CN113340873B - SERS system for simultaneously detecting multiple toxic substances - Google Patents
SERS system for simultaneously detecting multiple toxic substances Download PDFInfo
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- CN113340873B CN113340873B CN202110905323.5A CN202110905323A CN113340873B CN 113340873 B CN113340873 B CN 113340873B CN 202110905323 A CN202110905323 A CN 202110905323A CN 113340873 B CN113340873 B CN 113340873B
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- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000010526 radical polymerization reaction Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 229910000404 tripotassium phosphate Inorganic materials 0.000 description 1
- HIZCIEIDIFGZSS-UHFFFAOYSA-L trithiocarbonate Chemical group [S-]C([S-])=S HIZCIEIDIFGZSS-UHFFFAOYSA-L 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 238000001392 ultraviolet--visible--near infrared spectroscopy Methods 0.000 description 1
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Abstract
The invention relates to a SERS system for simultaneously detecting a plurality of toxic substances, which comprises the following substances A and B: the substance A is an anti-fouling magnetic microbead coupled with more than two toxic substance haptens, which is expressed as MB @ P-hap, wherein MB is a magnetic microbead, P is an anti-fouling polymer brush, and hap is a toxic substance hapten and is obtained by coupling the toxic substance haptens on the magnetic microbead grafted with the anti-fouling polymer brush; the B substance is a mixture of more than two toxic substance monoclonal antibody labeled nano-probes, wherein the toxic substance monoclonal antibody labeled nano-probes are expressed as SERS substrate @ mAb-RP, the SERS substrate represents a surface enhanced Raman scattering substrate, the mAb represents a toxic substance monoclonal antibody, and the RP represents a Raman Probe (Raman Probe). The detection sensing system constructed by the invention is executed based on a sample solution in a competitive immunoassay form, has satisfactory detection accuracy and sensitivity, and can be used for detecting trace toxic substances in complex matrixes such as food and the like.
Description
Technical Field
The invention relates to the field of SERS sensors, in particular to a SERS system for simultaneously detecting multiple toxic substances.
Background
The rapid, accurate and low-cost multi-target detection has important significance in the aspects of early diagnosis of diseases, food safety supervision, environmental monitoring and the like. However, the current detection method has limited sensitivity and weak multiplexing detection capability, so that the wide application of the method is limited. The Surface Enhanced Raman Spectroscopy (SERS) technology is simple, high in sensitivity and strong in coding capability, is considered as an ideal means for manufacturing a multi-channel sensing platform, and still has some obstacles in the aspects of total quantity, reliability, sensitivity and repeatability in practical application. The intensive study of interface chemistry to control target recognition and signal transduction properties is crucial to the development of high performance sensing platforms. Currently, non-contaminating polymer brushes have been used as sensing or binding interfaces. The unique microstructure and excellent properties of the polymer brush not only improve the recognition ligand density on the sensing interface, but also reduce non-specific binding. Non-contaminating polymer brushes as functional ligands have been utilized as specific adsorption materials to achieve efficient target recovery, thus demonstrating their superiority in constructing sensing interfaces. On the other hand, the manufacturing of the uniform and high-sensitivity SERS encoder is also the key for realizing high-performance multi-path SERS detection. However, the demand for obtaining uniform, stable, plasmonic nanostructures with high density hot spots in a simple way is still high.
Diphacinone (DPN), Bromadiolone (BRD) and phenthoate (TET) as highly toxic rodenticides have caused a number of accidental or deliberate food poisoning events worldwide. Although instrument-based analytical methods such as GC-MS, LC-MS have been well-established for the detection of these rodenticides, the need for rapid and early detection tools for on-site screening of suspected foods and biological samples, in order to improve diagnostic efficiency, timely medical care, and particularly in rural areas where laboratory equipment and trained personnel are lacking, is high and has not yet been met.
SERS is one of the detection means of multiple targets in food and biological samples, has simple technology, high sensitivity and strong coding capacity, but still has some obstacles in the aspects of totality, reliability, sensitivity and repeatability. And due to non-specific adsorption (fouling) and cross-reaction on the sensing interface, the detection accuracy is affected by the coexisting interference in the background matrix. At present, single immunodetection methods for DPN, BRD and TET exist, but a simultaneous on-site rapid detection method for multiple targets is not reported. Therefore, there is a need to develop a reliable on-site screening technology to meet the requirement of simultaneously detecting multiple targets in a complex matrix rapidly, sensitively and accurately.
Disclosure of Invention
The invention aims to construct a multiple competitive immunoassay method integrating hapten-modified anti-pollution magnetic beads and an SERS coding mixture. A detection system constructed by multiple hapten modified polymer magnetic beads and multiple SERS probes can be used for simultaneously detecting multiple targets in a complex matrix. The invention designs an SERS sensing system, combines MB @ P-CyM-hap with an SERS coding mixture, and can simultaneously and rapidly detect three high-toxicity rodenticides such as DPN, BRD and TET. The signal-to-noise ratio and the interference of false positive are obviously improved, the detection limits of DPN, BRD and TET in a complex matrix are respectively 0.5 ng/mL, 0.5 ng/mL and 0.2 ng/mL, the labeling recovery rate is 69.8% -133.0%, and the variation coefficient is 0.24% -25.06%. The method can be applied to rapid, accurate and low-cost multi-target detection of early disease diagnosis, food safety supervision and environmental monitoring.
The purpose of the invention is realized by the following technical scheme:
the SERS system for simultaneously detecting a plurality of toxic substances comprises the following substances A and B:
the substance A is an anti-fouling magnetic microbead coupled with more than two toxic substance haptens, which is represented as MB @ P-hap, wherein MB is a magnetic microbead, P is an anti-fouling polymer brush, hap is a toxic substance hapten, and MB @ P-hap is obtained by coupling the toxic substance haptens on the magnetic microbead grafted with the anti-fouling polymer brush;
the B substance is a mixture of more than two toxic substance monoclonal antibody labeled nano-probes, wherein the toxic substance monoclonal antibody labeled nano-probes are expressed as SERS substrate @ mAb-RP, the SERS substrate represents a surface enhanced Raman scattering substrate, the mAb represents a toxic substance monoclonal antibody, and the RP represents a Raman Probe (Raman Probe).
The proportion relation of the A substance and the B substance meets the following conditions: the hapten in substance A is capable of binding to all the monoclonal antibodies in substance B, i.e. the hapten of substance A is in excess of the monoclonal antibodies in substance B. Therefore, in the system to be detected, if the related toxic substances to be detected do not exist, the B substance with the Raman signal, namely the SERS probe is completely combined by the A substance, the Raman signal cannot be detected in the system after magnetic separation, and the signal-to-noise ratio is high.
