ultrapure water was added to 100 ml, the resulting solution was cooled to room temperature, and stored at 4° C. away from light.

[0042] (2) Preparation of a Solid-Phase Substrate Material by Self-Assembly of AgNPs Prepared by a Seed Mediated Two-Step Growth Method.

[0043] The present disclosure proposes a one-step method for preparing self-assembled silver nanoparticle metallic film at the water-cyclohexane liquid-liquid interface under the action of cetyltrimethylammonium bromide (CTAB) surfactant. Ethanol is used to adjust the charge density on the surface of silver nanoparticles to create an equilibrium state on the surface of water and cyclohexane. And a flexible silver nanoparticle metallic film can be obtained after a simple evaporation process of the cyclohexane phase.

[0044] A glass container and a silicon wafer were soaked and cleaned with aqua regia (a volume ratio of concentrated hydrochloric acid to concentrated nitric acid of 3:1) to remove impurities on the surface, washed with deionized water several times, and dried with nitrogen for use. 2.5 ml of deionized water as a water phase was added into a 10 ml small beaker, 1 ml of cyclohexane as an oil phase was added along the wall of the beaker, mixed, and followed by standing for several seconds to form a clearly visible layered oil-water interface. 10 ml of the AgNPs treated with a seed mediated two-step growth method prepared in the previous step was taken, enriched by centrifugation, and then suspended in absolute ethanol by ultrasonic dispersion. The resulting suspension was injected to a water-cyclohexane oil-water interface with a pipette. It can be observed with naked eyes that the AgNPs dispersed in ethanol will be drawn to the liquid-liquid interface by surface tension and self-assembled together in constant motion. The entire self-assembly process was very rapid, and it can be observed that the AgNPs formed a thin film with a monomolecular arrangement floating on a liquid surface after volatilization of cyclohexane. The cleaned glass slide or silicon wafer was deep below the liquid surface by tweezers, a good and complete silver film was found and attached to the silicon wafer by an inclined inserting and pulling method, and dried under nitrogen to obtain the AgNPs@SASP substrate material, which was the SERS substrate.

[0045] 1. Characterization of SERS Substrate

[0046] 1). Preparation of AgNPs by a Seed Mediated Two-Step Growth Method

[0047] The AgNPs was prepared by a seed mediated two-step growth method. The AgNPs seed solution prepared in the first stage was gray-green, and the AgNPs treated with a seed mediated two-step growth method was yellow-brown. The strongest absorption peak in the UV-Vis spectrum of the AgNPs seed solution prepared in the first stage is located at 416 nm (panel A-1 in FIG. 2); the strong absorption peak in the UV-Vis spectrum of the AgNPs treated with a seed mediated two-step growth method is located at 435 nm (panel B-1 in FIG. 2), and the ultraviolet absorption peak is red-shifted and more rounded and sharp, indicating that the particle size uniformity and shape of AgNPs have been optimized. Combined with a laser particle size analyzer, the particle size analysis of the AgNPs sol was analyzed, the average particle size of the AgNPs seed solution prepared in the first stage is about 70.86 nm, the average particle size of the AgNPs treated with a seed mediated two-step growth method is about 90.62 nm, and the particle size range of AgNPs treated with a seed mediated two-step growth method is more concentrated between 80-100 nm. From the results of SEM image (panel C-1 in FIG. 2 and panel C-2 in FIG. 2), it can be visually observed that the particle size uniformity and shape integrity of AgNPs treated with a seed mediated two-step growth method have been well improved. The results show that the AgNPs prepared by a seed mediated two-step growth method can prepare nanomaterials with good particle size uniformity, which effectively avoids the problem of the unstable SERS detection signals caused by the inability of AgNPs to maintain relatively uniform shape and size during synthesis.

