CN108051424B - SERS substrate based on shell surface, preparation method and application thereof - Google Patents

Abstract

The invention relates to an SERS substrate based on a shell surface, a preparation method and application thereof, and belongs to the field of biological detection. The SERS substrate is obtained by covering a precious metal nanoparticle layer on the surface of a shell structure. The invention further provides application of the SERS substrate in detecting microorganisms.

Description

SERS substrate based on shell surface, preparation method and application thereof

Technical Field

The invention relates to an SERS substrate based on a shell surface, a preparation method and application thereof, and belongs to the field of biological detection.

Background

The raman spectrum can provide fingerprint information of molecules, and the molecular fingerprint information has high specificity when being used for detecting sample molecules, but the raman signal detected by the traditional means is very weak, so that the application of the molecular fingerprint information in the trace detection of the sample molecules is limited. Until 1974, scientists discovered that the raman signal of sample molecules was orders of magnitude improved when they were in close proximity to the surface of some noble metal nanoparticles, such as silver, gold, copper, and the like, a phenomenon known as Surface Enhanced Raman Scattering (SERS). SERS has very high sensitivity, can even reach the detection level of single molecule under certain conditions, combines the high specificity of Raman to the detection of sample molecules, and plays an important role in the detection of tumor markers, environmental pollutants, pathogenic microorganisms and the like.

The prepared high-activity Raman enhancement substrate has important significance for SERS detection, the traditional Raman enhancement substrate comprises silver, gold, copper and other noble metal nano particles which are generally prepared into sol, the sol is mixed with sample molecules during detection, and nano agglomerates are searched in a solution through a microscope for Raman signal detection. Due to the fact that the nano agglomerates are different in size and are distributed unevenly in the solution, the detected Raman signal is poor in reproducibility; after the laser is focused on the agglomerates, the nano agglomerates are easy to float in the solution, so that the subsequent detection is not focused accurately. In order to solve this problem, researchers cover or deposit the noble metal nanoparticles on a solid substrate having a three-dimensional structure, so that the nanoparticles are fixed on the substrate and do not move any more, thereby greatly improving the reproducibility of SERS detection. However, the preparation of the three-dimensional solid substrate requires special expensive instruments such as a magnetron sputtering instrument, a laser etching instrument, an electrochemical deposition instrument and the like, and is time-consuming, labor-consuming and high in cost.

The nano particles are covered or deposited on the material by utilizing certain materials with natural three-dimensional structures, and the natural materials have wide sources, low price and environmental protection, so that the difficulty in manufacturing the solid substrate in the traditional technology is well solved. And many natural materials have hydrophobic effect, can concentrate and enrich nano particles or sample molecules, and further improve detection sensitivity. For example, researchers use natural rose petals, taro leaves, butterfly wings and the like as solid substrates to provide three-dimensional structures for modification of the noble metal nanoparticles, and the noble metal nanoparticles are used as SERS substrates.

Many shells have a structure of a pearl layer, and the surface looks colorful due to a structure similar to a grating. The shell pearl layer is formed by stacking a plurality of calcium carbonate lamellar structures to form a plurality of grooves, and has a good three-dimensional structure for covering nano particles. The shell pearl layer also has hydrophobic property, can concentrate nano particles and analytes, and further improves detection sensitivity. The nano particles on the groove have higher Raman enhancement effect, and the natural grating structure has certain regularity, so that the detection reproducibility can be improved. Furthermore, shell substrates have better mechanical strength relative to other kinds of natural materials.

Disclosure of Invention

The invention aims to prepare a shell-based Raman enhancement substrate which has wide source, low price, high sensitivity, high mechanical strength, environmental protection and hydrophobicity and is used for identifying different pathogenic bacteria.

The invention provides a shell-based SERS substrate, wherein the SERS substrate is obtained by covering a precious metal nanoparticle layer on the surface of a shell structure.

In one embodiment, the shell has a nacreous layer, including, but not limited to, mussels, abalone shells, pearl shells, and the like. The SERS substrate is obtained by covering a precious metal nanoparticle layer on the surface of a shell pearl layer.

