CN113702355B - Preparation method and application of AgNPs@PDMS porous microporous filter membrane SERS detection platform - Google Patents
Preparation method and application of AgNPs@PDMS porous microporous filter membrane SERS detection platform Download PDFInfo
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Abstract
The invention discloses a preparation method and application of an AgNPs@PDMS microporous filter membrane SERS detection platform, wherein a uniform and stable multi-hot-spot structure can be formed by carrying out material preparation and assembly through a self-assembly technology and a microporous structure. The invention reasonably utilizes self-assembly technology and microporous structure to prepare the SERS detection platform which is not only favorable for the fixation and enrichment of microorganism samples, but also has a plurality of detection sites, thereby expanding the practical range while enhancing the stability of the SERS detection system. The AgNPs@PDMS microporous filter membrane SERS detection platform provided by the invention is an integrated platform for efficient qualitative and quantitative detection, which is orderly arranged by nano materials, stable in structure, free of sample fixing time, favorable for simultaneous detection of multiple samples and enrichment of the samples, high in SERS detection sensitivity and good in reproducibility.
Description
Technical Field
The invention belongs to the field of nano materials, and particularly relates to a preparation method and application of an AgNPs@PDMS porous microporous filter membrane SERS detection platform.
Background
Surface Enhanced Raman Scattering (SERS) is an ultrasensitive selective analysis technique that uses noble metal nanoparticles attached to or near the surface of target detection molecules for signal enhancement purposes, thereby distinguishing individual components of a sample through a clear molecular-specific spectrum, and is an ideal technique for rapid detection of multiple analytes. SERS substrates are mainly classified into liquid phase substrate materials based on metal nanosols and solid phase substrate materials based on roughened metal surface materials. With the increasing development of SERS technology in rapid detection, the development of SERS substrate materials has become the focus of research in the field of raman detection.
Current studies indicate that metal substrates with rich nanoscale gap ("hot spot") structures exhibit good SERS activity. Nano silver (AgNPs) is simple to prepare, easy to operate and has a good SERS enhancement effect, and the main disadvantage is uncontrolled aggregation of nano materials. The self-assembly technology can fix the metal nano material on the solid matrix to form the solid phase nano structure substrate, the preparation process is relatively simple, and the uncontrollable aggregation of the nano sol can be effectively solved. Polydimethylsiloxane (PDMS) is widely used as a high-molecular polymer, has the advantages of good elasticity, heat resistance, stability, low cost, easy manufacturing and forming and the like, and can be used as a solid SERS substrate carrier platform with multiple detection sites through a microporous structure design. In addition, the problem of difficult enrichment of the analyte limits the potential of application of SERS techniques in quantitative detection of samples.
Disclosure of Invention
The invention aims to provide a preparation method and application of an AgNPs@PDMS porous microporous filter membrane SERS detection platform, wherein a uniform and stable multi-hot-spot structure can be formed by carrying out material preparation and assembly through a self-assembly technology and a microporous structure. 0.22 The microporous filter membrane with the diameter of mu m has good filtering effect, and can fix and adsorb bacteria on the microporous filter membrane, so that the sample fixing time is saved, the enrichment of bacteria is facilitated, and the aim of efficient quantitative detection is achieved. The SERS substrate material prepared by the invention has high sensitivity and good repeatability, and can be used for high-efficiency qualitative and quantitative detection of bacteria.
In order to achieve the above object, the present invention adopts the following technical scheme:
a preparation method of an AgNPs@PDMS microporous filter membrane SERS detection platform comprises the following steps:
(1) Synthesis of AgNPs sol: agNO is to be carried out 3 Adding into ultrapure water, heating to boil, and heating to boilRapidly adding sodium citrate aqueous solution under vigorous 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 placing the AgNPs sol in a refrigerator for preservation at 4 ℃ for later use;
(2) Immersing and pulling a round microporous filter membrane with the diameter of 3.5 and mm in AgNPs sol for a period of time, so that AgNPs interact with each other in Van der Waals to uniformly and irreversibly adsorb nano particles on the microporous filter membrane and self-assemble the nano particles on the microporous filter membrane; rapidly drying the soaked and pulled microporous filter membrane by 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, and performing vacuum 1-h to remove small bubbles generated in the stirring process so as to obtain a colloid; slowly pouring the colloid into a PDMS (polydimethylsiloxane) reverse mould, putting the PDMS reverse mould into an oven, drying at 70 ℃ for 48 h until the PDMS colloid is completely solidified and molded, and demolding and molding to obtain a PDMS plate (the diameter of an upper cylinder is 3.5 mm, the diameter of a lower cylinder is 3 mm) with a plurality of identical cylindrical grooves which are wide at the upper side, namely a PDMS multi-hole plate;
(4) Firstly, a round copper mesh with the diameter of 3.5 mm is placed in an orifice of a PDMS porous plate, namely an upper layer cylinder, to serve as a supporting material, and then an AgNPs-microporous filter membrane is placed on the round copper mesh, so that an AgNPs@PDMS porous microporous filter membrane SERS detection platform is obtained.