[0048] 2. Characterization of AgNPs@SASP Substrate Materials

[0049] A solid-phase substrate material was prepared by self-assembly of AgNPs prepared by a seed mediated two-step growth method (denoted as AgNPs@SASP). The present disclosure uses cyclohexane as an organic phase and ethanol as an inducer, and uses the interfacial tension of the organic/water interface for self-assembly to arrange the nanoparticles in a single layer. The addition of a low-dielectric solvent as an inducer reduces the charge density on the nanoparticles. As a result, the van der Waals force combined with the reduction of the Coulomb repulsion force cause the nanoparticles to reassemble at the organic/water interface to form a new equilibrium, and a SERS platform of AgNPs with a two-dimensional array is prepared. The SEM image of AgNPs@SASP is shown in FIG. 3. The results show that the AgNPs successfully self-assembled tightly into a layer of AgNPs dot array with a large area, and the self-assembled AgNPs with a two-dimensional array are evenly distributed on the silicon wafer. As a new SERS platform, the AgNPs@SASP with a two-dimensional AgNPs hotspot array has higher sensitivity and reproducibility, and is more suitable for practical detection applications.

[0050] 2. Enhancement Effect and Reproducibility of AgNPs@SASP Substrate Material

[0051] In order to understand the enhancement effect of AgNPs@SASP as the SERS substrate material, a laser Raman spectrometer was used to collect the SERS spectra of single samples of the three spores and the three spores on AgNPs@SASP respectively. The comparison results of the enhancement effect of AgNPs@SASP substrate materials are shown in FIG. 4 and Table 1. It can be seen from FIG. 4 that the Raman spectra of single samples of the three spores have a low peak intensity and few characteristic peaks, which makes it difficult to qualitatively identify different spores; Using AgNPs@SASP substrate material for signal enhancement, the Raman signals of the three spores are significantly enhanced, and the characteristic peaks with weaker signals can also be displayed in the SERS spectrum. The number increase of the Raman characteristic peaks is more conducive to distinguishing and identifying different spores. Therefore, AgNPs@SASP as a substrate material has a good signal enhancement effect on Raman spectra.

[0052] In order to quantify the SERS enhancement effect of AgNPs@SASP on the three spores, taking the common strongest peak band of 1570-1577 cm.sup.−1 (CaDPA) of the three spores as an example, an enhancement factor of AgNPs@SASP as the substrate material was calculated, the calculation formula is shown in formula (1), where C.sub.SERS and I.sub.SERS are the concentration and the intensity value of the single peak of the test sample when the substrate material is used, respectively; C.sub.RS and I.sub.RS are the concentration and the intensity value of the single peak of the test sample when the substrate material is not used, respectively. In order to better obtain the Raman spectrum of the simple sample, and better highlight the enhancement effect of the substrate material, the concentration of the simple sample is 100 times that of the enhanced detection in the detection of the present disclosure, i.e., C.sub.RS=100 C.sub.SERS. The calculation results of the SERS enhancement factor of AgNPs@SASPS on the three spores are shown in Table 1. The results show that AgNPs@SASP has a good SERS enhancement effect on all the three spores, with an enhancement factor up to 1.79×10.sup.4, which has a good SERS enhancement effect.

[00001]$\begin{array}{cc}\mathrm{AEF}=\frac{I_{\mathrm{SERE}}/C_{\mathrm{SERS}}}{I_{\mathrm{RS}}/C_{\mathrm{RS}}}& \left(1\right)\end{array}$

TABLE-US-00001 TABLE 1 Calculation results of the SERS enhancement factor of AgNPs@SASPS on three spores SERS I.sub.RS I.sub.SERS Target substrate (a.u.) (a.u.) AEF value C. perfringens AgNPs@SASP 340.23 9536.61 9.54 × 10.sup.3 spores B. subtilis spores 337.84 12150.82 1.22 × 10.sup.4 B. cereus spores 272.81 17882.87 1.79 × 10.sup.4

[0053] In order to test the reproducibility of substrate material for SERS detection of three spores, the three spores were attached to the AgNPs@SASP solid phase substrate respectively, and after drying with nitrogen, a laser confocal Raman spectrometer was used to randomly scan 10 times with a scanning range of 400-3200 cm.sup.−1. The reproducibility results of the substrate material for SERS detection of three spores are shown in FIG. 5. The 10 SERS spectra of each spore show good consistency. The results show that the SERS enhancement method based on the AgNPs@SASP prepared by self-assembly at a liquid-liquid interface as the substrate material has high stability and good reproducibility, which provides strong technical support for the rapid detection of food-borne pathogens based on SERS technology.