In another embodiment, the noble metal nanoparticles include silver, gold, silver-coated gold, and the like noble metal nanoparticles. Further, the noble metal nanoparticles are silver-coated gold nanoparticles (Au @ Ag NPs). Furthermore, in the silver-coated gold nanoparticles, the particle size of the gold core is between 15 and 50nm, and the thickness of the silver shell is between 3 and 7 nm.

In a specific embodiment, the noble metal nanoparticles are silver-coated gold nanoparticles, the particle size of the gold core is 30nm, and the thickness of the silver shell is 5 nm.

The second aspect of the invention provides a method for preparing the shell-based SERS substrate, which comprises the following steps:

(1) pretreatment of shells: removing meat from shell, and sequentially treating shell with ethanol and water by ultrasonic treatment;

(2) preparing a substrate: and (3) dropwise adding the noble metal nano particle solution on the shells obtained in the step (1) to obtain the shell.

In one embodiment, the noble metal nanoparticle solution is prepared as follows:

(1) and (3) synthesis of gold core: HAuCl was added under magnetic stirring₄(0.1M) into 50mL of boiling ultrapure water, after stirring well, adding 0.75mL of sodium citrate (1% by weight) solution into the boiling solution; stirring for 30 min to obtain wine red dispersion of gold nanoparticles, cooling to room temperature, and adding 0.22 μm solutionFiltering with a porous filter membrane, and collecting filtrate;

(2) 10mL of the filtrate obtained in step 1 and ascorbic acid (0.1M) solution were taken and mixed well under magnetic stirring. 1mM AgNO was added at room temperature while maintaining magnetic stirring₃Dropwise adding one drop every 30 seconds, adding the solution into the solution, and continuously stirring for 30 minutes after the dropwise adding is finished to obtain the noble metal nano particle solution.

A third aspect of the invention provides the use of the SERS substrate for detecting microorganisms.

The fourth aspect of the invention provides an application of the SERS substrate in preparation of a microorganism detection kit.

The fifth aspect of the invention provides a method for detecting microorganisms by using the SERS substrate, which comprises the following steps:

(1) dripping a sample containing microorganisms on the SERS substrate;

(2) detecting the SERS substrate dropwise added with the sample by a laser micro-Raman system, obtaining a Raman fingerprint spectrum by exciting the wavelength of 633nm, and identifying the microorganisms in the sample by a chemometrics method.

The invention discovers that the surface of the shell pearl layer is of a natural multistage micro-nano structure, the shell pearl layer is formed by staggered arrangement of a plurality of nano-scale lamella layers, a regular and ordered brick wall structure is embodied, and the lamella layers are mostly composed of calcium carbonate and conchiolin, so that the mechanical strength is high. Certain grooves are formed between the sheet layers, the shapes of the grooves can be combined into a Y shape, a V shape and a cross shape, the grooves have abundant three-dimensional space structures, and the grooves can be used as templates for modifying nano particles. The surface structure of the shell pearl layer is observed on a micron level, and the shell sheet layers are arranged into a regular stripe structure to form a structure similar to a grating, so that the shell surface presents the colorful color.

In addition, the surface of the shell pearl layer has better hydrophobicity, and after the precious metal nano solution and/or probe molecules or pathogenic bacteria are dripped on the hydrophobic surface of the shell, the liquid is not easy to spread on the surface of the shell, thereby playing the roles of concentration and enrichment.

More importantly, the shell pearl layer has better enhancement effect on Raman signals. The obtained SERS substrate based on shells has high sensitivity and good reproducibility.

