CN116148239A - Multiple SERS signal enhanced nano sandwich bacteria detection system and preparation method thereof - Google Patents

Multiple SERS signal enhanced nano sandwich bacteria detection system and preparation method thereof Download PDF

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CN116148239A
CN116148239A CN202310149926.6A CN202310149926A CN116148239A CN 116148239 A CN116148239 A CN 116148239A CN 202310149926 A CN202310149926 A CN 202310149926A CN 116148239 A CN116148239 A CN 116148239A
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solution
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李永强
王春妮
徐国鹏
任志远
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Shandong University
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Abstract

The invention discloses a nano sandwich bacteria detection system with multiple SERS signal enhancement and a preparation method thereof, belonging to the field of functional material preparation and Raman detection. The nano sandwich bacteria detection system comprises labeled nano particles D-Au@Ag-C with high bacterial adhesiveness, and has the structure that Au@Ag core-shell nano particles grow on the surface of dendritic mesoporous silica DMSN in situ, and meanwhile, concanavalin Con A and SERS labels are modified, and a capturing substrate Fe is adopted 3 O 4 The structure of the @ Au-Ab is as follows: fe (Fe) 3 O 4 Nanoparticle surfaceThe surface is provided with a gold coating, and the bacterial targeting antibody is modified. The nano sandwich bacteria detection system can identify staphylococcus aureus in aqueous solution or blood at a high degree, has extremely high detection accuracy and extremely low detection lower limit, and has great prospect in the field detection of clinical bacterial infection related diseases.

Description

Multiple SERS signal enhanced nano sandwich bacteria detection system and preparation method thereof
Technical Field
The invention belongs to the field of functional material preparation and Raman detection, and particularly relates to a nano sandwich bacteria detection system with multiple SERS signal enhancement and a preparation method thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Bacterial infections are a major public health threat worldwide, and it is important to develop a rapid, sensitive and effective diagnostic method. Traditional blood bacterial detection methods involve three main techniques including standard plate colony counts, polymerase Chain Reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). However, standard plate colony counting methods typically require the separation, incubation and counting of complex and time-consuming biochemical processes (typically days). The PCR method requires expensive specialized equipment and complicated processing procedures (cell lysis, nucleic acid extraction, etc.). The ELASA method is tedious, labor-intensive, time-consuming, has a plurality of external interference factors, and has poor repeatability. To overcome these drawbacks, there is an urgent need to develop a technique for detecting blood bacteria in bacterial infectious diseases with shorter detection time and simple operation.
With the continuous and intensive research, more and more means and techniques are applied to detection of blood bacteria. Based on the need for rapid sensing, surface Enhanced Raman Scattering (SERS), which benefits from Localized Surface Plasmon Resonance (LSPR) of noble metals, has become a well-established means of non-destructive detection of bacteria. SERS is a powerful analytical technique that makes its portable detection device simple and fast to operate. The primary advantage of detecting biomarkers is their inherent ability to provide specific high signal "fingerprint" spectra that can selectively detect pathogens in complex environments.
In recent years, detection of various pathogenic bacteria has been successfully achieved by constructing excellent SERS sensors. However, the preparation of high-sensitivity SERS sensors remains a serious challenge, and the detection sensitivity of the SERS sensors still does not achieve the desired effect.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a nano sandwich bacteria detection system with multiple SERS signal enhancement and a preparation method thereof.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
in a first aspect of the invention, there is provided a multiple SERS signal enhanced nano "sandwich" bacterial detection system comprising labeled nanoparticles D-au@ag-C of high bacterial adhesion, a capture substrate Fe 3 O 4 @Au-Ab；
The structure of the D-Au@Ag-C is as follows: the method comprises the steps of growing Au@Ag core-shell nanoparticles on the surface of dendritic mesoporous silica DMSN in situ, and modifying concanavalin Con A and SERS labels;
the Fe is 3 O 4 The @ Au-Ab is: fe (Fe) 3 O 4 The surface of the nano-particle is covered with a gold coating, and meanwhile, a bacterial targeting antibody is modified.
Preferably, the SERS tag is 4-mercaptobenzoic acid 4-MBA.
Preferably, the diameter of the dendritic mesoporous silica DMSN is 160-180nm.
Preferably, the bacterium is staphylococcus aureus.
The nano sandwich bacteria detection system of the invention can form D-Au@Ag-C/bacteria/Fe when detecting bacteria 3 O 4 The sandwich structure of the@Au-Ab has a multiple enhancement effect. Especially for the detection of staphylococcus aureus, the lower limit of detection is extremely low, the selectivity is good, and the detection is sensitive.
In a second aspect of the present invention, a method for preparing the above-described multiple SERS signal enhanced nano "sandwich" bacteria detection system is provided, comprising the steps of:
1) Preparation of D-Au@Ag-C:
modifying amino group on DMSN surface to make its surface positively charged so as to obtain DMSN-NH 2 The method comprises the steps of carrying out a first treatment on the surface of the With DMSN-NH 2 Gold is grown on the surface of the DMSN by a seed solution growth method to obtain DMSN-Au; SERS label modification is carried out on the DMSN-Au to obtain the DMSN-Au 4-MBA The method comprises the steps of carrying out a first treatment on the surface of the In DMSN-Au 4-MBA Silver Ag grows on the surface to obtain DMSN-Au@Ag; SERS label modification is carried out on the DMSN-Au@Ag to obtain the DMSN-Au@Ag 4-MBA The method comprises the steps of carrying out a first treatment on the surface of the For DMSN-Au@Ag 4-MBA Carrying out concanavalin Con A modification to obtain D-Au@Ag-C;
2)Fe 3 O 4 preparation of @ Au-Ab:
preparing a gold seed solution; fe is added to 3 O 4 The nano particles are subjected to surface modification to make the nano particles have positive potential, then are mixed with gold seed solution, and are grown on Fe by a seed solution growth method 3 O 4 Gold grows on the surface of the nano particle to obtain Fe 3 O 4 @Au；Fe 3 O 4 Modifying bacterial targeting antibody Ab on surface of@Au to obtain Fe 3 O 4 @Au-Ab。
Preferably, the preparation method of the dendritic mesoporous silica DMSN comprises the following steps: fully dissolving triethanolamine into ultrapure water, heating and stirring the obtained solution; adding sodium salicylate and cetyltrimethylammonium bromide into the solution, and stirring; dropwise adding tetraethoxysilane into the solution under stirring, and continuing stirring after the dropwise adding is finished; after the solution is cooled to room temperature, centrifugally collecting a product; washing; removing cetyl trimethyl ammonium bromide; and drying to obtain the dendritic mesoporous silica DMSN.