[0054] 3. SERS Spectra Analysis of Three Spores Using AgNPs@SASP as the Substrate Material

[0055] C. perfringens spores, B. subtilis spores, and B. cereus spores were scanned randomly using SERS technology using AgNPs@SASP as the substrate material for 10 times, after baseline correction, normalization and smoothing, SERS average spectra of the spores of the three food-borne pathogens and a tentative peak attribution of SERS spectra are shown in FIG. 6 and Table 2. A right side of FIG. 6 shows the observation result of C. perfringens spores, B. subtilis spores and B. cereus spores under a phase contrast microscope with a 100× oil lens. It can be seen from FIG. 6 that the numbers of Raman vibration peaks of the three spores are significantly different (C. perfringens spores>B. subtilis spores>B. cereus spores). The concentration of Ca.sup.2+-DPA in the spores is relatively high (estimated to be 5%-15% of a dry weight of the spores). In the SERS spectra of the spores of the three food-borne pathogen, it can be seen that the number of Raman vibration peaks and peak intensity of Ca.sup.2+-DPA are dominant, and the Raman vibration peaks are located at about 670-677 cm.sup.−1, 1016-1021 cm.sup.−1 and 1571-1578 cm.sup.−1, similar to previous studies. The difference in the Ca.sup.2+-DPA concentration of different spores results in different peak numbers and intensities, which can also be used as a basis for judging different spores. The comparison results of the SERS peak intensities of Ca.sup.2+-DPA of the three different spores are shown in FIG. 7, where the peak intensities of Ca.sup.2+-DPA of the three spores in the 1571-1578 cm.sup.−1 band are greater than the other two Ca.sup.2+-DPA peaks in the same spores, and the peak intensities of B. cereus spores in the 670-677 cm.sup.−1, 1016-1021 cm.sup.−1 and 1571-1578 cm.sup.−1 bands are greater than those of B. subtilis spores and C. perfringens spores. The SERS spectra of Ca.sup.2+-DPA of the three spores show obvious difference in peak intensity, which is the main characteristic peak of Ca.sup.2+-DPA and also the main characteristic peak to distinguish the three spores.

[0056] Different Raman vibration peaks are attributed to the different components and vibrational forms of chemical bonds of the spores. C. perfringens spores show unique Raman vibration peaks at 455 cm.sup.−1 (Polysaccharides), 534 cm.sup.−1 (Protein disulfide stretch), 1159 cm.sup.−1 (C—C/C—N stretch of proteins), 1250 cm.sup.−1 (C—H.sub.2 stretching vibration) and 1450 cm.sup.−1 (CH.sub.2CH.sub.3 deformation); B. subtilis spores show a unique Raman vibration peak at 793 cm.sup.−1 (Cytosine or uracil of nucleic acids); B. cereus spores show a unique Raman vibration peak at 1416 cm.sup.−1 (CH.sub.2 sciss of lipids). Peaks located at 948-954 cm.sup.−1 (Phospholipid N—C stretch) and 1299-1301 cm.sup.−1 (—CH.sub.2 deformation of Lipid) exist in both SERS spectra of B. subtilis spores and B. cereus spores, but not in the spectrum of C. perfringens spores. It can be seen that in the SERS spectra, the difference in the peak position and peak intensity of the Raman vibration peak can be observed in the spores of the three food-borne pathogens, indicating that the SERS technology using AgNPs@SASP as the substrate material has the ability to distinguish the spores of different food-borne pathogens.

