CN113702355B - Preparation method and application of AgNPs@PDMS porous microporous filter membrane SERS detection platform - Google Patents

Abstract

The invention discloses a preparation method and application of an AgNPs@PDMS porous microporous filter membrane SERS detection platform. Through self-assembly technology and multi-microporous structure for material preparation and assembly, a uniform and stable multi-"hot spot" structure can be formed. The invention rationally utilizes the self-assembly technology and the microporous structure to prepare a SERS detection platform that is beneficial to the fixation and enrichment of microbial samples and has multiple detection sites, which enhances the stability of the SERS detection system and expands the practicality. scope. The AgNPs@PDMS porous microporous membrane SERS detection platform provided by the present invention is a kind of nanomaterials arranged in an orderly manner, with a stable structure, eliminating the need for sample fixing time, and is conducive to efficient qualitative and quantitative simultaneous detection of multiple samples and sample enrichment Integrated detection platform, SERS detection has high sensitivity and good reproducibility.

Description

Preparation method and application of AgNPs@PDMS porous microporous membrane SERS detection platform

technical field

The invention belongs to the field of nanomaterials, and in particular relates to a preparation method and application of an AgNPs@PDMS porous microporous membrane SERS detection platform.

Background technique

Surface-enhanced Raman scattering (SERS) is an ultrasensitive selective analysis technique that uses noble metal nanoparticles attached to or close to the surface of target detection molecules to achieve signal enhancement, thereby distinguishing individual components of the sample through clear molecular-specific spectra. Components, an ideal technology for the rapid detection of multiple analytes. SERS substrates are mainly divided into two types: liquid-phase substrate materials based on metal nanosols and solid-phase substrate materials based on rough metal surface materials. With the increasing application of SERS technology in rapid detection, the development of SERS substrate materials has become the research focus in the field of Raman detection.

Existing studies have shown that metal substrates with abundant nanoscale interstitial ("hot spot") structures exhibit good SERS activity. Silver nanoparticles (AgNPs) are simple to prepare, easy to operate and have a good SERS enhancement effect, but their main disadvantage is the uncontrollable aggregation of nanomaterials. Self-assembly technology can fix metal nanomaterials on a solid substrate to form a solid-phase nanostructure substrate. The preparation process is relatively simple, and it can effectively solve the advantages of uncontrollable aggregation of nano-sols. As a widely used polymer, polydimethylsiloxane (PDMS) has the advantages of good elasticity, heat resistance, stability, low cost and easy molding. It serves as a solid-state SERS substrate carrier platform with multiple detection sites. In addition, the problem of difficult enrichment of analytes limits the application potential of SERS technology in quantitative detection of samples.

Contents of the invention

The purpose of the present invention is to provide a preparation method and application of a SERS detection platform based on AgNPs@PDMS porous microporous membrane, through self-assembly technology and multi-microporous structure material preparation and assembly, can form a uniform and stable multi-" "hot spot" structure. The 0.22 μm microporous membrane has a good filtration effect, and the bacteria can be immobilized and adsorbed on the microporous membrane, eliminating the need for sample fixation time, which is conducive to the enrichment of bacteria, so as to achieve the purpose of efficient quantitative detection. The SERS base material prepared by the invention has high sensitivity and good reproducibility, and can efficiently detect bacteria qualitatively and quantitatively.

In order to realize the above-mentioned purpose of the invention, the present invention adopts the following technical solutions:

A method for preparing an AgNPs@PDMS porous microporous membrane SERS detection platform, comprising the following steps:

(1) Synthesis of AgNPs sol: Add AgNO ₃ into ultrapure water, heat to boiling, quickly add sodium citrate aqueous solution under vigorous stirring, keep boiling for a period of time, and naturally cool to room temperature after the reaction to obtain AgNPs sol. The AgNPs sol was stored in the refrigerator at 4°C for later use;

(2) Soak a circular microporous filter membrane with a diameter of 3.5 mm in the AgNPs sol for a period of time, so that the AgNPs can interact with van der Waals to make the nanoparticles adsorb uniformly and irreversibly on the microporous filter membrane. It is self-assembled on the microporous membrane; the soaked and pulled microporous membrane is quickly dried with nitrogen to obtain AgNPs-microporous membrane;