Preferably, in step 1), the seed solution growth method specifically includes: taking DMSN-NH 2 Dispersing into deionized water, adding tetrachloroauric acid solution, and mixing with ultrasound; under intense stirring, rapidly injecting sodium borohydride solution into the mixed solution, stirring for reaction, centrifuging and washing with ultrapure water for multiple times to obtain DMSN-m-Au seed nano particles; resuspending the DMSN-m-Au seed solution into hydroxylamine hydrochloride solution, vigorously stirring at room temperature, and rapidly adding gold chloride solution; stirring and separatingCollecting heart to obtain DMSN-Au.
Preferably, the method is carried out on DMSN-Au 4-MBA The silver Ag grown on the surface is specifically as follows: DMSN-Au 4-MBA Dispersing into polyvinylpyrrolidone water solution, and stirring; adding silver nitrate and ascorbic acid water solution into the mixed solution for incubation, and reducing silver ions into metallic silver; centrifuging, washing to remove redundant reactants, and obtaining the DMSN-Au@Ag.
Preferably, the pair DMSN-Au@Ag 4-MBA The concanavalin Con A modification is specifically: activation of DMSN-Au@Ag with a mixture of tetramethylammonium hydroxide 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysulfosuccinimide sodium salt 4-MBA Carboxyl on 4-MBA; con A solution and activated DMSN-Au@Ag 4-MBA Mixing, and stirring at room temperature to obtain the D-Au@Ag-C.
Preferably, in the step 2), the preparation method of the ferroferric oxide nano-particles comprises the following steps: ferric trichloride hexahydrate and trisodium citrate TSC are dissolved in ethylene glycol, and ethylene glycol containing sodium acetate is added under magnetic stirring; stirring vigorously at room temperature; subsequently, the mixture was transferred to an autoclave, which was placed in a 200 ℃ oven and heated to obtain a black precipitate; washing, collecting by a magnet, and drying to obtain the ferroferric oxide nano particles.
Preferably, the Fe 3 O 4 The surface modification of the nano particles is specifically as follows: fe is added to 3 O 4 Dispersing the nano particles into a polyethyleneimine PEI aqueous solution, fully mixing, and forming a self-assembly mode on Fe 3 O 4 The surface of the nano particle is modified with a layer of PEI, and Fe 3 O 4 The nanoparticle is modified to a positive potential.
Preferably, in step 2), the Fe 3 O 4 Modifying bacterial targeting antibody Ab on surface of@Au to obtain Fe 3 O 4 The @ Au-Ab is specifically: polyethylene glycol PEG added to Fe 3 O 4 Ultrasonic mixing is carried out on the @ Au, and redundant PEG is removed by centrifugation, thus obtaining Fe 3 O 4 @ Au-PEG; activation of Fe 3 O 4 Carboxyl groups on the surface of the @ Au-PEG; after sonication at room temperature, the activated nanoparticles were resuspendedStirring in phosphate buffered saline containing bacterial targeting antibody Ab to obtain Fe 3 O 4 @Au-Ab。
In a third aspect of the present invention, a bacterial detection method is provided, which adopts the above-mentioned nano "sandwich" bacterial detection system with multiple SERS signal enhancement, and specifically comprises the following steps:
adding capture substrate Fe into sample to be detected 3 O 4 Incubating at @ Au-Ab, washing, adding high bacterial adhesion marked nanoparticle D-Au @ Ag-C, mixing, washing by magnetic separation method, transferring onto silicon wafer, and measuring at 1583cm -1 Is a raman signal of (c).
Preferably, the bacterium is staphylococcus aureus.
The beneficial effects of the invention are as follows:
the inventors of the present invention inspired the natural phenomenon that fruit-bearing branches are very close to each other, introducing dendritic mesoporous silica DMSN nanoparticles with pollen morphology into SERS-tagged nanoparticles. Due to multivalent interactions, nanoscale topologies can enhance interactions between nanoparticles and bacteria, thereby enhancing bacterial capture. And carrying the Au@Ag core-shell nanomaterial with excellent SERS performance on the surface of the DMSN in an in-situ growth mode, and simultaneously modifying concanavalin Con A and SERS label 4-MBA to obtain the marked nanoparticle D-Au@Ag-C with high bacterial adhesion. In addition, in order to enhance the intensity of the detection bacterial signal, in the conventional magnetic Fe 3 O 4 Innovations are made on the basis of nano materials. Magnetic substrate Fe with SERS enhancement function is synthesized by noble metal shell coating method 3 O 4 At the same time, bacteria targeting antibody modification is carried out on the@Au-Ab MNPs, which leads to the magnetic substrate Fe 3 O 4 the@Au-Ab has magnetic separation performance, SERS enhancement performance and stability.
The multiple SERS signal enhanced nano sandwich bacteria detection system of the invention finally forms D-Au@Ag-C/bacteria/Fe when bacteria detection is carried out 3 O 4 Sandwich structure of @ Au-Ab has multiple enhancement effects:
(1) The Au@Ag core-shell structure in the D-Au@Ag-C material can provide stronger coupling SERS signals compared with pure Au nanoparticles; the DMSN is introduced to play a role in space enrichment on Au@Ag, and a high coupling field can be generated between two adjacent Au@Ag nano particles.
(2) Magnetic Fe 3 O 4 The Au shell of the outer layer of the@Au MNPs has high SERS activity, and can generate signal enhancement on nearby Raman molecules; internal Fe 3 O 4 The core has good magnetic reaction, and the separation and enrichment of the target substance and other substances in the mixture are realized.