Further, in the step (1), the mass fraction of the sodium citrate aqueous solution is 1%, and 35-40 mg of AgNO 3 200 mL-10 mL of ultrapure water, mL, and 8 mL of sodium citrate aqueous solution are required.
And (3) further, keeping the boiling state in the step (1) for reacting for 20-30 min.
Further, 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 μm.
Further, the circular microporous filter membrane in the step (2) is soaked in AgNPs sol and pulled for 120-150s.
The AgNPs@PDMS porous microporous filter membrane SERS detection platform prepared by the preparation method disclosed by the invention.
The application of the AgNPs@PDMS microporous filter membrane SERS detection platform is based on the fact that the AgNPs@PDMS microporous filter membrane SERS detection platform is a SERS substrate, and laser confocal Raman is applied to detection analysis of the following three aspects. (1) The practical application of the 3 bacteria combined AgNPs@PDMS microporous filter membrane SERS detection platform compares the Raman signal enhancement effects before and after the 3 bacteria are combined with the detection platform; (2) Based on the practical application of the detection platform for rapidly identifying 3 bacteria; (3) Clostridium perfringens is used for SERS quantitative identification as an example.
Compared with the prior art, the invention has the advantages that:
the AgNPs@PDMS microporous filter membrane SERS detection platform provided by the invention is an integrated platform for efficient qualitative and quantitative detection, which is orderly arranged by nano materials, stable in structure, free of sample fixing time, favorable for simultaneous detection of multiple samples and enrichment of the samples, high in SERS detection sensitivity and good in reproducibility. The invention reasonably utilizes self-assembly technology and microporous structure to prepare the SERS detection platform which is not only favorable for the fixation and enrichment of microorganism samples, but also has a plurality of detection sites, thereby expanding the practical range while enhancing the stability of the SERS detection system.
Drawings
FIG. 1 is a schematic flow chart of an embodiment of a preparation method of an AgNPs@PDMS microporous filter membrane SERS detection platform according to the present invention;
FIG. 2 shows the UV-visible spectrum, laser granularity and TEM characterization results of the AgNPs sol prepared from sodium citrate in this example, (A) the UV-visible spectrum of the AgNPs sol, (B) the particle size distribution of the AgNPs sol, and (C) the transmission electron microscope image of the AgNPs sol;
FIG. 3 is a SEM characterization result of AgNPs-microporous filter membrane substrate in this example;
fig. 4 shows the actual application of the contrast of the enhancement effect of the 3 bacteria combined with the agnps@pdms microporous filter membrane SERS detection platform in this embodiment, the spectra before and after raman detection of clostridium perfringens by using the SERS detection platform are a and A1, the spectra before and after raman detection of bacillus subtilis by using the SERS detection platform are B1 and B2, and the spectra before and after raman detection of staphylococcus aureus by using the SERS detection platform are C and C1, respectively, and S represents the raman spectrum using the agnps@pdms microporous filter membrane SERS detection platform as a base material as a blank control.
FIG. 5 shows the results of SERS characterization of 3 bacteria using a laser confocal Raman spectrometer based on an AgNPs@PDMS microporous filter membrane SERS detection platform as a substrate in the present example,C. perfringensrepresents the average SERS spectrum of clostridium perfringens,B.subtilisrepresents the average SERS spectrum of bacillus subtilis,S. aureusmean SERS spectrum of staphylococcus aureus;
FIG. 6 shows the reproducibility of SERS detection of 3 bacteria based on AgNPs@PDMS microporous filter membrane SERS detection platform as substrate in this example;
fig. 7 is a raman spectrum of clostridium perfringens of different concentrations measured based on an agnps@pdms microporous filter membrane SERS detection platform in this example. Wherein 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 is a plot of the logarithmic value of clostridium perfringens concentration versus the Raman intensity at 1285 cm-1 in its SERS spectrum.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that the following examples are intended to illustrate the present invention and are not to be construed as limiting the scope of the invention, and that numerous insubstantial modifications and adaptations can be made by those skilled in the art in light of the foregoing disclosure.