(3) Preparation of PDMS porous plate: mix the polydimethylsiloxane prepolymer and curing agent evenly according to the volume ratio of 10:1, vacuum for 1 h to remove the small bubbles generated during the stirring process, and obtain the colloid; the colloid Slowly pour the PDMS inverted mold, put the PDMS inverted mold in an oven, and dry it at 70°C for 48 hours until the PDMS colloid is completely solidified and molded, and then demolded to obtain multiple identical "top width and bottom narrow" A PDMS plate with cylindrical grooves (the diameter of the upper cylinder is 3.5 mm, and the diameter of the lower cylinder is 3 mm), that is, the PDMS porous plate;

(4) First, a circular copper mesh with a diameter of 3.5 mm was placed in the upper cylinder at the hole of the PDMS porous plate as a support material, and then a layer of AgNPs-microporous filter membrane was placed on it to obtain AgNPs@PDMS polycarbonate. Porous microporous membrane SERS detection platform.

Further, in the step (1), the mass fraction of sodium citrate aqueous solution is 1%, 35-40 mg of AgNO ₃ requires 200 mL of ultrapure water, and 8 mL-10 mL of sodium citrate aqueous solution.

Further, in the step (1), the reaction is kept in a boiling state for 20-30 min.

Further, the circular microporous filter membrane with a diameter of 3.5 mm in the step (2) is made of polypropylene with a pore size of 0.22 μm.

Further, in the step (2), the circular microporous filter membrane is soaked and pulled in the AgNPs sol for 120-150s.

The AgNPs@PDMS porous microporous membrane SERS detection platform prepared by the preparation method of the present invention.

The application of the AgNPs@PDMS porous microporous membrane SERS detection platform of the present invention is based on the AgNPs@PDMS porous microporous membrane SERS detection platform as the SERS substrate, and laser confocal Raman is applied to the following three aspects of detection and analysis . (1) The practical application of three kinds of bacteria combined with the AgNPs@PDMS porous microporous membrane SERS detection platform, and the Raman signal enhancement effect of the three kinds of bacteria before and after the combination of the detection platform was compared; (2) Based on the detection platform, the Practical application of rapid identification of 3 kinds of bacteria; (3) Taking Clostridium perfringens as an example for quantitative identification of SERS.

Compared with the prior art, the advantages of the present invention are reflected in:

The AgNPs@PDMS porous microporous membrane SERS detection platform provided by the present invention is a kind of nanomaterials arranged in an orderly manner, with a stable structure, which eliminates the need for sample fixing time, and is conducive to the efficient qualitative and quantitative detection of multiple samples and sample enrichment at the same time. Integrated detection platform, SERS detection with high sensitivity and good reproducibility. The present invention rationally utilizes the self-assembly technology and the microporous structure to prepare a SERS detection platform that is not only beneficial to the fixation and enrichment of microbial samples, but also has multiple detection sites, which enhances the stability of the SERS detection system and expands the practicality. scope.

Description of drawings

Fig. 1 is a schematic flow diagram of an embodiment of the preparation method of the AgNPs@PDMS porous microporous membrane SERS detection platform of the present invention;

Figure 2 is the UV-visible spectrum, laser particle size and TEM characterization results of AgNPs sol prepared by sodium citrate in this example, (A) is the UV-visible spectrum of AgNPs sol, (B) is the particle size distribution of AgNPs sol, ( C) TEM image of AgNPs sol;

Fig. 3 is the SEM characterization result of AgNPs-microporous membrane substrate in the present embodiment;

Figure 4 is the actual application of the enhancement effect comparison of three kinds of bacteria combined with AgNPs@PDMS porous microporous membrane SERS detection platform in this example. A and A1 respectively use the SERS detection platform to perform Raman on Clostridium perfringens The spectra before and after the detection, B1 and B2 were the spectra before and after the Raman detection of Bacillus subtilis using the SERS detection platform, and the spectra of C and C1 were respectively before and after the Raman detection of Staphylococcus aureus using the SERS detection platform, S means The Raman spectrum of the AgNPs@PDMS porous microporous membrane SERS detection platform was used as a blank control.