Drawings

FIG. 1 ultraviolet absorption spectrogram for synthesizing Au @ Ag NPs with different thicknesses

FIG. 2 Transmission Electron Microscopy (TEM) images of Au @ Ag NPs synthesized in different thicknesses

FIG. 3 schematic of the preparation of a Shell-based SERS substrate

FIG. 4 Raman enhancement effect of Au @ Ag NPs with different shell thicknesses

FIG. 5a) shell nacreous layer photographed with a digital camera, b), c) study of shell nacreous layer hydrophobicity, d) measurement of shell nacreous layer contact angle, e) characterization and verification of successful nanoparticle modification onto shell substrate, f) effect of different excitation wavelengths on detection results

FIG. 6 is a micro-nano structure of a synthesized SERS substrate, the left image is a shell pearl layer shot by a digital camera, and the right image is a micrometer structure diagram shot by a scanning electron microscope

FIG. 7 Scanning Electron Microscope (SEM) study of the nanostructure of the shell substrate synthesized, a) lower magnification scanning electron microscope image of unmodified shell substrate, b) higher magnification scanning electron microscope image, c) lower magnification scanning electron microscope image of shell substrate modified with nanoparticles, d) higher magnification scanning electron microscope image of shell substrate modified with nanoparticles

FIG. 8 scanning electron micrograph of Shell surface modified with Au @ Ag NPs

FIG. 9 Raman scattering spectra of different concentrations of aqueous solutions of R6G molecules tested using Raman spectrometer under microscope system

FIG. 10 repeatability examination of SERS substrates, a) 100 μm on the surface of a shell²Randomly selecting 20 points to acquire Raman signals, wherein 20 spectrograms can be seen on the chart and have good reproducibility, b) taking the peak intensity at 613cm-1 as a histogram to obtain the RSD of 6.5%. c) Selecting 100 on the surface of the shellThe raman signal imaging was performed in the region of μm2 at intervals of 1 μm, and the results showed that the intensity of the raman signal collected from the shell surface was relatively uniform and the uniformity of the shell substrate was good.

FIG. 11a is a comparison of fresh rose petals, butterfly wings, taro leaves, and shells showing several materials intact; b, performing a tensile test on various materials, wherein the figure shows that after being pulled, only shells are kept intact, and other materials are all broken; c is a comparison of several materials after 12 hours at room temperature,

FIG. 12 modification of the Shell substrate of 5nm Shell thickness Au @ Ag NPs for the identification of different species of bacteria, a) Raman fingerprint of E.coli, b) Raman fingerprint of P.aeruginosa, c) Raman fingerprint of S.aureus, d) analysis of the obtained Raman fingerprints by PCA method

Detailed Description

The invention may be further understood by reference to the following examples, which illustrate some methods of making or using. However, it is to be understood that these examples do not limit the present invention. Variations of the invention, now known or further developed, are considered to fall within the scope of the invention as described herein and claimed below.

Example 1 Shell-based SERS substrate preparation

(1) Pretreatment of shells: the mussel shells bought in the market are fleshed, and the remaining shells are respectively treated with ultrasonic treatment for 10 minutes by using ethanol and deionized water to obtain clean mussel shells.

(2) Synthesis of 30nm gold core

All glassware was treated with 3:1 by volume HNO prior to use₃HCl for one hour, then sonicated with ultrapure water and dried in an oven. The synthesis method is to use sodium citrate to react with chloroauric acid (HAuCl)₄) Reducing to obtain 30nm gold nanoparticles. First, 0.125mL of HAuCl was added with magnetic stirring₄(0.1M) was added to 50mL of boiling ultrapure water, and after stirring well, 0.75mL of a sodium citrate (1% by weight) solution was added to the aboveIn boiling solution. Stirring for 30 minutes to obtain a wine red dispersion, namely the gold nanoparticle dispersion, cooling the dispersion to room temperature, filtering with a 0.22 micron microporous filter membrane, and storing the filtrate in a refrigerator at 4 ℃.