The invention relates to an anti-fouling magnetic microbead (MB @ P-hap) coupled with more than two toxic substance haptens, which has the following preferable technical scheme:
further, the toxic substance is selected from at least one of rodenticides such as Diphacinone (DPN), Bromadiolone (BRD), diphacinone (TET), Brommurine (BTF), thiabendazole (DFT), rodenticide (CMT), Cricet (CMF), flocmafen (FCF), Diphacinone (DPC), and chlorophacinone (CPC). Haptens of toxic substances are well known in the art and are typically small molecules of toxic substances-linker arms-reactive groups, the length of the linker arm being 3-6 carbon atoms, the reactive groups including carboxyl, amine, hydroxyl, thiol or anhydride.
Further, the toxic substance is selected from two or more of Diphacinone (DPN), Bromadiolone (BRD) and diphacinone (TET), and their haptens (hap) are
Further, the monomers of the anti-fouling polymer brush comprise at least one of cysteine methacrylate (CyM), 3- ((3-aminopropyl) dimethylammonium methacrylate) -propionate (CBMAA), 2-hydroxyethyl methacrylate (HEMA), 2-hydroxypropyl methacrylate (HPMA). Cysteine methacrylate (CyM) is preferred.
The polymer brush is the anti-fouling magnetic microbead grafted by the polymer brush, which is obtained by initiating polymerization after the magnetic microbead is connected with a chain transfer agent. The chain transfer agent is not particularly limited, and is generally a compound containing a trialkoxy group and a trithiocarbonate group, such as trimethoxysilyltrithiocarbonate ethyl ester.
Further, the magnetic microbead is SiO2Coated Fe3O4Magnetic beads, Fe3O4As a core, a diameter of 100-200nm, SiO2The protective layer has a thickness of 10-40 nm. The magnetic micro-beads are grafted with anti-fouling polymer brushes, and the thickness of the polymer brushes is 10-30 nm; the saturation magnetic susceptibility of the anti-pollution magnetic micro-beads is 20-40emu g-1。
Further, the hapten hap content of MB @ P-hap is 1-25. mu.g/g, preferably 9-22. mu.g/g, more preferably 11-14. mu.g/g. Although theoretically, the higher the hapten content in MB @ P-hap, the stronger the binding ability with the corresponding monoclonal antibody (mAb) to the toxic substance, the applicants have found that the content of hap cannot be too high, otherwise some non-specific adsorption occurs, affecting the detection result, and the hapten density of 11-14. mu.g/g is most suitable.
Further, the anti-fouling magnetic microbead (MB @ P-hap) coupled with the toxic substance hapten is obtained by a preparation method comprising the following steps:
(T1) adding a Chain Transfer Agent (CTA) into a suspension of magnetic Microbeads (MBs), and refluxing for 2-6h under an inert atmosphere to obtain the magnetic microbeads (CTA-MBs) connected with the chain transfer agent;
(T2) under an inert atmosphere, dispersing the magnetic microspheres and the initiator linked by the chain transfer agent into a solvent, heating (corresponding to the decomposition temperature of the initiator) to perform a polymerization reaction for 4-6h to obtain the polymer brush grafted anti-fouling magnetic microspheres (MB @ P).
(T3) two or more haptens of the toxic substance are covalently linked to the polymer brush grafted anti-fouling magnetic microsphere through coupling to obtain MB @ P-hap.
Further, in the above preparation of MB @ P-hap, in the step (T1), the mass ratio of CTA to MBs was 1: 1-2; in the step (T2), the initiator is not particularly limited, and there may be mentioned initiators conventionally used in radical polymerization in the art, such as AIBN, BPO, potassium persulfate, ammonium persulfate and the like. The mass ratio of the magnetic microbeads connected by the chain transfer agent to the monomers to the initiator is 1: 1-1.5: 0.05-0.2; in step (T3), coupling means of hapten and polymer brush grafted anti-fouling magnetic microspheres are well known in the art, such as coupling reaction after EDC/NHS activation.
For the mixture of the nanoprobes (SERS substrate @ mAb-RP) marked by the monoclonal antibodies of more than two toxic substances, the invention has the following preferred technical scheme:
further, the surface enhanced raman scattering substrate (SERS substrate) is gold nanoflowers (AuNFs) which are monodisperse and have a size of 40-70 nm.
The gold nanoflowers (AuNFs) are prepared by a hydrazine reduction method, and specifically, the gold nanoflowers (AuNFs) are obtained by uniformly dispersing a gold source and Tannic Acid (TA) in an alcohol-water solution and adding hydrazine for reaction. The gold source is HAuCl4(ii) a Gold source, TA and hydrazineIn a molar ratio of 10-20: 1-3: 2-5.
The obtaining of monoclonal antibodies to toxic substances is well known in the art and is obtained by immunization of animals (e.g. rabbits, mice, dogs, camels) by hybridoma, phage display technology.
The raman probe is a substance having characteristic peaks in a raman spectrum, such as organic substances with larger raman cross sections, such as thiosalicylic acid (TSA), bis (2-nitrobenzoic acid (DTNB), 2, 6-dimethylphenyl isocyanate (DMPI), rhodamine 6G, malachite green, crystal violet, mercaptonaphthalene, mercaptophenol, mercaptobenzoic acid, mercaptophenylboronic acid, 4-fluorobenzenethiol, benzene ring substitutes of thiophenol, thiocyanate, cyano and the like.
In the nano-probes marked by the monoclonal antibodies of different toxic substances, Raman probes which cannot interfere with each other in a Raman spectrum are adopted. The characteristic peaks of any two Raman probes have a difference of more than two half-peak widths, such as 100cm-1The above gap.
Further, the nano-probe labeled by the toxic substance monoclonal antibody (SERS substrate @ mAb-RP) is obtained by a preparation method comprising the following steps:
(P1) reacting gold nanoflowers (AuNFs) with a substance containing carboxyl groups to obtain carboxyl-terminated gold nanoflowers;
(P2) after carboxyl activation, the gold nanoflowers are coupled with monoclonal antibodies (mAbs) of toxic substances to obtain monoclonal antibody labeled gold nanoflowers (AuNFs @ mAbs);
(P3) reacting the gold nanoflowers marked by the monoclonal antibody with Raman Probe (Raman Probe) substances to obtain the nano-Probe (SERS substrate @ mAb-RP) marked by the monoclonal antibody of the toxic substances.