Figure 5 shows the results of SERS characterization of three bacteria using a laser confocal Raman spectrometer based on the AgNPs@PDMS porous microporous membrane SERS detection platform in this example. C. perfringens represents Clostridium perfringens Average SERS spectrum, B.subtilis represents the average SERS spectrum of Bacillus subtilis, S.aureus represents the average SERS spectrum of Staphylococcus aureus;

Figure 6 shows the reproducibility results of SERS detection of three bacteria based on the AgNPs@PDMS porous microporous membrane SERS detection platform in this example;

Figure 7 is the Raman spectrum of different concentrations of Clostridium perfringens determined based on the AgNPs@PDMS porous microporous membrane SERS detection platform in this example. Among them, a is 10 ⁸ cfu/mL; b is 10 ⁷ cfu/mL; c is 10 ⁶ cfu/mL; d is 10 ⁵ cfu/mL; e is 10 ⁴ cfu/mL; f is 10 ³ cfu/mL; g is 10 ² cfu/mL, h is 10 ¹ cfu/mL;

Fig. 8 The logarithmic value of different concentrations of Clostridium perfringens and its Raman intensity at 1285 cm-1 in the SERS spectrum correspond to the linear relationship diagram.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following examples are only used to illustrate the present invention rather than limit the scope of the present invention, and those skilled in the art can make some non-essential improvements and adjustments based on the content of the above invention.

Example 1

As shown in Figure 1, it is a schematic flow diagram of an embodiment of the preparation method of the AgNPs@PDMS porous microporous membrane SERS detection platform, including the following steps:

Step 1. Synthesis of AgNPs sol. Add 35 mg of AgNO ₃ to 200 mL of ultrapure water, heat to boiling, quickly add 8 mL of 1% sodium citrate aqueous solution under vigorous stirring, keep boiling for 30 min, cool naturally to room temperature, and the solution It is yellow-green. Cool the prepared solution and store it in the refrigerator at 4°C for later use. As shown in part (A) of Figure 1.

Step 2. Soak and pull a circular microporous filter membrane (polypropylene with a pore size of 0.22 μm) with a diameter of 3.5 mm in 5 mL of AgNPs sol for 120 s. Due to the presence of van der Waals interactions, the AgNPs nanoparticles can Uniform and irreversible adsorption on the microporous membrane to make it self-assembled on the microporous membrane. The soaked and pulled microporous membrane was quickly dried with nitrogen to obtain AgNPs-microporous membrane. As shown in Figure 1(B) part.

Step 3. Mix the main agent in the PDMS reagent: auxiliary agent (i.e. polydimethylsiloxane prepolymer: curing agent) in a test tube at a volume ratio of 10:1 and stir well with a glass rod, vacuum for 1 h Remove the small air bubbles generated during the stirring process to obtain the colloid; slowly pour the colloid into the PDMS inverted mold, put the PDMS inverted mold in an oven, and dry it at 70°C for 48 h until the PDMS colloid is completely solidified and molded. A PDMS plate with multiple identical "upper wide and lower narrow" cylindrical grooves (the diameter of the upper cylinder is 3.5 mm, and the diameter of the lower cylinder is 3 mm) is prepared, that is, a PDMS multi-hole plate. As shown in part (C) of Figure 1.

Step 4. First put a circular copper mesh with a diameter of 3.5 mm in the upper cylinder of the PDMS porous plate as a support material, and then put a layer of AgNPs-microporous filter membrane to obtain AgNPs@PDMS porous microporous filter. Membrane SERS detection platform.

The AgNPs sol and AgNPs-microporous membrane obtained in Step 1 and Step 2 were respectively characterized, and the characterization results are shown in Figure 2 and Figure 3 . Among them, the results in Figure 2(A)-(C) show that the AgNPs sol is in the form of spherical particles, the particle size is mainly distributed in the range of 40-80 nm, and the average size is about 69 nm. The results in Figure 3 show that the AgNPs successfully self-assembled on the microporous membrane into a compact two-dimensional array of AgNPs, and the AgNPs were evenly distributed on the microporous membrane, forming a rich "hot spot" structure, which is conducive to enhancing the SERS activity.