(3) Synthesis of Au @ Ag NPs with different thicknesses

All glassware was treated with 3:1 by volume HNO prior to use₃HCl for one hour, then sonicated with ultrapure water and dried in an oven. The synthesis method of Au @ Ag NPs mainly comprises the steps of reducing silver nitrate (AgNO3) by using ascorbic acid to form a thin silver shell on the surface of a gold core, and obtaining the silver shells with different thicknesses according to the amount of the added silver nitrate. In a 50mL flask, 10mL of the prepared gold nanoparticle solution and ascorbic acid (0.1M) solution were added and mixed well under magnetic stirring. While maintaining magnetic stirring, 1mM AgNO3 was added dropwise (one drop every 30 seconds) to the above solution at room temperature, and stirring was continued for 30 minutes after completion of the addition to obtain a solution of Au @ Ag NPs. Adding varying amounts of AgNO₃The Au @ Ag NPs solution with different thicknesses is obtained, the amount of AgNO3 added is respectively 0.5mL,1.5mL,2.5mL,3.5mL and 4.5mL, and the Au @ Ag NPs with the thicknesses of 1nm, 3nm, 5nm, 7nm and 9nm are obtained. The obtained Au @ Ag NPs of different thicknesses were stored in a refrigerator at 4 ℃ for further use.

Referring to the attached FIG. 1, which is an ultraviolet absorption spectrum diagram of synthesized Au @ Ag NPs with different thicknesses, it can be seen from the diagram that Au @ Ag NPs with the shell thickness of 1nm only have a plasma resonance absorption peak around 520nm, and as the shell thickness increases, the peak at 520nm gradually decreases, and the peak at 380nm gradually increases.

Referring to FIG. 2, which is a Transmission Electron Microscope (TEM) of the synthesized Au @ Ag NPs of different thicknesses, it can be seen that a is a TEM of the Au @ Ag NPs of 5nm shell thickness in a lower magnification view, from which it can be seen that the synthesized nanoparticles have a relatively uniform and spherical particle size distribution; b-f are TEM images of Au @ Ag NPs with different shell thicknesses in high-multiple visual field, and it can be seen from the images that a thin silver shell is uniformly wrapped on the surface of a gold core, and AgNO is synthesized₃The larger the dosage, the larger the shell thickness, the synthesized nano-particleThe larger the volume of the daughter.

(4) Modifying nano particles on shells to serve as SERS enhanced substrate

Referring to the attached figure 3, a drop of 20 microliter Au @ Ag NPs was dropped onto the pearl layer of mussel shell and naturally dried. And (3) dripping a drop of 20 microliter of probe molecule rhodamine 6G on the shell substrate modified with Au @ Ag NPs, and naturally airing for investigating the Raman enhancement performance of the synthesized substrate.

(5) Selection of different types of nanoparticles and different thicknesses of Au @ Ag NPs

Rhodamine 6G is used as a probe molecule, Raman enhancement effects of Au NPs, Ag NPs and Au @ Ag NPs with different shell thicknesses are respectively considered and modified, and referring to the attached drawing 4, it can be seen from a and b that the Raman enhancement effect gradually increases with the increase of the shell thickness from 1nm to 5nm, but when the shell thickness is larger than 5nm, the Raman enhancement effect is gradually smaller, and the correlation between the Raman enhancement effect of the synthesized shell substrate and the shell thickness of the Au @ Ag NPs is demonstrated. Meanwhile, the SERS enhancement effects of pure Au NPs and Ag NPs are considered, and the result shows that the SERS enhancement effect of the Au NPs is the weakest, and the enhancement effect of the Ag NPs is between 7nm and 3nm of Au @ Ag NPs. Therefore, the enhancement effect of Au @ Ag NPs with a shell thickness of 5nm is the best.

(6) Characterization of the synthesized Shell substrate

a) Study of shell pearl layer hydrophobicity and measurement of contact angle

Referring to fig. 5a, which is an unmodified mussel shell, the inset shows that when a drop is dropped on the shell, it shrinks into a drop, keeping a round shape, and the naked eye preliminarily judges that the nacreous layer of the shell has a certain hydrophobicity.