Further, in the above method for preparing SERS substrate @ mAb-R, the carboxyl group-containing substance in step (P1) is preferably carboxyl-linker-thiol, the linker is a polymer segment with hydrophilic groups, such as polyether, polyatomic alcohol, polyester, preferably carboxyl-PEG-thiol, and the molecular weight of PEG is 1000-; the method of carboxyl activation described in step (P2) is well known in the art, such as activation with EDC/NHS.
Further, gold nanoflowers (AuNFs), monoclonal antibodies (mAbs) to toxic substances, and Raman probe substances are used in amounts ranging from 0.3 to 0.5mmol:100-200 mg: 1-2 mmol.
When the nano-probe marked by other toxic substance monoclonal antibodies is prepared, other modes and conditions are unchanged, but different Raman probes need to be replaced, and when the nano-probe is prepared by adopting different toxic substance cloned antibodies, the characteristic peaks of Raman scattering spectra of the adopted Raman probe substances can not interfere with each other, and the positions of the general characteristic peaks are different by more than two half peak widths and can be considered as not interfering with each other.
The nano probe mixture marked by the toxic substance monoclonal antibody is prepared by mixing SERS substrate @ mAb-RP of more than two toxic substances according to a certain proportion.
In a preferred embodiment of the present invention, the toxic substance is selected from two or more of Diphacinone (DPN), Bromadiolone (BRD), and diphacinone (TET), and the raman probe substance is selected from two or more of thiosalicylic acid (TSA), bis (2-nitrobenzoic acid (DTNB), and 2, 6-dimethylphenyl isocyanate (DMPI).
In a more preferred embodiment of the present invention, when the toxic substance is DPN, the raman probe is DMPI; when the toxic substance is BRD, the Raman probe is TSA; when the toxic substance is TET, the Raman probe is DTNB.
In a still further preferred embodiment of the present invention, the SERS substrate is gold nanoflowers, and the obtained nanoprobes labeled with the three monoclonal antibodies against toxic substances are respectively AuNF @ DMPI-mabsDPN；AuNF@TSA-mAbBRD；AuNF@DTNB-mAbTET(ii) a The AuNF @ mAB-RP is suspended in BSA solution for storage, the concentration of the AuNF @ mAB-RP is prepared to be 0.2-1.0mM, preferably 0.3-0.5mM, the concentration of the gold atom can be digested according to aqua regia, and the system has the calculation of the number of the gold atoms after conversion.
Further, when preparing the nanoprobe mixture, AuNF @ PEG/DMPI-mAbDPN、AuNF@PEG/TSA-mAbBRDAnd AuNF @ PEG/DTNB-mAbTETAccording to a molar ratio of 1-2: 0.5-1.5:2-4; more preferably 1.5 to 2: 0.7-1: 2.5-3. The inventor finds that when the nanoprobe mixture is prepared according to the molar ratio and then Diphacinone (DPN), Bromadiolone (BRD) and diphacinone (TET) are detected simultaneously, the sensitivity and the accuracy are best.
In a preferred embodiment of the present invention, when the magnetic beads in the substance A are SiO2Coated Fe3O4When the SERS substrate in the substance B is gold nanoflowers (AuNFs), the dosage and the proportion of the substance A and the substance B are such that the concentration of Au in the final system is 0.1-1 mM, the concentration of Fe is 1-50 mM, preferably, the concentration of Au is 0.3-0.5mM, and the concentration of Fe is 15-25 mM.
The invention also provides a method for simultaneously detecting multiple toxic substances based on SERS, which comprises the following steps:
(S1) adding different amounts of standard toxic substances into a detection system respectively, wherein the detection system comprises more than two kinds of hapten-coupled anti-fouling magnetic microbeads (MB @ P-hap) of the toxic substances and a solution of nanoprobes (SERSssubstrate @ mAb-RP) marked by the monoclonal antibodies of the toxic substances, fully reacting, collecting free SERS probes through magnetic separation, carrying out Raman detection, drawing the characteristic peak intensities of different Raman probes on the SERSssubstrate @ mAb-RP and lgC, establishing a standard curve and a linear regression equation, wherein C is the concentration (molar concentration or mass concentration) of the toxic substances in the system, and lgC is the logarithm of the concentration (base 10) of the toxic substances in the system;
(S2) replacing another standard toxic substance and the corresponding nano probe marked by the toxic substance monoclonal antibody, and repeating the step (S1) to obtain a standard curve and a linear regression equation of the characteristic peak intensities of different toxic substances lgC and Raman probes;
(S3) repeating the step (S2) until a linear relationship between the concentration of all toxic substances and the intensity of the characteristic peak of the corresponding raman probe is established;
(S4) adding the substance A and the substance B into a sample solution to be detected, after full reaction, collecting free SERS probes in the solution through magnetic separation, carrying out Raman detection, detecting different Raman probe characteristic peak intensities, and qualitatively judging whether toxic substances exist in the sample according to the different toxic substance Raman probe characteristic peak intensities; or quantitatively testing the concentration of the toxic substance according to the linear relation between the intensity of the characteristic peak of the LiRaman probe of different toxic substances and the concentration of the toxic substance.
The principle that the invention can detect a plurality of toxic substances simultaneously is based on the combination of the monoclonal antibody of the toxic substance and the hapten and the competition between the monoclonal antibody of the toxic substance and the toxic substance, when no related toxic substance exists in a sample to be detected, all nano probes (SERSssubstrate @ mAb-RP) marked by the monoclonal antibody of the toxic substance can be combined with anti-fouling magnetic microbeads (MB @ P-hap) coupled with the hapten of the toxic substance, and through magnetic separation, because all SERS nano probes are combined with the magnetic microbeads and free SERS nano probes do not exist in the system, the characteristic peak signal of the Raman probe on the SERS probe can not be detected. When related toxic substances exist in the system, when reaction balance is achieved, the monoclonal antibody on the SERS probe can be preferentially combined with the toxic substances and cannot be combined by the magnetic beads, therefore, after magnetic separation, free SERS probes can exist in the solution system, the SERS probes are collected for Raman detection, and whether related toxic substances exist in a sample to be detected or not and the content of the toxic substances can be qualitatively or quantitatively tested according to the linear relation between the concentration of the toxic substances and the characteristic peak intensity of the Raman probe substances through the characteristic peak intensity of the Raman probe substances.