Example 2

Clostridium perfringens, Bacillus subtilis and Staphylococcus aureus stored in -80°C refrigerator were activated three times on tryptone-sulfite-cycloserine agar and nutrient agar respectively. Wash 3 times with sterile deionized water and resuspend. Clostridium perfringens, Bacillus subtilis and Staphylococcus aureus were dropped onto glass slides and AgNPs@PDMS porous microporous membrane SERS detection platform respectively, and SERS scanning was performed immediately. The excitation wavelength is 632.8 nm, and the scanning time is 20s. Using a 100× objective lens, the scanning range is 400 cm ^-1 to 1800 cm ^-1 . The spectra were smoothed and baseline corrected using the automatic functions of the LabSpec 6.0 software that comes with the instrument. The SERS enhancement effects of the AgNPs@PDMS porous microporous membrane SERS detection platform on the three bacteria were compared, and the results are shown in Figure 4. When the SERS platform was not used for detection, the Raman spectrum intensities of Clostridium perfringens, Bacillus subtilis and Staphylococcus aureus were very low, and it was difficult to distinguish the three bacteria through Raman characteristic peaks; after detection by the SERS platform , the Raman signals of the three bacteria were significantly enhanced and the characteristic peak differences in the SERS spectra were obvious, indicating that the AgNPs@PDMS porous microporous membrane SERS detection platform has good Raman spectral signal enhancement capabilities.

Example 3

According to the method in Example 2, three kinds of bacteria were cultivated and purified to prepare SERS detection samples. Add 10 μL of Clostridium perfringens, Bacillus subtilis and Staphylococcus aureus dropwise on the AgNPs@PDMS porous microporous membrane SERS detection platform, and immediately perform SERS scanning, and randomly measure 10 Raman strips for each sample spectrum. Raman detection parameters and data processing methods are the same as in Example 2. Calculate each average spectrum for difference comparison and reproducibility comparison for 10 spectra of each sample, the results are shown in Figure 5 and Figure 6. In Figure 5, it can be observed that the Raman vibration peak positions and peak intensities in the SERS spectra of the three bacteria are significantly different, and the different Raman vibration peaks are attributed to different components and chemical bond vibration forms of the bacteria. It can be seen that the SERS technology based on the AgNPs@PDMS porous microporous membrane SERS detection platform as the base material has the ability to distinguish different species of bacteria, and can realize the rapid identification of different bacteria. Figure 6 shows the reproducibility results of SERS detection of three bacteria based on the AgNPs@PDMS porous microporous membrane SERS detection platform in this example. The results show that the 10 random SERS spectra of each bacterium have good Consistency, based on AgNPs@PDMS porous microporous membrane SERS detection platform as the substrate material, the SERS enhanced method has high stability and good reproducibility, which provides a solid foundation for the rapid detection of foodborne pathogens based on SERS technology. Strong technical support.

Example 4

Use the SERS detection platform to quantitatively detect Clostridium perfringens, and the specific steps are as follows: Culture Clostridium perfringens stored in a -80°C refrigerator on tryptone-sulfite-cycloserine agar and nutrient agar The base is activated three times. The colonies on the plate were picked and cultured in liquid thioglycolate liquid medium for 24 h, and the bacterial count was carried out by the dilution coating plate method. The bacteria were washed three times with ultrapure water and resuspended, and the OD 600 value of the enrichment solution was measured with a microplate reader. After measurement and counting, when the OD 600 of the enrichment solution was 1.92, the concentration of the bacteria solution was on the order of 10 ⁸ cfu/mL. Add 10 μL of 10 ⁸ cfu/mL, 10 ⁷ cfu/mL, 10 ⁶ cfu/mL, 10 ⁵ cfu/mL, 10 ⁴ cfu/mL to the AgNPs@PDMS porous microporous membrane SERS detection platform in turn. , 10 ³ cfu/mL, 10 ² cfu/mL and 10 ¹ cfu/mL Clostridium perfringens suspensions were tested by SERS. The Raman detection parameters are the same as those shown in Example 2. Figure 7 and Figure 8 are respectively the SERS spectra of different concentrations of Clostridium perfringens and the linear relationship between the logarithmic values of different concentrations of Clostridium perfringens and the Raman intensity at 1285 cm ^-1 in the SERS spectrum. From the SERS spectra of different concentrations of Clostridium perfringens enhanced by the AgNPs@PDMS porous microporous membrane SERS detection platform, it can be seen that Clostridium perfringens has the strongest SERS signal at 1285 cm ^-1 , which will The Raman peak at 1285 cm-1 was used as the characteristic peak for estimating the concentration of bacterial cells, and the intensity of the Raman peak at 1285 cm ^-1 , the strongest characteristic peak of Clostridium perfringens, was used to quantify the logarithmic value of different concentrations equation. Figure 7 shows that under the same measurement conditions, the Raman peak intensity of Clostridium perfringens at 1285 cm ^-1 decreases with the decrease of bacterial concentration. At concentrations lower than 10 ² cfu/mL, the main characteristic peak of Clostridium perfringens at 1285 cm ^-1 could not be clearly identified. Therefore, the detection limit of this method for Clostridium perfringens was estimated to be 10 ² cfu/mL. In Figure 8, the logarithmic value (lg C) of different Clostridium perfringens concentrations is taken as the abscissa, and the peak intensity at the strongest characteristic peak at 1285 cm ^-1 is taken as the ordinate, and a quantitative equation y=555.82x ² - 2614.91x+4053.70, correlation coefficient R ² =0.961. It can be seen that the SERS technology based on the AgNPs@PDMS porous microporous membrane SERS detection platform has good quantitative analysis ability. In addition, due to the bacterial enrichment effect of the microporous membrane, the minimum detection limit of Clostridium perfringens can reach 10 ² cfu/mL, and the SERS enhancement performance is good.