Referring to fig. 5b, a contact angle measuring instrument is used to measure the hydrophobicity of the shell surface before and after modification of the 5nm shell thickness Au @ Ag NPs, and the result shows that the contact angle before modification of the nanoparticle by the shell pearl layer is 100.5 °, which indicates that the shell pearl layer has hydrophobicity, the contact angle after modification of the 5nm shell thickness Au @ Ag NPs by the shell pearl layer is 99.6 °, which indicates that the hydrophobicity does not change greatly, which indicates that the shell surface still has hydrophobicity after modification of the nanoparticle.

b) Characterization and confirmation of successful modification of nanoparticles onto shell substrates

Referring to the attached figure 5c, a black curve is an ultraviolet spectrogram of the shell substrate before modification of 5nm shell thickness Au @ Ag NPs, an absorption peak is formed around 390nm and is an ultraviolet absorption peak of the shell substrate, a red curve is an ultraviolet absorption spectrogram of the shell substrate after modification of 5nm shell thickness Au @ Ag NPs, and the ultraviolet absorption peak is shown to be added at 510nm, which indicates that Au @ Ag NPs are successfully modified on the pearl shell layer. Referring to the attached figures e and f, which are energy spectra before and after the shell substrate is modified with 5nm shell thickness Au @ Ag NPs, respectively, the results show that the shell has no Au and Ag elements before the modification of the Au @ Ag NPs, and the existence of the two metal elements can be detected after the modification of the Au @ Ag NPs. The results mutually verify that Au @ Ag NPs are successfully modified on the shell.

c) Selection of excitation wavelength

Different excitation wavelengths have influence on the detection result, and referring to SERS spectrograms detected by 532nm and 633nm excitation light in fig. 5d, 633nm is selected as the optimal excitation wavelength because the background fluorescence at 532nm is too strong.

d) Micro-nano structure of SERS substrate synthesized by research institute

Referring to fig. 6, the left image is a shell nacreous layer photographed with a digital camera, on which it can be seen that the shell nacreous layer is colorful in appearance; the right picture is a micrometer structure picture of the shell pearl layer shot by a scanning electron microscope, and the picture can show that the surface of the shell has periodic stripe structures which are similar to grating structures and can diffract natural light, so that the shell pearl layer displays colorful colors under the natural light. Importantly, the regular stripe structures enable the shell substrate to have better uniformity, and are beneficial to improving the reproducibility of subsequent SERS detection.

The nanostructure of the synthesized shell substrate was studied using Scanning Electron Microscopy (SEM). Referring to fig. 7, a is a scanning electron microscope image of a shell substrate without modification at a lower multiple, and b is a scanning electron microscope image of a shell substrate at a higher multiple, and the scanning electron microscope image shows that the shell substrate is composed of a plurality of polygonal lamellae, and grooves are formed between the lamellae to form a template with a three-dimensional structure, which can be used for modifying nanoparticles. In addition, the lamellar structure gives the shell substrate high mechanical strength, and the rough surface is also the reason for the hydrophobicity of the shell surface. c is a scanning electron microscope image of the shell substrate modified with the nano particles under a lower multiple, d is a scanning electron microscope image of the shell substrate modified with the nano particles under a higher multiple, and as can be seen from the images, the nano particles are uniformly distributed on each sheet layer of the shell. The surface of the shell has hydrophobicity, so that more nano particles are gathered on the surface unit area of the shell.

e) FDTD simulates local electric field distribution of the metal nanoparticles modified on the shell, so that the contribution of the metal nanoparticles at different positions on the three-dimensional template of the shell to SERS physical enhancement is obtained

We performed FDTD simulations based on the distribution of Au @ Ag NPs on the shell substrate by scanning electron microscopy. Referring to fig. 8, a is a scanning electron microscope image of the shell surface modified with Au @ Ag NPs, and it can be seen that the grooves formed by the shell surface lamellar structure have three shapes, which are respectively marked with yellow lines and are divided into "V" shapes, "Y" shapes and "cross" shapes, and b is a schematic diagram of the three different types of grooves. FDTD simulation was performed with a trench formed between the two sheets of 220nm wide and two nanoparticles on the sheets of 27nm apart. FDTD simulation is carried out according to different nano particle arrangement forms formed by the three different similar grooves, c-e are simulation results, the nano particles on the grooves have higher electromagnetic enhancement effect, and the natural groove structure of the shell is proved to have good SERS enhancement effect after the nano particles are modified theoretically. In addition, it can be seen from the figure that the electromagnetic enhancement effects of the nanoparticles are not greatly different in the groove structures of different modes, which theoretically shows that the shell substrate has a local electromagnetic distribution with high uniformity. f is the cross section of the shell groove, and the electromagnetic enhancement effect of the nano particles at the upper end of the groove is stronger than that of the nano particles at the bottom of the groove.