The nano sandwich bacteria detection system with the enhanced multiple SERS signals successfully realizes the target identification of staphylococcus aureus in aqueous solution or blood, has extremely high accuracy, and has great prospect in the field detection of clinical bacterial infection related diseases.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a schematic diagram of the synthesis and schematic mechanism of a multiple SERS signal enhanced nano "sandwich" bacterial detection system of the present invention; a) The synthesis schematic diagram is D-Au@Ag; b) Is Fe 3 O 4 Schematic diagram of Au synthesis; c) The principle mechanism drawing for the multi-SERS signal enhanced nanometer 'sandwich' bacteria detection system in bacteria detection shows typical 3 types of enhanced 'hot spots' generated in the sandwich structure, wherein 1 is weaker 'hot spot' generated on the surface of a plasma nanoparticle (Au@Ag), and 2 is strong 'hot spot' formed by a nanometer gap generated by mutually approaching between Au@Ag nanoparticles under the constraint of DMSN; 3 is Fe 3 O 4 Coupling "hot spot" between Au (part of the enrichment module) and au@ag (part of the signal module);
FIG. 2 shows DMSN-Au@Ag, fe prepared by the embodiment of the invention 3 O 4 TEM image of @ Au; wherein a) is DMSN NaRepresentative TEM image of nanoparticles, b) representative TEM image of labeled nanoparticles with high bacterial adhesion DMSN-Au@Ag, c) capture substrate Fe 3 O 4 Representative TEM pictures of @ Au;
FIG. 3 is a graph showing the UV-visible absorption spectrum of different nanoparticles prepared according to an embodiment of the present invention; wherein a) is the ultraviolet visible absorption spectrum of DMSN, DMSN-m-Au, DMSN-Au and DMSN-Au@Ag nano particles, b) is Fe 3 O 4 、Fe 3 O 4 -PEI、Fe 3 O 4 -Au and Fe 3 O 4 Ultraviolet visible absorption spectrum of the @ Au nanoparticle;
FIG. 4 is a zeta potential plot of different nanoparticles prepared according to an example of the present invention; wherein a) is the zeta potential of DMSN, DMSN-m-Au, DMSN-Au@Ag and D-Au@Ag-C nano particles, and b) is Fe 3 O 4 、Fe 3 O 4 -PEI、Fe 3 O 4 -Au、Fe 3 O 4 @ Au and Fe 3 O 4 Zeta potential of the @ Au-Ab nanoparticle;
FIG. 5 shows the D-Au@Ag-C and Fe prepared according to an embodiment of the present invention 3 O 4 Hemolysis property of the @ Au-Ab nanoparticle;
FIG. 6 is an SEM image of capture of Staphylococcus aureus by SERS tags and capture substrates, wherein 2 is the SERS tag, with a size of about 170nm;3 is a capture substrate, about 80nm in size; 1 is staphylococcus aureus, about 1 micron in size;
FIG. 7 shows the Raman spectra of DMSN-Au, DMSN-Au@Ag, and D-Au@Ag-C prepared in the example of the invention;
FIG. 8 shows the stretched noodle spectra of different nanoparticles prepared according to the example of the present invention at 1583cm -1 Intensity of raman peak; wherein, a) is Raman spectrum of Au@Ag and DMSN-Au@Ag, b) is Raman spectrum of Au@Ag and DMSN-Au@Ag corresponding to 1583cm -1 At the intensity of the Raman peak, C) is D-Au@Ag-C/bacterium and D-Au@Ag-C/bacterium/Fe 3 O 4 Raman spectra of the @ Au-Ab "Sandwich" bacteria detection System, D) D-Au@Ag-C/bacteria and D-Au@Ag-C/bacteria/Fe 3 O 4 The @ Au-Ab "sandwich" bacteria detection system corresponds to 1583cm -1 The raman peak at the site is strong;
FIG. 9 is a diagram of the detection of different bacteria by the "sandwich" bacteria detection system of the present invention; wherein a) is specific detection of staphylococcus aureus, b) is the repeatability of detection of staphylococcus aureus;
FIG. 10 is a graph showing the results of a "sandwich" bacteria detection system of the present invention for different concentrations of Staphylococcus aureus in an aqueous solution; wherein a) is the detection performance of staphylococcus aureus in aqueous solution, b) is 1583cm in graph a) -1 A raman peak intensity fitting map;
FIG. 11 is a graph showing the results of a "sandwich" bacterial detection system of the present invention for different concentrations of Staphylococcus aureus in a 1% blood solution; wherein a) is the detection performance for staphylococcus aureus in 1% blood solution, b) is 1583cm in graph a) -1 A raman peak intensity fitting map;
FIG. 12 is a diagram of a "sandwich" bacterial detection system of the present invention for detecting blood bacterial specimens from infected mice; wherein a) is the detection Raman spectrum of a bacterial sample in the blood of an infected mouse, b) is 1583cm according to FIG. a) -1 Comparison of the calculated bacterial numbers of raman peak intensities with the results obtained from plate culture, the inset in b) is the recovery of labeled nanoparticles and capture substrate with high bacterial adhesion.
Detailed Description
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
Example 1
A method for preparing a multiple SERS signal enhanced nano "sandwich" bacterial detection system, comprising the steps of:
1. materials: tetraethyl orthosilicate (TEOS), hydrochloric acid (HCl), acetone, cetyl trimethylammonium bromide (CTAB), sodium salicylate (NaSal), triethanolamine (TEA), sodium borohydride (NaBH 4), phosphate Buffered Saline (PBS), tetramethylammonium hydroxide 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide sodium salt (NHS), purchased from national pharmaceutical chemicals company (Shanghai, china). HexahydrateFerric chloride (FeCl) 3 ·6H 2 O), anhydrous sodium acetate (NaAc), sodium citrate, ethylene Glycol (EG), polyvinylpyrrolidone K30 (PVP-K30), silver nitrate (AgNO) 3 ) Ammonia (28%), 4-mercaptobenzoic acid (4-MBA), (3-aminopropyl) triethoxysilane (APTES), hydroxylammonium chloride (NH) 2 OH HCl), ascorbic Acid (AA), gold (III) chloride trihydrate (HAuCl) 4 ·3H 2 O), ethanol, bovine Serum Albumin (BSA) and concanavalin (Con a) were purchased from source She Shiji limited (shanghai, china). All other chemicals were obtained from Adamas beta and used without further purification. Deionized (DI) water (Millipore Milli-Q grade, 18.2 M.OMEGA.) was used for all experiments.