The above descriptions are only preferred specific embodiments of the present invention, and are not intended to limit the scope of the invention. Anyone familiar with the technical field within the technical scope disclosed in the present invention, according to the technical scheme of the present invention and its inventive concepts to make equivalent replacements or changes shall fall within the scope of protection of the present invention.

Claims (8)

1. A preparation method of an AgNPs@PDMS microporous filter membrane SERS detection platform is characterized by comprising the following steps of: the method comprises the following steps:

(1) Synthesis of AgNPs sol: agNO is to be carried out ₃ Adding the solution into ultrapure water, heating to boil, rapidly adding sodium citrate aqueous solution under intense stirring, keeping boiling state for reaction for a period of time, naturally cooling to room temperature after the reaction is finished to obtain AgNPs sol, and storing the AgNPs sol in a refrigerator at 4 ℃ for later use;

(2) Soaking and pulling the round microporous filter membrane in AgNPs sol for 120 s, and rapidly drying the soaked and pulled microporous filter membrane with nitrogen to obtain an AgNPs-microporous filter membrane;

(3) Preparing a PDMS porous plate: uniformly mixing a polydimethylsiloxane precursor and a curing agent according to a volume ratio of 10:1 to obtain a colloid, slowly pouring the colloid into a PDMS (polydimethylsiloxane) reverse mould, putting the PDMS reverse mould into an oven, drying 48-h until the PDMS colloid is completely solidified and molded at 70 ℃, and demolding to obtain the PDMS porous plate;

(4) And (3) placing a round copper mesh with the diameter of 3.5 and mm at the orifice of the PDMS porous plate as a supporting material, and placing a layer of AgNPs-microporous filter membrane, thus obtaining the AgNPs@PDMS porous microporous filter membrane SERS detection platform.

2. The method for preparing the AgNPs@PDMS microporous filter membrane SERS detection platform according to claim 1, which is characterized by comprising the following steps: in the step (1), the mass fraction of the sodium citrate aqueous solution is 1%, and 35-40 mg of AgNO ₃ 200-mL of ultrapure water and 8-10 mL of sodium citrate aqueous solution are required.

3. The method for preparing the AgNPs@PDMS microporous filter membrane SERS detection platform according to claim 1, which is characterized by comprising the following steps: and (3) keeping the boiling state in the step (1) for reacting for 20-30 min.

4. The method for preparing the AgNPs@PDMS microporous filter membrane SERS detection platform according to claim 1, which is characterized by comprising the following steps: the round microporous filter membrane with the diameter of 3.5 and mm in the step (2) is made of polypropylene, and the pore diameter is 0.22 mu m.

5. The method for preparing the AgNPs@PDMS microporous filter membrane SERS detection platform according to claim 1, which is characterized by comprising the following steps: and (3) immersing and pulling the round microporous filter membrane with the diameter of 3.5 and mm in the step (2) in AgNPs sol for 120-150s.

6. An agnps@pdms microporous filter membrane SERS detection platform prepared according to the preparation method of any one of claims 1-5.

7. The application of the AgNPs@PDMS microporous filter membrane SERS detection platform according to claim 6, which is characterized in that: bacteria are rapidly identified based on the detection platform.

8. The use according to claim 7, characterized in that: clostridium perfringens is used for SERS quantitative identification as an example.

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