According to the invention, the excellent SERS enhancement factor of the gold nanoflowers and the anti-fouling capability of the magnetic microbeads are endowed by the polymer brushes in the shape of the high-molecular brushes, so that the sensitivity and accuracy of detection are ensured. When the detection substances are Diphacinone (DPN), Bromadiolone (BRD) and texin (TET), the detection Lines (LOD) of the three toxic substances are respectively as low as 0.42ng/mL, 0.18ng/mL and 0.76 ng/mL. And various toxic substance haptens are coupled on the magnetic microspheres, and simultaneously, SERS probes of various toxic substances are constructed in a matched manner, so that bright related toxic substances in a complex substrate sample can be tested simultaneously. The operation is simple and convenient, the operation is quick, and a conventional Raman detection instrument is used without a large instrument.
The invention achieves the following beneficial effects:
firstly, the invention constructs a pollution-free polymer brush, namely magnetic beads (MB @ P-CyM-hap) grafted with triple haptens, which are used as substrates for competitive combination of DPN, BRD and TET. The result shows that the binding capacity of the MB @ P-CyM-hap and the SERS (surface enhanced Raman scattering) coder marked by the monoclonal antibody is obviously enhanced due to the high-molecular hair-shaped three-dimensional nano interface, so that the signal-to-noise ratio (S/N) and the linear relation are obviously improved.
The invention also provides a method for synthesizing the Au nanoflower by the one-step method, and the obtained Au nanoflower is a single-layer SERS substrate with excellent performance and has the characteristics of high Raman probe Enhancement Factor (EF), simplicity in preparation, good repeatability, low cost and the like. The multiple SERS sensing platform improved by the Au nanoflower ensures the sensitivity, reliability and rapid convenience of on-site detection capability, has good popularization potential, and can be easily expanded to immune competitive detection analysis of other small molecules.
The detection sensing system constructed by the invention is implemented by combining the SERS probe mixture coupled with the monoclonal antibody and MB @ P-CyM-hap into a sample solution based on a competitive immunoassay form, has satisfactory detection accuracy and sensitivity, and can be used for detecting trace toxic substances in complex matrixes such as food and the like.
Drawings
FIG. 1 shows the chemical structures of three rodenticides TET, BRD, DPN (FIG. 1A) and their respective haptens (FIG. 1B) for the substances to be detected.
FIG. 2 is a schematic diagram of the scheme for the synthesis of MB @ P-CyMbal-hap.
FIG. 3 is the nanostructure of MBs and MB @ P-CyM obtained in preparation example 1.
Fig. 4A is an infrared spectrum of each magnetic bead, and fig. 4B is a thermogravimetric analysis (TGA) of each magnetic bead.
FIG. 5 is a graph of the saturation susceptibility of MB, MB @ P-CyM, MB @ P-CyM-hap.
FIG. 6 is a graph showing the binding capacity and nonspecific adsorption of MB @ P-CyM-hap at various hapten densities.
Fig. 7 is a TEM image of the resulting gold nanoparticles.
FIG. 8 is an AuNF @ PEG/TSA-mAbBRD、AuNF@PEG/DTNB-mAbTETAnd AuNF @ PEG/DMPI-mAbDPNAnd SERS signals of mixtures thereof.
FIG. 9 shows the results of the detection of different concentrations of standard rodenticides (DPN, BRD, and TET) by the three SERS probes coupled to different monoclonal antibodies of example 1.
FIG. 10 shows that the SERS detection system of the present invention is 1034cm for seven kinds of interference rodenticides-1The raman intensity of (a).
Fig. 11 is a magnetic frame for use in magnetic separation according to the present invention.
Detailed Description
The technical solution of the present invention is further explained with reference to the following embodiments, but it should be noted that the embodiments are only an embodiment and explanation of the technical solution of the present invention, and should not be construed as a limitation to the scope of the present invention.
The reagents used in the present invention and together with them, if not otherwise specified, are commercially available reagents and instruments.
Carboxy-polyethylene glycol-thiol was purchased from Sigma-Aldrich, where PEG molecular weight is 1000.
Preparation of monoclonal antibody 4G5 to Dihydronaphthalenone (DNP) reference is made to Li, H., Liu, S., Dong, B., Li, C., Yang, H., Zhang, X, Wen, K, Yu, X, Yu, W, Shen, J., Li, J., Wang, Z, Production of a specific monomeric antibody and a reactive immunological assay for the detection of a pathological in biological samples 2019, 411 (25), 6755-6765.
Monoclonal antibodies 15C1 Li, H., Wen, K., Dong, B., Zhang, J., Bai, Y., Liu, M., Li, P., Mujtaba, M.G., Yu, X., Yu, W., Ke, Y., Shen, J., Wang, Z, Novel inner fi lter effect-based fluorescent specimen with a microbial assay with gold nanoparticles for a bromoiodide detection in human um, Sensors and activators B Chemical 2019, 297.
The rat-fast (TET) monoclonal antibody 1G6 was obtained from the donation of the Wang war Chi professor of animal medical college of Chinese agricultural university.
UV-vis-nir spectrophotometers using Shimadzu UV3600, nanomorphs were purchased from Malvern instruments by JEM-2100F projection electron microscope (JEOL ltd, japan), hydrodynamic diameter and zeta potential testing instruments, and thermogravimetric analysis (TGA) was performed using TGA Q500 from room temperature to 800 ℃ under nitrogen at a heating rate of 10 ℃/min. The Fourier infrared is Bruker Vertex 70 FT-IR. AuNFs and MBs concentrations were measured using ICP-MS (Agilent 8800) and ICP-OES (IRIS Advantage, Thermo Scientific).
SiO2Coated Fe3O4Magnetic Beads (MBs) and ammonia-modified magnetic beads (NH)2MBs) (Deng, Y.; Cai, Y.; Sun, Z.; Liu, J.; Liu C.; Wei, J.; Li, W.; Liu, C.; Wang, Y.; Zhao, D.J. Am. Chem. Soc. 2010, 132, 8466-.
The concentration of Au in the SERS sensor is obtained by ICP-MS test after the Au is digested by aqua regia; the concentration of Fe atoms in the magnetic microbeads was obtained by ICP-OES test.
Preparation example 1Chain Transfer Agent (CTA) trimethoxysilyltrithiocarbonate ethyl ester
CTA is described in literature (Qu, Z.; Hu, F.; Chen, K.; Duan, Z.; Gu, H.; Xu, H.,J. Colloid and Interface Sci.2013, 398, 82-87). Specifically, 1-propanethiol (6.6 mmol) is added to K3PO4(1.02 g, 6.6 mmol) in a stirred suspension of anhydrous acetone (15 mL) and stirred for about half an hour. Addition of CS2(1.1 mL, 18 mmol) the solution turned bright yellow. After stirring for another 10 min, (4- (chloromethyl) phenyl) -trimethoxysilane (1.43 mL, 6.6 mmol) was added, stirred at room temperature under nitrogen for 13 h, diluted with dichloromethane and filtered. After removal of the solvent from the filtrate under reduced pressure, purification by petroleum ether/ethyl acetate gradient column chromatography on silica gel gave a bright yellow oil, the product ethyl trimethoxysilyltrithiocarbonate.