2. Experimental protocol
1. Preparation of Dendritic Mesoporous Silica (DMSN) nanoparticles and amino modification:
first, 0.068g of TEA was sufficiently dissolved in 25mL of ultrapure water, and the resulting solution was heated to 80℃and stirred with continued heating for 0.5h. Subsequently, 168mg of sodium salicylate NaSal and 380mg of CTAB were added to the reaction solution. After stirring for 1h, 4mL of TEOS solution was added dropwise to the above solution under gentle stirring (300 rpm) and stirring was continued gently for 2h. After cooling the solution to room temperature, the product was collected by high-speed centrifugation (20000 rpm, 15 min). Wash three times with water and ethanol each to remove excess reactants. Finally, the material was resuspended with a mixed solution of hydrochloric acid and ethanol (HCl: ethanol=1:9), and refluxed at 80 ℃ for 6h. The extraction was repeated 3 times to remove the surfactant (CTAB). The final purified DMSN was dried under vacuum at 50 ℃ overnight.
In order to positively charge the DMSN surface, amino groups are modified on the surface. 100. Mu.L APTES was added to 10mL of DMSN solution (10 mg/mL), heated to 80℃and stirred for 12h to give DMSN-NH 2 And (3) nanoparticles. And (5) after washing for many times with ultrapure water, preserving at 4 ℃ for standby.
2. Preparation of DMSN-Au@Ag nanoparticles:
50mg of the prepared DMSN-NH is taken 2 NPs were dispersed in 5mL deionized water. Then HAuCl is added 4 The solution (500. Mu.L, 20 mM) was sonicated for 0.5h. Fresh preparation under vigorous stirringNaBH of (B) 4 The solution (5 mL, 0.1M) was quickly injected into the mixture. After stirring for 1h, the mixture was centrifuged and washed with ultrapure water several times to give DMSN-NH 2 Au nano particles with the particle size of about 3nm grow on the surface of the NPs to obtain the DMSN-m-Au seed nano particles. 4mL of DMSN-m-Au NPs seed solution (5 mg/mL) was resuspended in hydroxylamine hydrochloride solution (12.5 mL,2.8 mM), vigorously stirred at room temperature, and 150. Mu. LHAuCl was added rapidly 4 Solution (50 mM). After stirring for 0.3h, DMSN-Au NPs were collected by centrifugation and resuspended in ethanol. Subsequently, 50. Mu.L of 4-MBA (10 mM) solution was added. Stirring for two hours, centrifuging and washing for several times to obtain DMSN-Au 4-MBA Nanoparticles and stored at 4 ℃. In addition, in order to promote SERS activity of the nano-particles, DMSN-Au is prepared 4-MBA And Ag shells grow on the surfaces of the nano particles. Will 6mg DMSN-Au 4-MBA NPs were dispersed in 60mL PVP-K30 aqueous solution (2 mg/mL) and stirred well for 30min. Then, 600. Mu.L of silver nitrate (10 mM) and 600. Mu.L of ascorbic acid (10 mM) in water were added to the mixed solution, and the mixture was incubated for 1 hour to reduce silver ions to metallic silver. Centrifuging at 9000rpm for 15min, and washing with ultrapure water for multiple times to remove excessive reactants, thereby obtaining purified DMSN-Au@Ag nanoparticles.
3. Preparation of highly bacterial-adherent labeled nanoparticles (D-au@ag-C nanoparticles):
6mL of DMSN-Au@Ag ethanol solution was mixed well with 60. Mu.L of 4-MBA (10 mM) solution and stirred at room temperature for 2h. The 4-MBA is modified on the nanoparticle by the formation of Ag-S bond with the thiol group and Ag. Then, the excess 4-MBA is removed by centrifugation, and the obtained sample is resuspended in 2mL PBS solution to obtain monodisperse DMSN-Au@Ag 4-MBA Nanoparticle solutions. Activation of DMSN-Au@Ag with EDC/NHS Mixed solution 4-MBA Carboxyl on 4-MBA in nanoparticle. 200. Mu.L Con A solution was mixed with the activated nanoparticles and stirred at room temperature for 12h. Subsequently, 200 μl of 10wt% bsa solution was added and stirring was continued for 1h to block excess amino groups to reduce non-specific binding of the nanoparticles. The prepared D-Au@Ag-C nanoparticles were resuspended in PBS buffer (10 mM) for further use.
4. Preparation of 3nm Au nanoparticles:
trisodium citrate (1.5 mL,1 wt%) and aqueous tetrachloroauric acid (148.5 mL,0.01 wt%) were thoroughly mixed and stirred vigorously at room temperature for 15min. Subsequently, a freshly prepared ice water solution of sodium borohydride (4.5 ml,0.1 m) was rapidly poured into the mixed solution. Stirring is continued for 4 hours to obtain colloidal gold solution with the particle size of 3 nm.
5. Ferroferric oxide (Fe) 3 O 4 ) Preparation of nanoparticles:
1.62g of ferric trichloride hexahydrate and 0.5g of trisodium citrate TSC were dissolved in 40mL of ethylene glycol, and ethylene glycol (40 mL) containing 6.64g of sodium acetate (NaAc) was added with magnetic stirring. Stirring vigorously at room temperature for 0.5h. Subsequently, the mixture was transferred to a polytetrafluoroethylene-lined autoclave having a capacity of 100 mL. The autoclave was placed in an oven at 200℃and heated for 10 hours to obtain a black precipitate. Then washed 4 times with ethanol and water each with manual shaking, collected by magnet and dried under vacuum at 60 ℃ for 6h for further use.
6、Fe 3 O 4 Preparation of @ Au core-shell nanoparticles:
10mg Fe 3 O 4 The nanoparticles were dispersed in 20mL of PEI aqueous solution (5 mg/mL) and thoroughly mixed and sonicated for 2h. By self-assembly, in Fe 3 O 4 A layer of PEI is modified on the surface of the nano particles, and the material is modified to positive potential to obtain Fe 3 O 4 -PEI nanoparticles. Fe to be obtained 3 O 4 Mixing PEI nanoparticles with 3nm Au seeds, and performing ultrasonic treatment for 30min to prepare Fe 3 O 4 -Au seed nanoparticles. The product was magnetically collected and washed 4 times with deionized water under manual shaking to remove excess Au seeds. Fe obtained by preparation 3 O 4 Re-dispersing of Au nanoparticles to HAuCl 4 To the solution (50 mL,0.4 mM), 0.5mL NH was added rapidly under ultrasound 2 Aqueous OH HCl and 0.15g PVP-K30. Magnetic collection is carried out after 15min of reaction to obtain the final product Fe 3 O 4 At Au, washed multiple times and resuspended in 10mL deionized water for use.
