阅读说明：本技术 一种基于荧光和拉曼双信号增强的肠毒素光谱分析方法 (Enterotoxin spectral analysis method based on fluorescence and Raman double-signal enhancement ) 是由 赵媛 施丽霞 于 2020-06-24 设计创作，主要内容包括：本发明提供了一种基于荧光和拉曼双信号增强的肠毒素光谱分析方法,属于光谱分析技术领域。本发明包括：将铬、铒、镱元素掺杂在镓锗酸锌基质得到长余辉纳米粒子(ZGGO:Cr,Er,Yb NPs)。在长余辉纳米粒子和金锥纳米粒子表面分别修饰肠毒素的一抗和二抗,当存在肠毒素时,抗体捕获了抗原形成双抗体夹心结构。抗体的刚性结构为金锥和ZGGO:Cr,Er,Yb NPs之间提供了合适的距离,金锥引起的电磁场增强促进了荧光物质的辐射衰减速率,导致荧光增强。此外ZGGO:Cr,Er,Yb NPs金锥之间存在电荷转移,导致SERS信号增强。本发明以荧光信号和拉曼信号共同作为检测信号,提高了检测的准确性。(The invention provides a fluorescence and Raman dual-signal enhancement based enterotoxin spectral analysis method, and belongs to the technical field of spectral analysis. The invention comprises the following steps: the long afterglow nano particle (ZGGO: Cr, Er, Yb NPs) is obtained by doping chromium, erbium and ytterbium elements in the gallium zinc germanate matrix. The primary antibody and the secondary antibody of the enterotoxin are respectively modified on the surfaces of the long-afterglow nano particles and the gold cone nano particles, and when the enterotoxin exists, the antibody captures the antigen to form a double-antibody sandwich structure. The rigid structure of the antibody provides a proper distance between the gold cone and ZGGO, Cr, Er and Yb NPs, and the electromagnetic field enhancement caused by the gold cone promotes the radiation attenuation rate of the fluorescent substance, so that the fluorescence is enhanced. In addition, charge transfer exists between ZGGO, Cr, Er and Yb NPs gold cones, and the SERS signal is enhanced. The invention takes the fluorescence signal and the Raman signal as the detection signal, thereby improving the accuracy of detection.)

1. A fluorescence and Raman dual signal enhancement based enterotoxin spectral analysis method is characterized by comprising the following steps: and (2) carrying out surface enterotoxin antibody modification on the long-afterglow nano particles to obtain ZGGO (chromium, Er, Yb/SEC-1) solution, mixing with Au NBPs @4-ATP/SEC-2 solution obtained by carrying out surface enterotoxin antibody modification on the gold cone nano particles, then adding enterotoxin SEC, uniformly mixing, and respectively measuring the fluorescence spectrum and the Raman spectrum of the solution.

2. The enterotoxin spectroscopic analysis method based on fluorescence and raman dual signal enhancement of claim 1, wherein the analysis method comprises the following specific steps:

(1) preparing long afterglow nano particle ZGGO, Cr, Er, Yb NPs and modifying surface SEC-1:

preparing long afterglow nano particle ZGGO, Cr, Er and Yb NPs:

mixing Cr (NO)₃)₂，Zn(NO₃)₂，Yb(NO₃)₂，Er(NO₃)₂With ammonium germanate solution to Ga (NO)₃)₂Mixing to obtain solution 1, and mixingAdding CTAB into the solution 1, adjusting the pH value of the solution to 7-9, carrying out hydrothermal reaction after uniform mixing, and heating for 14-16h at 110-; cooling to room temperature, carrying out solid-liquid separation to obtain a precipitate, washing and drying to obtain the long-afterglow nano particles ZGGO, Cr, Er and Yb NPs;

② amination of long afterglow nano particle ZGGO, Cr, Er, Yb NPs: dispersing the obtained ZGGO, Cr, Er and Yb NPs into NaOH solution, uniformly mixing, carrying out solid-liquid separation to obtain precipitate, dispersing the precipitate into N, N-dimethylformamide reagent, adding 3-aminopropyltriethoxysilane APTES reagent, heating in 75-85 ℃ water bath, carrying out solid-liquid separation to obtain a solid phase after the reaction is finished, and dispersing the solid phase into water to obtain an aminated ZGGO, Cr, Er and Yb NPs solution;