Example 1
Fig. 1 is a schematic flow chart of an embodiment of a method for preparing an agnps@pdms microporous filter membrane SERS detection platform, comprising the steps of:
Step 2. A round microporous filter membrane (polypropylene, pore size of 0.22 μm) with diameter of 3.5 and mm is soaked in 5 mL AgNPs sol to lift 120 s, and AgNPs nano particles can be uniformly and irreversibly adsorbed on the microporous filter membrane due to Van der Waals interaction, so that the AgNPs nano particles are self-assembled on the microporous filter membrane. And (5) rapidly drying the soaked and pulled microporous filter membrane by using nitrogen to obtain the AgNPs-microporous filter membrane. As shown in part (B) of fig. 1.
Step 3, mixing a main agent and an auxiliary agent (namely polydimethylsiloxane precursor polymer and a curing agent) in a PDMS reagent in a test tube according to a volume ratio of 10:1, fully and uniformly stirring the mixture by using a glass rod, and removing small bubbles generated in the stirring process in vacuum of 1 h to obtain a colloid; slowly pouring the colloid into a PDMS (polydimethylsiloxane) reverse mould, putting the PDMS reverse mould into an oven, drying at 70 ℃ for 48 and h until the PDMS colloid is completely solidified and molded, and demolding and molding to obtain the PDMS plate (the diameter of an upper cylinder is 3.5 mm, and the diameter of a lower cylinder is 3 mm) with a plurality of identical cylindrical grooves, namely the PDMS multi-hole plate. As shown in part (C) of fig. 1.
And 4, firstly, putting a round copper mesh with the diameter of 3.5 mm into an upper layer cylinder of the PDMS porous plate as a supporting material, and then putting a layer of AgNPs-microporous filter membrane, thus obtaining the AgNPs@PDMS porous microporous filter membrane SERS detection platform.
And (3) respectively characterizing the AgNPs sol and the AgNPs-microporous filter membrane obtained in the step (1) and the step (2), and obtaining characterization results shown in the figure 2 and the figure 3. The results in fig. 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 and nm, and the average size is about 69 nm. The results of FIG. 3 show that AgNPs are successfully self-assembled on the microporous filter membrane to form a layer of compact AgNPs two-dimensional point array, and AgNPs are uniformly distributed on the microporous filter membrane to form a rich 'hot spot' structure, thereby being beneficial to enhancing SERS activity.
Example 2
Clostridium perfringens, bacillus subtilis and staphylococcus aureus stored in-80 ℃ refrigerator were activated three times on tryptone-sulfite-cycloserine agar and nutrient agar medium, respectively. The suspension was resuspended after 3 washes with sterile deionized water. And respectively dripping clostridium perfringens, bacillus subtilis and staphylococcus aureus on a glass slide and an AgNPs@PDMS microporous filter membrane SERS detection platform, and immediately carrying out SERS scanning. The excitation wavelength was 632.8 nm and the scan time was 20 s. Using a 100 x objective lens, the scan range was 400 cm -1 ～1800 cm -1 . The spectra were smoothed and baseline corrected using the automated functionality of the instrument with LabSpec 6.0 software. The SERS enhancement effect of the agnps@pdms microporous filter membrane SERS detection platform on 3 bacteria was compared and the results are shown in fig. 4. When the SERS platform is not used for detection, the Raman spectrum intensity of clostridium perfringens, bacillus subtilis and staphylococcus aureus is very low, and 3 bacteria are difficult to distinguish through Raman characteristic peaks; after detection by using the SERS platform, the Raman signals of 3 bacteria are obviously enhanced, and the obvious characteristic peak difference in the SERS spectrum shows that the AgNPs@PDMS microporous filter membrane SERS detection platform has good Raman spectrum signal enhancement capability.
Example 3
Cultivation and purification of 3 bacteria were performed according to the method in example 2 to prepare SERS detection samples. And respectively dripping 10 mu L clostridium perfringens, bacillus subtilis and staphylococcus aureus on an AgNPs@PDMS microporous filter membrane SERS detection platform, immediately carrying out SERS scanning, and randomly measuring 10 Raman spectra of each sample. The raman detection parameters and the data processing method are the same as in example 2. The average spectra were calculated for differential comparison and 10 spectra for each sample were compared reproducibly, the results being shown in figures 5 and 6. In fig. 5, it can be observed that the raman vibrational peaks in SERS spectra of 3 bacteria have significant differences in peak-out positions and peak-out intensities, and different raman vibrational peaks are assigned to different components and chemical bond vibrational forms of the bacteria. Therefore, the SERS technology based on the AgNPs@PDMS microporous filter membrane SERS detection platform as the substrate material has the capability of distinguishing bacteria of different genera, and can realize rapid identification of different bacteria. Fig. 6 shows the repeatability results of SERS detection on 3 bacteria based on the agnps@pdms microporous filter membrane SERS detection platform in the example, and the results show that 10 SERS spectra of each bacteria taken randomly have good consistency, and the SERS enhancement method based on the agnps@pdms microporous filter membrane SERS detection platform as a substrate material has high stability and better repeatability, and provides powerful technical support for rapid detection of food-borne pathogenic bacteria based on SERS technology.