7. Capturing substrate (Fe) 3 O 4 @ Au-Ab nanoparticle) preparation:
1mg polyethylene glycol PEG added to Fe 3 O 4 @ Au (5 mL).The solution was sonicated for 3 hours and then centrifuged at 9000rpm for 10 minutes to remove excess PEG. Subsequently, the modified nanoparticles were resuspended in PBS buffer (5 mL; pH 7.4) containing EDC (200. Mu.g/mL) and NHS (200. Mu.g/mL) to activate the carboxyl groups on the surface. After 30min of sonication at room temperature, the activated nanoparticles were resuspended in PBS (1 mL) containing 20 μg of bacterial targeting antibody (Ab) and stirring was continued for 2h. Finally, 500. Mu.L of 10wt% BSA in water was added to block the excess binding sites. Fe obtained 3 O 4 The @ Au-Ab nanoparticle was stored at 4deg.C for further use.
8. Highly bacterial adherent labeled nanoparticles and morphology of capture substrate:
taking 100 mu L of DMSN, DMSN-Au@Ag nano particles and Fe 3 O 4 Diluting the nano-particles @ Au with deionized water to 1mL, sucking 10 mu L of diluted nano-particles to drop on a copper mesh, drying overnight in an electronic moisture-proof box, shooting with a Transmission Electron Microscope (TEM), and observing the morphology of the nano-particles.
9. Highly bacterial-adherent labeled nanoparticles and capture ultraviolet visible absorption spectra of the substrate:
and respectively diluting the DMSN, the DMSN-Au and the DMSN-Au@Ag nano particles to the same concentration by deionized water. Placing the solutions in a quartz cuvette, detecting ultraviolet-visible absorption spectrums of the solutions at the wavelength range of 380-700 nm by using an ultraviolet-visible spectrophotometer, and performing contrast treatment to verify the synthesis of SERS labels D-Au@Ag-C nanoparticles.
Fe is added to 3 O 4 、Fe 3 O 4 -PEI、Fe 3 O 4 -Au、Fe 3 O 4 The @ Au nanoparticles were each diluted to the same concentration with deionized water. Placing the solutions in a quartz cuvette, detecting ultraviolet-visible absorption spectrum of each solution at a wavelength range of 300-700 nm by using an ultraviolet-visible spectrophotometer, and performing contrast treatment to verify the capture substrate Fe 3 O 4 Synthesis of @ Au-Ab nanoparticles.
10. Characterization of zeta potential of labeled nanoparticles and capture substrates for high bacterial adhesion:
taking 100 mu L of DMSN and DMSN-NH 2 The nanoparticles of DMSN-m-Au, DMSN-Au@Ag and D-Au@Ag-C were diluted to 2mL with deionized water, the zeta potential of the nanoparticles in the aqueous environment was detected with a nanoparticle sizer, repeated three times, and the data were recorded.
Taking 100 mu L of Fe 3 O 4 、Fe 3 O 4 -PEI、Fe 3 O 4 -Au、Fe 3 O 4 @ Au and Fe 3 O 4 The @ Au-Ab nanoparticles were diluted to 2mL with deionized water, the zeta potential of the nanoparticles in an aqueous environment was measured with a nanoparticle sizer, repeated three times, and the data recorded.
11. High bacterial adhesion labeled nanoparticles and hemolysis experiments of capture substrates:
1.2 ml of blood was drawn from the eyes of healthy seven-week old mice and thoroughly mixed with ethylenediamine tetraacetic acid EDTA solution. Centrifugation was carried out at 2000rpm for 10 minutes, the supernatant was removed, and the pellet was washed with PBS, to finally obtain pure red blood cells. The resulting 20% red blood cell suspensions were then diluted with PBS buffer to a series of different concentrations (containing 25, 50, 100, 200. Mu.g mL -1 DMSN and Fe of (A) 3 O 4 Nanoparticles) D-Au@Ag-C/Fe 3 O 4 The @ Au-Ab material was mixed. Blood samples in PBS and deionized water served as negative and positive controls, respectively. All samples incubated at 37℃for 2 hours were centrifuged and finally, the supernatant was taken to measure absorbance.
12. Bacterial culture:
staphylococcus aureus, escherichia coli, salmonella plague, bacillus subtilis, MRSA and pseudomonas aeruginosa are adopted in the experiment. These bacteria were grown overnight in LB medium at 37℃and obtained by centrifugation during the growth phase. The concentration of bacteria in the PBS solution can be obtained by measuring the optical density at 600 nm. OD value of Staphylococcus aureus and Escherichia coli is 1, and corresponding concentrations are 4×10 respectively 9 And 2X 10 9 。
13. High bacterial adhesion of the labeled nanoparticle and capture ability of the capture substrate to bacteria:
to detect the bacterial preference of SERS tagsThe adhesion properties of the surface topography to bacteria were studied using scanning electron microscopy. D-Au@Ag-C and Fe 3 O 4 Mixing at Au-Ab NPs and incubation of bacteria in a shaker for 30min, fixation with 2.5% glutaraldehyde for 2h, then gradient dehydration with a series of aqueous ethanol solutions (50%, 70%, 90% and 100%) for 10min, finally dropping the sample on a silicon wafer, drying and then spraying platinum, imaging with SEM.
14. Investigation of SERS properties of labeled nanoparticles with high bacterial adhesion:
the DMSN-Au, DMSN-Au@Ag and D-Au@Ag-C nanoparticles were diluted to the same concentration with deionized water. And (3) absorbing 10 mu L of diluted nano particles to drop on a silicon wafer, measuring the Raman spectrum of the nano particles by using a microscopic Raman instrument, and repeating the experiment three times.
15. Research on SERS coupling enhancement performance by DMSN and Fe 3 O 4 Research on SERS coupling properties by Au-Ab:
diluting DMSN-Au@Ag and Au@Ag nano particles to the same concentration, taking 10 mu L of the mixture to drop on a silicon wafer, and measuring 1000-1700 cm -1 Raman spectrum in the range.
D-Au@Ag-C and 10 6 CFU/mL staphylococcus aureus is incubated for 30min and divided into two parts, one part is kept still, and 100 mu L of Fe is added into the other part 3 O 4 After incubation for 30min, the nano Au-Ab particles are resuspended in 100 mu LPBS by centrifugation and magnetic enrichment, and tested for 1000-1700 cm -1 Raman spectrum in the range.