③ Long persistence nanoparticle surface enterotoxin antibody (SEC-1) modification by glutaraldehyde and NaBH₄Adding the solution into an aminated ZGGO (chromium, iron and boron) NPs solution, stirring, carrying out solid-liquid separation, washing the precipitate, redissolving the precipitate by using a PBS (phosphate buffer solution), adding an SEC-1 solution, carrying out oscillation incubation, centrifuging, resuspending the product by using a BSA (bovine serum albumin) solution, storing at room temperature for 4-5 hours, collecting the precipitate after the reaction is finished, washing by using water, finally uniformly dispersing the precipitate in the PBS buffer solution again, and obtaining a ZGGO (chromium, iron and Yb/SCE-1 solution after the reaction is finished.

(2) Preparing a gold cone nano Au NBPs solution and modifying surface SEC-2:

① preparation of gold cone nano Au NBPs solution by adding HAuCl₄Cetyl trimethyl ammonium chloride CTAC, adding NaBH to citric acid solution₄Stirring the solution at room temperature for 1-3 min; heating the solution in 75-85 deg.C oil bath, stirring for 85-95min to obtain gold seed solution, adding CTAB and HAuCl₄、AgNO₃Adding the gold seed solution into a mixed solution of HCl and AA ascorbic acid, and incubating for 1.8-2.2h to obtain a gold cone nano Au NBPs solution;

preparing Au NBPs @ 4-ATP: adding a 4-ATP solution into the gold cone nano Au NBPs solution, carrying out solid-liquid separation after the reaction is finished, taking a solid phase, washing with water, and dissolving the solid phase in water to obtain an Au NBPs @4-ATP solution;

③ modification of surface enterotoxin antibody SEC-2: adding enterotoxin antibody SEC-2 diluted by TBE buffer solution into AuNBPs @4-ATP solution for reaction, performing solid-liquid separation after the reaction is finished to obtain precipitate, using BSA solution to resuspend the precipitate, storing at room temperature for 2-3h, and blocking the binding site of unbound antibody; after the reaction is finished, centrifugally collecting precipitates, washing the precipitates with water, and finally re-dispersing the precipitates in a PBS buffer solution to obtain an Au NBPs @4-ATP/SEC-2 solution;

(3) constructing a fluorescence Raman double spectrum analysis method:

mixing the Au NBPs @4-ATP/SEC-2 solution with the prepared SEC standard solution, and incubating for 5-6h at room temperature; then adding ZGGO (chromium oxide), Cr, Er and Yb/SEC-1 solution, and incubating for 5-6h at room temperature; after the reaction is finished, a 250-one 260nm ultraviolet lamp is used for irradiation, the luminescence spectrum of each group of solution is tested, the logarithm of the SEC concentration is taken as the abscissa, and the luminescence recovery degree F-F of the probes ZGGO, Cr, Er, Yb/SEC-1 is detected₀A standard curve of ordinate; testing the Raman spectrum of each group of solution to obtain Raman signal change degree I-I by taking SEC concentration logarithm as abscissa and detecting probe Au NBPs @4-ATP/SEC-2₀A standard curve on the ordinate, in which F represents the fluorescence intensity in the presence of enterotoxin; i represents the Raman intensity in the presence of enterotoxin; f₀Represents the fluorescence intensity in the absence of enterotoxin; i is₀Representing the raman intensity in the absence of enterotoxin.

3. The method for enterotoxin spectroscopic analysis based on fluorescence and raman dual signal enhancement of claim 2 wherein said Cr (NO) in step (1) ①₃)₂、Zn(NO₃)₂、Yb(NO₃)₂、Er(NO₃)₂Ammonium germanate and Ga (NO)₃)₂The molar ratio of 0.004-0.006:1.2-1.4:0.02-0.03:0.002-0.003:0.2-0.3: 1.4-1.6; CTAB is used in an amount of 15-17 mg.