The synthetic route is as follows:
preparation example 2Preparation of MB @ P-Cym, MB @ P-CyM-hap and MB @ hap
1. Preparation of MB @ P-Cym
Anti-fouling polymer brush-grafted magnetic beads (MB @ P-CyM) were prepared by typical surface initiated polymerization (Qu, Z.; Hu, F.; Chen, K.; Duan, Z.; Gu, H.; Xu, H.; J. Colloid and Interface Sci. 2013, 398, 82-87.). The method comprises the following specific steps: 50 mg of CTA was added to 100mL of MBs absolute ethanol (1.0 mg. multidot.mL)-1) The suspension was refluxed for 5 hours under nitrogen, and the resulting CTA-MBs were collected and washed 3 times with ethanol. The CTA-MBs were suspended in ethanol for further use. CTA-MBs (0.1g), AIBN (20mg) and cysteine methacrylate (CyM, 0.1g) were dispersed in 10mL of degassed water/methanol (1:1) solution for grafting CyM polymer brushes. After bubbling nitrogen for 30min, the system was closed and heated at 80 ℃. After 5 h of reaction, MB @ P-CyM was collected and washed 3 times with DMF.
2. Preparation of MB @ P-CyM-hap
The triple hapten is covalently connected to MB @ P-CyM through a typical EDC coupling processMB@P-CyM-hap. Triple hapten mixture (molar ratio of hapten to TET, BRD, DPN 1:1) in 1.0mL of methanol was NHS (10 mg mL-1) And EDC (20mg mL)-1) And (4) activating. After 60 min of reaction, MB @ P-CyM (0.1g, 5mL of methanol) was added to the solution for coupling. Reacting at room temperature for 10 h, and taking supernatant. The resulting MB @ P-CyM-hap was washed repeatedly with acetone and finally dispersed in PBS buffer for further use.
To achieve different hapten competition matrix densities, three different hapten mixture concentrations (1: 1:1 molar ratio) of 10, 5 and 1 mmol. multidot.L in 1mL of methanol were used-1Covalent linkage of hap to MB @ P-CyM high density (MB @ P-CyM-hap1), medium density (MB @ P-CyM-hap2), and low density (MB @ P-CyM-hap3) were prepared, respectively. Detection of coupling ratio by Mass Spectrometry, MB @ P-CyM-The hapten density in hap1 was 18.3 g/. mu.g, the hapten density in MB @ P-CyM-hap2 was 11.4 g/. mu.g, and the hapten density in MB @ P-CyM-hap3 was 2.6 g/. mu.g.
3.Preparation of MB @ hap
In addition, hapten-conjugated MBs (MB @ hap) were also prepared for comparison. The specific method is 1mL of 1 mmol.L-1 The hapten (hap) mixture of (2) was put into NHS10 mg mL-1) EDC (10 mg. and 5mL methanol, with 0.1g Magnetic Beads (MBs) added, after shaking for ten hours on a shaker, was washed with magnetic recovery to give the final product MB @ hap. When used, MB @ P-CyM and MB @ hap had the same concentration of iron atoms.
FIG. 1 shows the chemical structures of three rodenticides TET, BRD, DPN (FIG. 1A) and their respective haptens (FIG. 1B) for the substances to be detected.
FIG. 2 is a schematic diagram of the scheme for the synthesis of MB @ P-CyMbal-hap.
FIG. 3 is a nanostructure of MBs and MB @ P-CyM. Wherein FIG. 3A is a TEM image of MBs, FIG. 3B is a TEM image of MB @ P-CyM, and FIG. 3C is a partially enlarged TEM image of MB @ P-CyM. It can be seen that both MBs and MB @ P-CyM exhibit good dispersibility and uniform morphology. MB @ P-CyM was internally Fe with a diameter of about 110nm3O4A core of about 20nm SiO in the middle2The protective layer, the outside, was a P-CyM polymer brush with a thickness of about 15 nm.
Fig. 4A is an infrared spectrum of each magnetic bead, and fig. 4B is a thermogravimetric analysis (TGA) of each magnetic bead. As can be seen from FIG. 4A, in the IR spectrum of MB @ P-CyM, strong carboxyl group stretching vibration of-COOH and C-O symmetrical stretching band appeared at 1720 cm-1And 1340 cm- 1. Amide at about 1625 cm-1About 2950 cm-1Peak value of and-CH2The stretching vibration of the group of P-CyM, notably due to the SiO in MBs and MB @ P-CyM2Layer present at 1080 cm-1There is a strong peak near, corresponding to the Si-O bond. TGA analysis of FIG. 4B shows that MB @ P-CyM has a significant mass loss, approximately 14%, which is significantly higher than MB @ hap without the polymer brush P-CyM.
FIG. 5 is a graph showing the saturation magnetic susceptibility of each magnetic bead, and it can be seen that MB @ P-CyMSaturation magnetic susceptibility of 30.5emu g-1And the magnetization curve shows symmetry and passes through the origin, so that the MB @ P-CyM prepared by the method is easily separated from a sample matrix and can be conveniently reused.
FIG. 6 is a graph showing the binding capacity and nonspecific adsorption of MB @ P-CyM-hap at various hapten densities. Wherein the combination of MB @ P-CyM-hap1 and MB @ P-CyM-hap2 with HSA is ten times the amount of HSA adsorbed actually. MB @ P-CyM-hap has abundant amino groups on the surface, and can conveniently have adjustability of hapten density. Different amounts of the three haptens were mixed in a molar ratio of 1:1:1 and MB @ P-CyM were coupled to give low density (MB @ P-CyM-hap1), medium density (MB @ P-CyM-hap2), and high density (MB @ P-CyM-hap3), respectively, and tested for their binding ability to three monoclonal antibodies (TET, monoclonal antibodies to BRD and DPN) and to a non-specific protein (HSA). The results show that as hapten density increases, the total monoclonal antibody binding capacity increases significantly, eventually reaching saturation adsorption. Under physiological conditions, medium density (MB @ P-CyM-hap2) and high density (MB @ P-CyM-hap3) showed nearly total monoclonal antibody adsorption. In contrast, MB @ hap had no polymer brush CyM thereon, and thus no soil resistance. MB @ P-CyM-hap exhibited some anti-fouling ability, both at low and high densities. MB @ P-CyM-hap2 showed the smallest amount of non-specific protein adsorption of 0.13 mg g-1While the nonspecific protein adsorption amount of MB @ P-CyM-hap3 increased to 0.64 mg g-1. Tests prove that MB @ P-CyM-hap2 can reach saturated adsorption within 30min, and the flexible polymer brush interface can reduce steric hindrance and promote synergistic affinity. Furthermore, no significant decrease in adsorption was observed for MB @ P-CyM-hap2 when stored at 4 ℃ for 6 months, indicating long-term chemical stability. In general, magnetic beads (MB @ P-CyM-hap2) of moderate hapten density (11-14. mu.g/g) were selected for subsequent testing.