16. Exploration of high bacterial adhesion labeled nanoparticles and capture substrate sandwich systems for specific detection of staphylococcus aureus:
preparation of 1mL of different concentrations (20, 50, 10) with deionized water, respectively 2 、10 3 And 10 4 CFU mL -1 ) Is a bacterial sample of (a). Taking 50 mu L of Fe 3 O 4 The @ Au-Ab NPs nanosystems, and were added to each sample. Incubating for 30 minutes. After multiple washes with PBS, the mixture was incubated with gentle shaking for 30 minutes with the addition of D-Au@Ag-C NPs. Finally, the obtained D-Au@Ag-C/S.aureus/Fe 3 O 4 The @ Au-Ab complex was washed three times by magnetic separation and transferred to a silicon wafer and measured at 1583cm -1 Is a raman signal of (c). The above experiments were repeated three times.
17. Exploration of high bacterial adhesion labeled nanoparticles and capture substrate sandwich systems for specific and reproducible detection of staphylococcus aureus:
for selective analysis, 1mL of the solution was used at a concentration of 10 3 cell/mL staphylococcus aureus and 5 concentrations of 10 5 The cell/mL of the interfering bacteria (E.coli, salmonella typhimurium, bacillus subtilis, MRSA and Pseudomonas aeruginosa) were incubated with the detection nanosystems for 30min, respectively, with gentle shaking (250 rpm,37 ℃). Finally, the mixture was washed three times and magnetically enriched. SERS signal measurements of six different species of bacteria were performed on the final enriched trace solutions and plotted at 1583cm -1 A raman intensity histogram.
In order to explore the detection repeatability of staphylococcus aureus, 10 batches of nano materials are respectively prepared, and the repeated measurement concentration is 10 5 cell/mL staphylococcus aureus and plotted at 1583cm -1 A raman intensity histogram. The SERS detection experiment was repeated three times for each batch of material described above.
18. Detection of bacteria in aqueous solutions by highly bacteria-adherent labeled nanoparticles and capture-substrate sandwich systems:
1mL of staphylococcus aureus samples (0, 20, 50, 10) at different concentrations were prepared with deionized water 2 、10 3 And 10 4 CFU/ml). Then, 50. Mu.L of Fe was taken 3 O 4 @Au-Ab NPs, and added to each sample. Incubating for 30 minutes. After PBS washing, the mixture was gently shaken by adding D-Au@Ag-C NPs. Finally, the obtained D-Au@Ag-C/S.aureus/Fe 3 O 4 The @ Au-Ab complex was washed three times by magnetic separation and transferred to a silicon wafer and measured at 1583cm -1 Is a raman signal of (c). The above experiments were repeated three times.
19. Detection of bacteria in 1% blood by high bacterial adhesion labeled nanoparticle and capture substrate sandwich system:
diluting fresh mouse blood with deionized water to obtain 1% blood concentration solutionIs used. 1mL of each of the different Staphylococcus aureus concentrations (0, 10) was prepared with 1% blood solution 2 、10 3 、10 4 And 10 5 CFU/ml). Then, 50. Mu.L of Fe was taken 3 O 4 @Au-Ab NPs, and added to each sample. Incubating for 30 minutes. After PBS washing, the mixture was gently shaken by adding D-Au@Ag-C NPs. Finally, the obtained D-Au@Ag-C/S.aureus/Fe 3 O 4 The @ Au-Ab complex was washed three times by magnetic separation and transferred to a silicon wafer and measured at 1583cm -1 Is a raman signal of (c). The above experiments were repeated three times.
20. Detection of bacteria in infected mouse blood samples by high bacterial adhesion labeled nanoparticles and capture substrate sandwich system:
to detect bacteria in the blood of mice, 100 microliters of bacterial PBS suspension (5×10 9 CFU individual staphylococcus aureus) was injected into the blood of Balb/c mice via tail vein to establish a bacterial infection mouse model. 1 hour after infection, 10 microliters of blood was drawn from the mouse orbit and diluted to 1% blood sample with PBS. Subsequently, 50. Mu.L of Fe 3 O 4 Incubation of the @ Au-Ab NPs with the detection blood for 30min at 37℃and addition of 50. Mu.L of D-Au@Ag-C. Finally, magnetic enrichment is performed and multiple washes are performed to remove unbound bacteria and blood cells. The complex is transferred to a silicon wafer to detect the corresponding raman signal, the number of bacteria is calculated according to the intensity in the standard curve, and the result of bacteria electroplating is compared.
In the experiment, female Balb/c mice, 6 weeks old, weighing 20 grams, were from Jinan Pengyue laboratory animal breeding Co. Adaptation was performed in the laboratory for 1 week prior to the experiment. All animal experiments meet the requirements of the Shandong university laboratory animal center.
3. Experimental results
1. Appearance characterization of labeled nanoparticles with high bacterial adhesion and capture substrate sandwich system:
as shown in FIG. 2 a), the diameter of the synthesized DMSN nanoparticle is about 170nm, the size is uniform, the dispersibility is good, and obvious dendritic mesoporous morphology can be observed on the surface, thus proving that the DMSN nanoparticle is successfully synthesized.
The morphology of the DMSN-Au@Ag nanoparticles is shown in fig. 2 b), the Au@Ag nanoparticles growing in situ can be obviously observed on the surfaces of the DMSN nanoparticles, and the successful loading of the Au@Ag core-shell nanoparticles with SERS performance on the surfaces of the DMSN nanoparticles is proved to be obtained. The nanoparticle has stable morphology, good dispersibility, and no large change between the size and the DMSN nanoparticle, and is about 175nm.
As shown in FIG. 2 c), the synthesized Fe 3 O 4 The diameter of the@Au nano-particles is about 80nm, the size is uniform, the dispersibility is good, a relatively obvious Au shell is observed on the surface energy, and the successful synthesis of Fe is proved 3 O 4 The @ Au captures the substrate core shell nanoparticle.
2. Highly bacterial-adherent labeled nanoparticles and capture the uv-visible absorbance spectrum of the substrate sandwich system:
as shown in FIG. 3 a), the DMSN-Au@Ag nanoparticles express characteristic peaks of Au and Ag nanoparticles at 520nm and 450nm, which proves that the Au@Ag nanoparticles are successfully loaded in the DMSN nanoparticles, and the DMSN-Au@Ag nanoparticles are obtained.