Example 4
The SERS detection platform is used for quantitatively detecting clostridium perfringens, and the specific steps are as follows: clostridium perfringens stored in a-80 ℃ freezer was activated three times on tryptone-sulfite-cycloserine agar and nutrient agar medium. Colonies on the plates were picked and cultured in liquid thioglycolate liquid medium for 24 h, and bacterial counts were performed using the dilution spread plate method. Bacteria were washed with ultrapure water three times for resuspension, and the OD 600 value of the bacteria-increasing liquid was measured by using an enzyme-labeling instrument. When the OD 600 = 1.92 of the enriched liquid is obtained after measurement and counting, the concentration order of the bacterial liquid is 10 8 cfu/mL. Sequentially adding 10 mu L of 10-concentration agents on an AgNPs@PDMS microporous filter membrane SERS detection platform 8 cfu/mL、10 7 cfu/mL、10 6 cfu/mL、10 5 cfu/mL、10 4 cfu/mL、10 3 cfu/mL 10 2 cfu/mL and 10 1 cfu/mL clostridium perfringens suspension was subjected to SERS testing. The raman detection parameters are as shown in example 2. FIGS. 7 and 8 are SERS spectra of clostridium perfringens at different concentrations and logarithmic values of clostridium perfringens concentrations at different concentrations, respectively, and 1285 cm in their SERS spectra -1 Raman intensity at the point corresponds to a linear plot. As can be seen from the SERS spectra of clostridium perfringens at different concentrations under the enhancement of an AgNPs@PDMS microporous filter membrane SERS detection platform, clostridium perfringens at 1285 cm -1 With the strongest SERS signal, the raman peak at 1285 cm-1 was used as the characteristic peak for estimating bacterial cell concentration, and the clostridium perfringens strongest characteristic peak 1285 cm -1 Establishing quantitative equation between the Raman peak intensity and the logarithmic value of different concentrations. FIG. 7 shows Clostridium perfringens at 1285 cm under identical measurement conditions -1 The raman peak intensity at this point decreases with decreasing bacterial concentration. At below 10 2 At a concentration of cfu/mL, clostridium perfringens at 1285 cm could not be clearly identified -1 Main characteristic peaks at the location. Therefore, the detection limit of the method on clostridium perfringens was estimated to be 10 2 cfu/mL. In FIG. 8, the logarithmic values (lg C) of the different clostridium perfringens concentrations are taken as the abscissa, and the strongest characteristic peak 1285 cm is taken as the maximum characteristic peak -1 The peak intensity at the position is taken as an ordinate, and a quantitative equation y= 555.82x is established 2 -2614.91x+4053.70, correlation coefficient R 2 =0.961. Therefore, the SERS technology based on the AgNPs@PDMS microporous filter membrane SERS detection platform has good quantitative analysis capability. In addition, the minimum detection limit of clostridium perfringens can reach 10 due to the bacterial enrichment function of the microporous filter membrane 2 cfu/mL, SERS enhancement performance is good.
The foregoing description is only of the preferred embodiments of the present invention and is not intended to limit the scope of the invention. It is intended that all technical solutions according to the present invention and their inventive concepts be replaced or modified within the scope of the present invention as disclosed by those skilled in the art.
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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| CN109781697A (en) * | 2018-12-27 | 2019-05-21 | 西安交通大学 | A kind of application of flexibility SERS substrate and preparation method thereof and the detection of hydrogen peroxide SERS spectra |
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| WO2021083169A1 (en) * | 2019-10-30 | 2021-05-06 | 江南大学 | Method for preparing polyurethane-based nano-silver sers substrate |
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| CN109781697A (en) * | 2018-12-27 | 2019-05-21 | 西安交通大学 | A kind of application of flexibility SERS substrate and preparation method thereof and the detection of hydrogen peroxide SERS spectra |
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| CN112798571A (en) * | 2020-12-29 | 2021-05-14 | 中国检验检疫科学研究院 | Preparation method of SERS substrate, SERS substrate and application of SERS substrate |
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