Preparation example 3Preparation of gold nanoflowers (AuNFs)
Prepared by hydrazine reduction method, specifically 2.0 mL of 50.0 mM HAuCl4Mixed with 0.5mL of 10.0 mM TA ethanol solution in 100mL deionized water. After stirring for 5 minutes0.5mL of fresh N2H4Injected into the solution quickly. The color of the solution immediately turned blue, indicating the formation of AuNFs. After standing at room temperature for 2 hours, the AuNFs were purified by ultracentrifugation (8000 rpm, 5 minutes) and washed twice with water and finally dispersed in 10mL of deionized water.
Fig. 7A is a TEM image of AuNFs obtained in preparation example 2, and fig. 7B is a TEM image of AuNFs @ PEG-mAb mixture obtained in preparation example 3. As can be seen, AuNFs are monodisperse and have a size of 40-70 nm; whereas the AuNFs @ PEG-mAb showed a pale-colored organic coating around gold nanoflowers (AuNFs). PEG can effectively improve the stability of these SERS nanoprobes in complex biological media, as evidenced by no significant aggregation for more than 1 month in PBS and 10-fold diluted human serum.
The applicant also prepares other gold nanometer substrate shapes except gold nanoflowers, as shown in fig. 7C, the gold nanoflowers are uniform in shape, strong in SERS enhancement signal, high in sensitivity and good in repeatability as a SERS substrate, and show better SERS substrate performance than other shapes of the gold nanoflowers, possibly due to the fact that the gold nanoflowers have more special slit shapes.
Preparation example 4SERS (surface enhanced Raman scattering) hybrid sensor for constructing monoclonal antibody marker
Purified AuNFs (0.5mM) were incubated with carboxy-PEG-sulfhydryl (1.0 μ M) for 30 minutes. After centrifugation, the carboxy-PEG terminated AuNFs were activated by EDC/NHS for 30 minutes, followed by the addition of monoclonal antibody (mAb) solutions of DPN, BRD and TET, respectively (0.1 mg/mL), and coupling was performed overnight while maintaining a temperature of 4 ℃, and finally mAb-labeled AuNFs (AuNFs @ PEG-mAb) were centrifuged at 8000rpm for 5 minutes to remove unbound monoclonal antibodies and washed with PBS buffer. Subsequently, 10. mu.L of thiosalicylic acid (TSA, 1.0mM), 5' -dithiobis (2-nitrobenzoic acid (DTNB,1.0mM), and 2, 6-dimethylphenyl isocyanate (DMPI,1.0mM) were added to 1.0mL of the purified AuNFs @ PEG-mAb solution, respectively, to give different SERS sensors, i.e., AuNF @ PEG/TSA-mAb, respectivelyBRD、AuNF@PEG/DTNB-mAbTETAnd AuNF @ PEG/DMPI-mAbDPNFinally, it was resuspended in 300. mu.L0.1% BSA solution and stored refrigerated at 4 ℃ until use. The concentration of the added amount of each material is based on the concentration of each substance in the final system.
FIG. 8 is an AuNF @ PEG/TSA-mAbBRD、AuNF@PEG/DTNB-mAbTETAnd AuNF @ PEG/DMPI-mAbDPNAnd SERS signals of mixtures thereof. Three typical AuNF @ PEG/TSA-mAbs for simultaneous screening of multiple rodenticidesBRD、AuNF@PEG/DTNB-mAbTETAnd AuNF @ PEG/DMPI-mAbDPNUnder the excitation of laser, the Raman signals respectively generate unique Raman signals of 1034cm-1，1332cm-1And 2174cm-1Moreover, the signals of the three SERS sensors can be seen in the mixture to be sensitive and not interfere with each other, which indicates that the combined SERS mixed sensor can be used as an effective tool for detecting multiple targets.
Example 1Detection of DPN, BRD and TET in PBS buffer
In order to test the accuracy of the SERS probe prepared by the invention, 1.0-10.0 μ L of standard toxins (DPN, BRD and TET) are respectively added into MB @ P-CyM-hap2 or MB @ hap solution (50 μ L, MB @ P-CyM-hap2 or MB @ hap is added in an amount of 25mM of Fe concentration in the final system) prepared in preparation example 1, and then SERS probes (AuNF @ PEG/TSA-mABBRD, AuNF @ PEG/DTNB-mAbTET and AuNF @ PEG/DMPI-mABDPN are respectively added in an amount of 0.5mM of Au concentration in the final system). The reaction was stirred at room temperature for 30 minutes. The free SERS probe solution is collected by magnetic separation by arranging a magnet at the bottom of the test tube, and the way of arranging the magnet is not particularly limited as long as the magnetic beads in the test tube system can be adsorbed at the bottom of the test tube, for example, the free SERS probe solution can be obtained by a magnetic rack (as shown in fig. 11). After magnetic separation, the solution on the upper part of the test tube is transferred to a quartz cell for Raman detection. The results were analyzed by linear regression and used to verify the accuracy of the immunoassay.