As shown in fig. 3 b), fe 3 O 4 The @ Au nanoparticles are compared to Fe 3 O 4 Au nanoparticles with a distinct blue shift of the characteristic peak around 600nm, demonstrated on Fe 3 O 4 The Au shell is synthesized on the surface, and the Fe is successfully prepared 3 O 4 @au core-shell nanoparticles.
3. Characterization of high bacterial adhesion labeled nanoparticles and capture substrate sandwich system potential:
as shown in FIG. 4 a), successful preparation of D-Au@Ag-C nanoparticles was demonstrated by stepwise modification and coating, with the zeta potential of the D-Au@Ag-C nanoparticles constantly changing.
As shown in FIG. 4 b), fe is modified and coated stepwise 3 O 4 The zeta potential of the @ Au-Ab nanoparticle is continuously changed, which proves that Fe 3 O 4 Successful preparation of Au-Ab nanoparticles.
4. High bacterial adhesion labeled nanoparticles and capture hemolysis of the substrate sandwich system:
as shown in fig. 5, all but the deionized water positive control samples exhibited good hemolysis performance, demonstrating good blood compatibility of the sandwich strategy.
5. Evaluation of the Capture ability of the Capture substrate for bacteria by labeled nanoparticles with high bacterial adhesion:
as shown in FIG. 6, it is evident from the SEM image that the surface of Staphylococcus aureus adsorbs D-Au@Ag-C nanoparticles and Fe 3 O 4 The @ Au-C nanoparticle proves that the sandwich strategy has good capability of capturing staphylococcus aureus.
6. Evaluation of SERS properties of labeled nanoparticles with high bacterial adhesion:
as shown in FIG. 7, with the synthesis of D-Au@Ag-C nanoparticles, the particles were located at 1073cm -1 And 1583cm -1 The raman peak intensity is gradually enhanced, and the synthesized D-Au@Ag-C nanoparticle has more excellent SERS performance compared with a simple DMSN-Au nanoparticle.
7. Research on SERS coupling enhancement performance by DMSN and Fe 3 O 4 Research on SERS coupling properties by Au-Ab:
as shown in fig. 8 a) and fig. 8 b), the introduction of DMSN effectively improves SERS performance of au@ag nanomaterials.
As shown in FIGS. 8 c) and 8 d), fe can be seen in the final sandwich structure 3 O 4 The @ Au-Ab has a good SERS enhancement effect on the D-Au@Ag-C label nano material enriched on the surface of bacteria, and proves that the invention successfully prepares a nano 'sandwich' bacteria detection system with multiple SERS signal enhancement.
8. Labeled nanoparticle and capture substrate sandwich system with high bacterial adhesion performance evaluation for staphylococcus aureus specificity and reproducibility detection:
as shown in FIG. 9 a), 1583cm of a sample of Staphylococcus aureus -1 The strong peak was clearly distinguishable from other bacteria, demonstrating the good selectivity of the SERS "sandwich" system against staphylococcus aureus.
As shown in fig. 9 b), the detection results of the SERS sandwich detection system prepared by 10 batches of different times on staphylococcus aureus are uniform, and the RSD value is 4.5%, which proves that the SERS sandwich structure has good repeatability.
9. Application evaluation of labeled nanoparticles with high bacterial adhesion and sandwich system composed of capture substrate for detection of staphylococcus aureus in aqueous solution:
as shown in fig. 10 a), the raman peak intensity is a significant trend of enhancement with increasing concentration of staphylococcus aureus. As shown in FIG. 10 b), 1583cm -1 The peak intensity at the position and the bacterial concentration show a good linear relation, the square value of R is 0.982, and the linear equation is y= -3598+3046.4x. The LOD value was calculated by the formula lod=3σ/S and found to be 7CFU. The sandwich detection strategy has great application prospect and extremely low detection lower limit in the application of bacteria detection in aqueous solution.
10. Evaluation of high bacterial adhesion labeled nanoparticles and capture substrate sandwich system for detection of 1% bacteria in blood:
as shown in fig. 11 a), the raman peak intensity is a significant trend of enhancement with increasing concentration of staphylococcus aureus in 1% blood solution. As shown in FIG. 11 b), 1583cm -1 The peak intensity at this point showed a very good linear relationship with bacterial concentration, with a square R value of 0.964 and a detection Limit (LOD) of 97CFU. The sandwich detection strategy has great application prospect in the application of bacteria detection in blood.
11. Detection of bacteria in infected mouse blood samples by high bacterial adhesion labeled nanoparticles and capture substrate sandwich system:
as shown in fig. 12 a), blood collected from bacteria-infected mice was quantified using the multiple SERS signal enhanced nano "sandwich" bacteria detection system proposed by the present invention. The SERS signal of blood bacterial samples of the three groups of mice was substantially uniform. Furthermore, as shown in fig. 12 b), the nano "sandwich" bacterial detection system based on multiple SERS signal enhancement quantitatively analyzed the corresponding bacterial concentrations and then compared with the measurement results of the standard plate bacterial culture method, showing high consistency. Recovery rates of highly bacterial-adherent labeled nanoparticles and capture substrates were also calculated to determine the accuracy of the results obtained from both detection of bacteria together, ranging from 91 to 102, as illustrated in the inset of FIG. 12 b).
These results strongly demonstrate that the multiple SERS signal enhanced nano "sandwich" bacterial detection system developed by the present invention has extremely high accuracy in blood sample analysis and has broad application prospects in clinical applications of hypersensitive bacterial detection.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A multiple SERS signal enhanced nano 'sandwich' bacteria detection system is characterized by comprising labeled nano particles D-Au@Ag-C with high bacterial adhesiveness and a capture substrate Fe 3 O 4 @Au-Ab；
The structure of the D-Au@Ag-C is as follows: the method comprises the steps of growing Au@Ag core-shell nanoparticles on the surface of dendritic mesoporous silica DMSN in situ, and modifying concanavalin Con A and SERS labels;
the Fe is 3 O 4 The @ Au-Ab is: fe (Fe) 3 O 4 The surface of the nano-particle is covered with a gold coating, and meanwhile, a bacterial targeting antibody is modified.
2. The multiple SERS signal enhanced nano "sandwich" bacterial detection system of claim 1 wherein the SERS tag is 4-mercaptobenzoic acid 4-MBA.
3. The multiple SERS signal enhanced nano "sandwich" bacterial detection system of claim 1 wherein the dendritic mesoporous silica DMSN is 160-180nm in diameter.