f) SERS performance of modified Au @ Ag NPs nano particle shell substrate

At 633nm ofAt the incident wavelength, raman scattering spectra of different concentrations of R6G molecular water solutions were tested under a microscope system using a raman spectrometer. Referring to FIG. 9, at 10^-3～10^-9The Raman spectrogram of the M R6G solution after the test by taking the shell substrate modified with 5nm shell thickness Au @ Ag NPs as the SERS enhanced substrate can be seen from the graph, and the lower limit of the detection of the prepared SERS substrate on the R6G solution can reach 10^-9M, indicating a very high sensitivity.

And meanwhile, the repeatability of the SERS substrate is investigated. Referring to FIG. 10, a is a 100 μm mark on the surface of the shell²The range of the Raman spectrum is randomly selected from 20 points to acquire Raman signals, 20 spectrograms visible on the Raman spectrum have good reproducibility, and the b chart is 613cm^-1The peak intensity was histogram-plotted and the RSD was found to be 6.5%. Drawing c selecting 100 μm on the surface of the shell²The raman signal imaging was performed at 1 μm intervals in the area (d), and the results showed that the intensity of the raman signal collected from the shell surface was relatively uniform and the uniformity of the shell substrate was good.

And meanwhile, the durability of the SERS substrate is considered. Referring to fig. 11, a is a comparison of fresh rose petals, butterfly wings, taro leaves, and shells, showing that several materials are intact; b, performing a tensile test on various materials, wherein the figure shows that after being pulled, only shells are kept intact, and other materials are all broken; c is a comparison of several materials after 12 hours at room temperature, and shows that only shells and butterfly wings are kept in the original state, and rose petals and taro leaves are withered. Indicating that seashells are more durable than other natural materials that have been reported.

Comparing the synthesized substrate with other natural materials, the synthesized shell substrate has the advantages. Reference is made to attached table 1.

Literature (1) Huang, j.a.; zhang, y.l.; zhao, y.q.; zhang, XL.; sun, m.l.; zhang, W.J.Nanoscale 2016,8, 11487-.

Document (2) Lv, m.y.; teng, h.y.; chen, z.y.; zhao, y.m.; zhang, x.; liu, l.; wu, z.l.; liu, l.m.; xu, H.J.sensor.Actuat.B-chem.2015,209,820-827.

Document (3) Xu, b.b.; zhang, y.l.; zhang, w.y.; liu, x.q.; wang, j.n.; zhang, x.l.; zhang, d.d.; jiang, h.b.; zhang r.; sun, h.b.adv.opt.mater.2013,1,56-60.

Document (4) Song, g.f.; zhou, h.; gu, j.j.; liu, q.l.; zhang, w.; su, h.l.; su, y.s.; yao, q.h.; zhang, d.j.mater.chem.b,2017,5,1594.

Example 2 modification of Shell substrate with 5nm Shell thickness Au @ AgNPs for identification of different species of bacteria

(1) The shell substrate modified with 5nm shell thickness Au @ Ag NPs is used as an SERS enhanced substrate to collect Raman signals of different bacteria.

Different kinds of bacteria have unique Raman fingerprints, and the different kinds of bacteria can be identified by collecting the Raman fingerprints. Referring to fig. 12, 12 spectral lines are collected from each bacterium, wherein a is a raman fingerprint of escherichia coli, b is a raman fingerprint of pseudomonas aeruginosa, and c is a raman fingerprint of staphylococcus aureus, and differences of the raman fingerprints of the three bacteria can be seen visually.