TABLE-US-00002 TABLE 2 Tentative attributions of SERS spectral Raman shifts of spores of three food-borne pathogens C. perfringens B. subtilis B. cereus Spores Spores Spores peak attribution Raman shift (cm.sup.−1) 455 Polysaccharides 456 534 Protein disulfide 540 stretch 670 675 677 Ca.sup.2+-DPA 662 793 Cytosine or uracil 780-795 (nucleic acids) 954 948 Phospholipid 930-957 N—C stretch 1016 1021 1018 Ca.sup.2+-DPA 1017 1093 1088 O═P—O 1092 (nucleic acid) 1159 C—C/C—N stretch 1158 (proteins) 1209 C—H.sub.2 stretching 1220 vibration 1250 protein amide III 1253 1301 1299 —CH2 deformation 1297 (Lipid) 1313 Amide III 1312 1347 1338 1343 Adenine, amide III 1338 1416 CH.sub.2 sciss (lipids) 1415 1450 CH.sub.2CH.sub.3 1449 deformation 1528 1547 Amine II 1550 1571 1578 1576 Ca.sup.2+-DPA 1570-1580

[0057] 4. Multivariate Statistical Analysis

[0058] In order to better realize the qualitative identification of spores of different food-borne pathogens, and avoid the interference of similar Raman vibration spectra on the classification and identification of microorganisms, combined with hierarchical cluster analysis (HCA) and linear discriminant analysis (LDA) in the multivariate statistical analysis method, the classification and comparative analysis of the SERS detection of spores of three food-borne pathogens using AgNPs@SASP as the substrate material were conducted from a statistical point of view. HCA can cluster the SERS spectra of the three spores and then judge and analyze them to construct a dendrogram to judge the similarities and differences between the research object populations. LDA is a simple linear recognition algorithm and a supervised learning dimensionality reduction technology, which can produce categorization output for each sample with a data set, and can be used to distinguish and evaluate the degree of discrimination of different bacterial populations. The results are shown in FIG. 8, FIG. 9, and Table 3.

[0059] It can be seen from the HCA analysis results (FIG. 8) that the 30 SERS spectra are divided into 3 clusters without cross-interference between each cluster. The three pathogens can be clearly divided into three clusters at a cluster distance of 7. B. subtilis spores and B. cereus spores are clustered into one cluster at a cluster distance of 15. The SERS characteristic Raman spectra of B. subtilis spores and B. cereus spores, which are clustered into one cluster at a cluster distance of 15, are similar, because B. subtilis spores and B. cereus spores all belong to the spores of the genus Bacillus with a relatively similar biochemical structure and composition, so they are distinct from the genus Clostridium of C. perfringens spores in terms of structure and composition, similar to the research of Peter et al. The HCA analysis not only effectively realize the distinction between the three spores, but also realize the distinction between Clostridium spores and Bacillus spores. It can be seen from FIG. 9 and Table 3 that the established LDA model has an accuracy rate of 100%, and can completely distinguish the SERS spectra of the spores of the three food-borne pathogens. The three food-borne pathogens are clustered into three independent clusters, and all samples of each food-borne pathogen are concentrated in a relatively small area near the centroid point without any overlapping area, with good sensitivity and specificity of 100%. The above results show that the multivariate statistical analysis method combining HCA and LDA can realize the rapid and accurate identification of spores of different food-borne pathogens.

TABLE-US-00003 TABLE 3 Using the LDA method to distinguish and assess the degree of discrimination of different bacterial populations prediction group sensitivity specificity sample 1 2 3 total (%) (%) 1 10 0 0 10 2 0 10 0 10 100 100 3 0 0 10 10

[0060] The basic principles an main features of the present disclosure an the advantages of the present disclosure have been shown and described above. Those skilled in the prior art should understand that the present disclosure is not limited by the above embodiments. The above embodiments and descriptions only illustrate the principles of the present disclosure, without departing from the spirit and scope of the present disclosure, there will be various changes and improvements in the present disclosure, and these changes and improvements fall within the scope of the claimed disclosure. The scope of protection claimed by the present disclosure is defined by the appended claims and their equivalents.