FIG. 9 is a graph of example 1 using MB @ P-CyM-hap2 in PBS buffer solution with SERS probes (AuNF @ PEG/TSA-mABBRD, AuNF @ PEG/DTNB-mABTET, and AuNF @ PEG/DMPI-mABDPN) coupled with three monoclonal antibodies, respectively, versus different concentrations of standard rodenticides (DPN, BRD, and TE)T) the result of the detection. FIG. 9A is the nanoprobe AuNF @ PEG/TSA-mAbBRDRaman spectrum 1034cm at different concentrations of rodenticide BRD-1The peak intensity variation of (c); FIG. 9B is a Raman spectrum 1034cm-1Peak intensity and base 10 logarithm of BRD concentration (lgC)BRD) Plotted, BRD concentrations from 0.5 to 150 ng/mL, and linear regression equation I1034=2357× lgCBRD+1488，R2= 0.989. Similarly, FIGS. 9C and 9D are AuNF @ PEG/TSA-mAbsTETAt 1332cm of rodenticide TET at various concentrations-1Curve of peak intensity variation and linear regression equation, I1332=2367× lgCTET+2573，R2= 0.988. FIGS. 9E and 9F are AuNF @ PEG/TSA-mAbsDPNAt 2174cm of rodenticide DPN at various concentrations-1Equation of peak intensity variation and linear regression, I2174=2125× lgCDPN+2082，R2= 0.987. Based on the standard deviation of the triple control signal, a BRD limit of detection (LOD) of 0.42ng/mL, a TET LOD of 0.18ng/mL, and a DPN LOD of 0.76ng/mL were calculated. The LOD is already well below the detection standard for trace amounts of rodenticide in food. And after the MB @ hap is used as a competitive substrate to match with the same SERS probe mixture, the LOD and linear relation of the competitive substrate are not the same as those of the competitive substrate obtained by the MB @ P-CyM-hap2 and the same SERS probe mixture. The advantage of constructing high-capacity SERS detection by using the non-fouling polymer brush magnetic competitive substrate MB @ P-CyM-hap2 is shown, and the special P-CyM of the three-dimensional hair-like structure (3D-hair like) is beneficial to the coupling of high-density hapten and can reduce the steric hindrance of competitive combination of an SERS probe on a nano interface, so that the sensing sensitivity and accuracy are enhanced, and when a target substance is detected, the LOD is extremely low, and a trace amount of target substance can be detected; good linear relation, R2High stability and reliability.
In order to test the excellent selectivity of the detection system and method, seven different interferents, namely, brodifacoum Bromide (BTF), thiabendazole (DFT), raticide (CMT), Cricetera (CMF), fluoromuron (FCF), Diphacinone (DPC) and chlorophacinone (CPC), are respectively added into the sensing system, the concentrations of the interferents are all 1 mug/mL, and the subsequent SERS measurement is consistent with the procedure. The results are shown in FIG. 10, and it can be seen that the amount of these interfering substances is 1034cm-1The raman signals of the point are all very weak, only about 400, and the intensity is basically consistent with the signal intensity of the background peak. And for the toxic substances to be detected, the Raman signal intensity can reach 12000 under the same concentration, which shows that the detection system and the detection method have good selectivity and cannot be interfered by other toxic substances.
Example 2Detection of DPN, BRD and TET in spiked samples and complex matrices
The invention takes diluted human serum, urine, milk and muscle as raw materials to prepare food and biological samples. The solid sample was diluted with PBS buffer (1.0 mL/g), homogenized for 5 min, and centrifuged at 1200 rpm for 2 min to remove large residues. The supernatant was frozen for use. The samples to be tested were diluted 5-fold with Phosphate Buffered Saline (PBS) buffer prior to analysis.
Specifically, for example, for a human serum sample, 2.0 mL of plasma was vortex extracted with ethyl acetate (10 mL) and centrifuged at 3500 g for 5 min. The supernatant was separated by 5mL and dried at 50 ℃ under a nitrogen stream. Then, PBS buffer (2 mL, 0.01 mol L)-1pH 7.4) the dried residue was redissolved and 50. mu.L of the pretreated sample was sent to the detection system.
AuNF @ PEG/TSA-mAb prepared in preparation example 3 was addedBRD、AuNF@PEG/DTNB-mAbTET and AuNF @ PEG/DMPI-mAbDPNPreparing a SERS mixed sensor (150 mu L, the concentration of Au in a mixed final system is 0.5mM) by mixing according to a final molar ratio of 2:1:3, then adding standard toxins (DPN, BRD and TET) and MB @ CyM-hap2 (50 mu L, MB @ CyM-hap2 is 25mM of Fe in the mixed final system) in sequence, reacting the reaction system at room temperature for a period of time, collecting a free SERS probe mixture solution by magnetic separation, and transferring the free SERS probe mixture solution into a quartz cell for Raman detection. Negative human serum and urine samples were provided by the disease prevention and control center of Beijing, China. Negative food samples include milk, chicken purchased locally. Adding BRD, DPN and TET standard into the above real sample (human serum, urine, milk, chicken), and making sampleThe final toxin concentrations in (1) were 5.0 ng/mL, respectively. After the reaction system reacts for a period of time at room temperature, the free SERS probe mixture solution is collected through magnetic separation and transferred to a quartz cell for Raman detection. The results are shown in table 1 below:
the results in Table 1 show that the method of the invention has excellent accuracy and precision. The convenience of operation is more advantageous. The drying time of the sample was extended due to the nitrogen flush compared to the commercial ELISA kit method. For liquid samples, the method of the invention does not need centrifugation and can detect the liquid samples after direct dilution. The method has simple sample extraction steps and good anti-interference capability on complex matrixes such as food, blood and the like. On the one hand, the unique property of MB @ P-CyM-hap, which effectively prevents interference with samples that may be contained in the solution; in addition, the SERS nano-probe has magnetism, can be easily separated from a sample substrate by using an external magnet after detection, is favorable for capturing SERS signals, and further resists interference from complex substrates.
Claims (10)
1. The SERS system for simultaneously detecting a plurality of toxic substances comprises the following substances A and B:
the substance A is an anti-fouling magnetic microbead coupled with more than two toxic substance haptens, which is represented as MB @ P-hap, wherein MB is a magnetic microbead, P is an anti-fouling polymer brush, hap is a toxic substance hapten, and MB @ P-hap is obtained by coupling the toxic substance haptens on the magnetic microbead grafted with the anti-fouling polymer brush;
the B substance is a mixture of more than two toxic substance monoclonal antibody labeled nano-probes, wherein the toxic substance monoclonal antibody labeled nano-probes are expressed as SERS substrate @ mAb-RP, the SERS substrate represents a surface enhanced Raman scattering substrate, the mAb represents a toxic substance monoclonal antibody, and the RP represents a Raman probe;
the proportion relation of the A substance and the B substance meets the following conditions: the hapten in the substance A can be combined with all the monoclonal antibodies in the substance B.