(2) The three bacteria were statistically differentiated using Principal Component Analysis (PCA)

Referring to FIG. 12d, it is shown that the three bacteria can be clearly separated, and the results presented by the PCA method are more intuitive than observing the differences between the three bacteria from a fingerprint.

This summary merely illustrates some embodiments which are claimed, wherein one or more of the features recited in the claims can be combined with any one or more of the embodiments, and such combined embodiments are also within the scope of the present disclosure as if they were specifically recited in the disclosure.

Claims (5)

1. A shell-based SERS substrate is obtained by covering a precious metal nanoparticle layer on the surface of a shell structure; the shell is a shell with a pearl layer, and comprises mussel, abalone shell and pearl shell; the noble metal nanoparticles are silver-coated gold nanoparticles (Au @ Ag NPs), wherein the particle size of a gold core in the silver-coated gold nanoparticles is 30nm, and the thickness of a silver shell is 5 nm;

the preparation method of the substrate comprises the following steps:

(1) pretreatment of shells: removing meat from shell, and sequentially treating shell with ethanol and water by ultrasonic treatment for 10 min;

(2) preparing a substrate: dropwise adding a noble metal nanoparticle solution on the shells obtained in the step (1) to obtain the shell;

the preparation method of the noble metal nanoparticle solution comprises the following steps:

(a) and (3) synthesis of gold core: 0.1M HAuCl was added with magnetic stirring₄Adding the mixture into 50mL of boiling ultrapure water, and adding 0.75mL of sodium citrate solution with the weight of 1% after uniformly stirring; continuously stirring for 30 minutes to obtain a wine red dispersion liquid, namely the dispersion liquid of the gold nanoparticles, then cooling the dispersion liquid to room temperature, filtering by using a 0.22 micron microporous filter membrane, and collecting filtrate;

(b) taking 10mL of the filtrate obtained in step (a) and 0.1M ascorbic acid solution, mixing well under magnetic stirring, keeping magnetic stirring, and adding 1mM AgNO at room temperature₃Dropwise adding one drop every 30 seconds, adding the solution into the solution, and continuously stirring for 30 minutes after the dropwise adding is finished to obtain the noble metal nano particle solution.

2. A method for preparing the shell-based SERS substrate of claim 1, comprising the steps of:

(1) pretreatment of shells: removing meat from shell, and sequentially treating shell with ethanol and water by ultrasonic treatment;

(2) preparing a substrate: dropwise adding a noble metal nanoparticle solution on the shells obtained in the step (1) to obtain the shell;

the preparation method of the noble metal nanoparticle solution comprises the following steps:

(a) and (3) synthesis of gold core: under magnetic stirring, 0.1M HAuCl4 was added to 50mL of boiling ultrapure water, and after stirring well, 1% by weight of 0.75mL of sodium citrate solution was added; continuously stirring for 30 minutes to obtain a wine red dispersion liquid, namely the dispersion liquid of the gold nanoparticles, then cooling the dispersion liquid to room temperature, filtering by using a 0.22 micron microporous filter membrane, and collecting filtrate;

(b) taking 10mL of the filtrate obtained in step (a) and 0.1M ascorbic acid solution, mixing well under magnetic stirring, keeping magnetic stirring, and adding 1mM AgNO at room temperature₃Dropwise adding one drop every 30 seconds, adding the solution into the solution, and continuously stirring for 30 minutes after the dropwise adding is finished to obtain the noble metal nano particle solution.

3. Use of the SERS substrate of claim 1 to detect microorganisms.

4. The use of the SERS substrate of claim 1 in the manufacture of a kit for detecting microorganisms.

5. A method for detecting microorganisms using the shell-based SERS substrate of claim 1, comprising:

(1) dripping a sample containing microorganisms on the SERS substrate;

(2) detecting the SERS substrate dropwise added with the sample by a laser micro-Raman system, obtaining a Raman fingerprint spectrum by exciting the wavelength of 633nm, and identifying the microorganisms in the sample by a chemometrics method.

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