4. The multiple SERS signal enhanced nano "sandwich" bacterial detection system of claim 1 wherein the bacteria is staphylococcus aureus.
5. A method of making a multiple SERS signal enhanced nano "sandwich" bacterial detection system according to any one of claims 1 to 4, comprising the steps of:
1) Preparation of D-Au@Ag-C:
modifying amino group on DMSN surface to make its surface positively charged so as to obtain DMSN-NH 2 The method comprises the steps of carrying out a first treatment on the surface of the With DMSN-NH 2 Gold is grown on the surface of the DMSN by a seed solution growth method to obtain DMSN-Au; SERS label modification is carried out on the DMSN-Au to obtain the DMSN-Au 4-MBA The method comprises the steps of carrying out a first treatment on the surface of the In DMSN-Au 4-MBA Silver Ag grows on the surface to obtain DMSN-Au@Ag; SERS label modification is carried out on the DMSN-Au@Ag to obtain the DMSN-Au@Ag 4-MBA The method comprises the steps of carrying out a first treatment on the surface of the For DMSN-Au@Ag 4-MBA Carrying out concanavalin Con A modification to obtain D-Au@Ag-C;
2)Fe 3 O 4 preparation of @ Au-Ab:
preparing a gold seed solution; fe is added to 3 O 4 The nano particles are subjected to surface modification to make the nano particles have positive potential, then are mixed with gold seed solution, and are grown on Fe by a seed solution growth method 3 O 4 Gold grows on the surface of the nano particle to obtain Fe 3 O 4 @Au；Fe 3 O 4 Modifying bacterial targeting antibody Ab on surface of@Au to obtain Fe 3 O 4 @Au-Ab。
6. The preparation method of the dendritic mesoporous silica DMSN according to claim 5, comprising the following steps:
fully dissolving triethanolamine into ultrapure water, heating and stirring the obtained solution; adding sodium salicylate and cetyltrimethylammonium bromide into the solution, and stirring; dropwise adding tetraethoxysilane into the solution under stirring, and continuing stirring after the dropwise adding is finished; after the solution is cooled to room temperature, centrifugally collecting a product; washing; removing cetyl trimethyl ammonium bromide; and drying to obtain the dendritic mesoporous silica DMSN.
7. The method of claim 5, wherein in step 1), the seed solution growth method specifically comprises: taking DMSN-NH 2 Dispersing into deionized water, adding tetrachloroauric acid solution, and mixing with ultrasound; under intense stirring, rapidly injecting sodium borohydride solution into the mixed solution, stirring for reaction, centrifuging and washing with ultrapure water for multiple times to obtain DMSN-m-Au seed nano particles; resuspending the DMSN-m-Au seed solution into hydroxylamine hydrochloride solution, vigorously stirring at room temperature, and rapidly adding gold chloride solution; stirring, and centrifugally collecting to obtain DMSN-Au;
or, the said DMSN-Au 4-MBA The silver Ag grown on the surface is specifically as follows: DMSN-Au 4-MBA Dispersing into polyvinylpyrrolidone water solution, and stirring; adding silver nitrate and ascorbic acid water solution into the mixed solution for incubation, and reducing silver ions into metallic silver; centrifuging, washing to remove redundant reactants, and obtaining DMSN-Au@Ag;
or, the pair DMSN-Au@Ag 4-MBA The concanavalin Con A modification is specifically: activation of DMSN-Au@Ag with a mixture of tetramethylammonium hydroxide 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysulfosuccinimide sodium salt 4-MBA Carboxyl on 4-MBA; con A solution and activated DMSN-Au@Ag 4-MBA Mixing, and stirring at room temperature to obtain the D-Au@Ag-C.
8. The method according to claim 5, wherein in the step 2), the method for preparing the ferroferric oxide nanoparticles comprises the following steps: ferric trichloride hexahydrate and trisodium citrate are dissolved in ethylene glycol, and ethylene glycol containing sodium acetate is added under magnetic stirring; stirring vigorously at room temperature; subsequently, the mixture was transferred to an autoclave, which was placed in a 200 ℃ oven and heated to obtain a black precipitate; washing, collecting by a magnet, and drying to obtain ferroferric oxide nano particles;
or, the Fe 3 O 4 NanoparticlesThe surface modification is specifically as follows: fe is added to 3 O 4 Dispersing the nano particles into a polyethyleneimine PEI aqueous solution, fully mixing, and forming a self-assembly mode on Fe 3 O 4 The surface of the nano particle is modified with a layer of PEI, and Fe 3 O 4 The nanoparticle is modified to a positive potential.
9. The process according to claim 5, wherein in step 2), the Fe is 3 O 4 Modifying bacterial targeting antibody Ab on surface of@Au to obtain Fe 3 O 4 The @ Au-Ab is specifically: polyethylene glycol PEG added to Fe 3 O 4 Ultrasonic mixing is carried out on the @ Au, and redundant PEG is removed by centrifugation, thus obtaining Fe 3 O 4 @ Au-PEG; activation of Fe 3 O 4 Carboxyl groups on the surface of the @ Au-PEG; after ultrasonic treatment at room temperature, the activated nano particles are resuspended in phosphate buffered saline containing bacterial targeting antibody Ab and stirred to obtain Fe 3 O 4 @Au-Ab。
10. A method for detecting bacteria, characterized in that a multiple SERS signal enhanced nano "sandwich" bacteria detection system according to any one of claims 1-4 is used, comprising the following steps:
adding capture substrate Fe into sample to be detected 3 O 4 Incubating at @ Au-Ab, washing, adding high bacterial adhesion marked nanoparticle D-Au @ Ag-C, mixing, washing by magnetic separation method, transferring onto silicon wafer, and measuring at 1583cm -1 Raman signal of (a);
preferably, the bacterium is staphylococcus aureus.
CN202310149926.6A 2023-02-21 2023-02-21 Multiple SERS signal enhanced nano sandwich bacteria detection system and preparation method thereof Pending CN116148239A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116593356A (en) * 2023-06-05 2023-08-15 南京工业大学 Method for detecting viscosity of micro-solution by stirring magnetic nano brush

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116593356A (en) * 2023-06-05 2023-08-15 南京工业大学 Method for detecting viscosity of micro-solution by stirring magnetic nano brush
CN116593356B (en) * 2023-06-05 2023-11-17 南京工业大学 Method for detecting viscosity of micro-solution by stirring magnetic nano brush

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