2. The SERS system for simultaneously detecting multiple toxic substances according to claim 1, wherein the toxic substances are selected from at least one of Diphacinone (DPN), Bromadiolone (BRD), tebufen (TET), Brommurine (BTF), thiabendazole (DFT), rodenticide ether (CMT), cricet-killing (CMF), fluoromurinone (FCF), Diphacinone (DPC) and chlorophacinone (CPC); when the toxic substance is selected from two or more of Diphacinone (DPN), Bromadiolone (BRD) and diphacinone (TET), their haptens hap are respectively
3. The SERS system according to claim 1, wherein the anti-fouling polymer brush monomers comprise at least one of cysteine methacrylate, 3- ((3-aminopropyl methacrylate) dimethylammonium) -propionate, 2-hydroxyethyl methacrylate, and 2-hydroxypropyl methacrylate; and/or
The magnetic micro-beads are SiO2Coated Fe3O4Magnetic beads, Fe3O4As a core, a diameter of 100-200nm, SiO2As a protective layer, the thickness is 10-40 nm; the magnetic micro-beads are grafted with anti-fouling polymer brushes, and the thickness of the polymer brushes is 10-30 nm; the saturation magnetic susceptibility of the anti-pollution magnetic micro-beads is 20-40emu g-1。
4. The SERS system for simultaneously detecting multiple toxic substances according to claim 1, wherein the content of hapten hap in MB @ P-hap is 9-22 μ g/g.
5. The SERS system for simultaneously detecting multiple toxic substances according to claim 4, wherein the content of hapten hap in MB @ P-hap is 11-14 μ g/g.
6. The SERS system for simultaneously detecting multiple toxic substances according to claim 1, wherein the anti-fouling magnetic microbead MB @ P-hap coupled with the hapten of the toxic substance is obtained by a preparation method comprising the following steps:
(T1) adding a chain transfer agent CTA into a suspension of the magnetic microbeads MBs, and refluxing for 2-6h under an inert atmosphere to obtain the magnetic microbeads CTA-MBs connected with the chain transfer agent;
(T2) under an inert atmosphere, dispersing the magnetic microspheres, the initiator and the monomer connected with the chain transfer agent in a solvent, heating to the decomposition temperature of the corresponding initiator, and carrying out polymerization reaction for 4-6h to obtain the polymer brush grafted anti-fouling magnetic microspheres MB @ P;
(T3) two or more haptens of the toxic substance are covalently linked to the polymer brush grafted anti-fouling magnetic microsphere through coupling to obtain MB @ P-hap.
7. The SERS system for simultaneously detecting multiple toxic substances according to claim 1, wherein the surface enhanced Raman scattering substrate is gold nanoflowers (AuNFs), and the gold nanoflowers are monodisperse and have a size of 40-70 nm; the gold nanoflowers are prepared by a hydrazine reduction method, and are obtained by uniformly dispersing a gold source and tannic acid in an alcohol-water solution and adding hydrazine for reaction; the gold source is HAuCl4(ii) a The molar ratio of the gold source to the tannic acid to the hydrazine is 10-20: 1-3: 2-5, wherein the amount of gold source substance is in terms of gold atoms; the Raman probes are substances with characteristic peaks in Raman spectra, and in the nano probes marked by the monoclonal antibodies of different toxic substances, the characteristic peaks of any two Raman probes have a difference of more than two half-peak widths.
8. The SERS system for simultaneously detecting multiple toxic substances according to claim 7, wherein the nanoprobe SERS substrate @ mAb-RP labeled with the monoclonal antibody of the toxic substance is obtained by a preparation method comprising the following steps:
(P1) reacting the gold nanoflowers AuNFs with a substance containing carboxyl groups to obtain carboxyl-terminated gold nanoflowers;
(P2) after carboxyl activation is carried out on the gold nanoflowers with the carboxyl end capping, the gold nanoflowers are coupled with monoclonal antibodies (mAbs) of toxic substances to obtain monoclonal antibody marked gold nanoflowers AuNFs @ mAbs;
(P3) reacting the gold nanoflowers marked by the monoclonal antibody with Raman probe substances to obtain nano-probes SERS substrate @ mAb-RP marked by the monoclonal antibody of the toxic substances.
9. The SERS system for simultaneously detecting multiple toxic substances according to claim 8, wherein when the toxic substance is DPN, the Raman probe is DMPI; when the toxic substance is BRD, the Raman probe is TSA; when the toxic substance is TET, the Raman probe is DTNB, and the obtained nanoprobes marked by the three toxic substance monoclonal antibodies are respectively AuNF @ DMPI-mAbDPN；AuNF@TSA-mAbBRD；AuNF@DTNB-mAbTET(ii) a And AuNF @ PEG/DMPI-mAbDPN、AuNF@PEG/TSA-mAbBRDAnd AuNF @ PEG/DTNB-mAbTETAccording to a molar ratio of 1-2: 0.5-1.5: 2-4, mixing;
when the magnetic micro-beads in the A substance are SiO2Coated Fe3O4When the SERS substrate in the substance B is gold nanoflowers AuNFs, the dosage and the proportion of the substance A and the substance B meet the requirements that the Au concentration is 0.3-0.5mM and the Fe concentration is 15-25 mM.
10. A method for simultaneously detecting multiple toxic substances by the SERS system as claimed in any of claims 1 to 9, comprising the steps of:
(S1) adding different amounts of standard toxic substances into a detection system respectively, wherein the detection system comprises anti-fouling magnetic microbeads MB @ P-hap coupled with more than two kinds of toxic substance haptens and a nano probe SERSssubstrate @ mAb-RP solution marked by the toxic substance monoclonal antibody, fully reacting, collecting free SERS probes through magnetic separation, carrying out Raman detection, drawing the characteristic peak intensities and lgC of different Raman probes on the SERSssubstrate mAb @ RP, and establishing a standard curve and a linear regression equation, wherein C is the concentration of the toxic substances in the system, and lgC is the logarithm of the concentration of the toxic substances in the system with the base of 10;
(S2) replacing another standard toxic substance and the corresponding nano probe marked by the toxic substance monoclonal antibody, and repeating the step (S1) to obtain a standard curve and a linear regression equation of the characteristic peak intensities of different toxic substances lgC and Raman probes;
(S3) repeating the step (S2) until a linear relationship between the concentration of all toxic substances and the intensity of the characteristic peak of the corresponding raman probe is established;
(S4) adding a substance A and a substance B into a sample solution to be detected, after full reaction, collecting free SERS probes in the solution through magnetic separation, carrying out Raman detection, detecting different Raman probe characteristic peak intensities, and qualitatively judging whether toxic substances exist in the sample according to the different toxic substance Raman probe characteristic peak intensities; or quantitatively testing the concentration of the toxic substance according to the linear relation between the Raman probe characteristic peak intensity of different toxic substances and the concentration of the toxic substance.
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