4. The enterotoxin spectroscopic analysis method based on fluorescence and Raman dual signal enhancement as claimed in claim 2, wherein the mass ratio of ZGGO: Cr, Er, Yb NPs and solid NaOH in step (1) is 8-12:0.48-1.2, and the volume ratio of N, N-dimethylformamide solvent to APTES solution is 3-5: 0.02-0.08.

5. The enterotoxin spectroscopic analysis method based on fluorescence and raman double signal enhancement of claim 2 wherein the mass concentration of glutaraldehyde in step (1) ③ is 20% -25%, NaBH₄The concentration of the solution is 24-26mM, the concentration of the BSA solution is 8-10mg/mL, the concentration of the SEC-1 solution is 10-14 mu M, and the aminated ZGGO is Cr, Er, Yb NPs solution, glutaraldehyde and NaBH₄The volume ratio of the solution, the SEC-1 solution and the BSA solution is 1-2:1-2:2-4:5-6: 8-10.

6. The method for enterotoxin spectroscopic analysis based on fluorescence and raman double signal enhancement of claim 2 wherein the HAuCl used in the preparation of the gold seed solution in step (2) ①₄CTAC, citric acid solution and NaBH₄In a molar ratio of 2.2 to 2.5: 495-505: 45-55: 6.1-6.5; CTAB and HAuCl in preparation of Au NBPs₄、AgNO₃The molar ratio of HCl to AA is 8-12:0.04-0.06:0.0008-0.0012:1-3: 0.07-0.09.

7. The enterotoxin spectroscopic analysis method based on fluorescence and raman double signal enhancement as claimed in claim 2, wherein the molar ratio of Au NBPs to 4-ATP in step (2) is 1: 50-1: 60.

8. the fluorescence raman duplex spectroscopic analysis method for detecting enterotoxin according to claim 2, wherein the SEC-2 solution concentration in the third step (2) is 10 to 14 μ M; the molar ratio of the SEC-2 solution to the Au NBPs @4-ATP solution is 1:100-1: 110.

9. The enterotoxin spectroscopic analysis method based on fluorescence and raman double signal enhancement as set forth in claim 2, wherein the concentration of the PBS buffer solution in steps (1), (2) and (3) is 8-12mM, and the pH is 7.2-7.6; TBE buffer concentration is 10-15. mu.M.

Technical Field

The invention belongs to the technical field of spectral analysis, and particularly relates to a fluorescence Raman dual spectral analysis technology for detecting enterotoxin.

Background

Enterotoxin is widely existed in nature, is an important clinical pathogenic bacterium, and can cause diseases such as bacterial food poisoning, bacteremia, skin tissue infection and the like. Enterotoxin infections are a worldwide public health problem due to their high thermal stability and pathogenicity. Current methods for enterotoxin detection are: mass spectrometry, PCR, electrochemical immunoassay, enzyme-linked immunoassay, etc., but has the disadvantages of expensive reagent equipment, time consumption, poor reproducibility, etc. Therefore, the development of a sensor capable of accurately, sensitively and specifically detecting the SEC content is urgently needed.

In recent years, nano materials have been rapidly developed in the fields of chemical engineering, biology and environmental science due to their unique optical, electrical, thermal and catalytic properties. The nano material is obviously superior to other traditional materials due to the dielectric confinement effect, the quantum size effect and the surface effect. The long afterglow nano material is a photoluminescence nano material which stores energy under the excitation light and can slowly release energy in the form of light after the excitation light is removed. The long afterglow material can effectively eliminate the autofluorescence background and light scattering interference in the detection environment because the in-situ excitation is not involved. Plasmonic metal nanomaterials, such as gold and silver nanomaterials, exhibit strong light absorption and light scattering in the visible region due to the Localized Surface Plasmon Resonance (LSPR) effect. In addition, the biosensor constructed by the nano material has sensitive reaction, good selectivity and low detection limit due to good biocompatibility and low cytotoxicity. Nanocomposites resulting from self-assembly of multiple component materials exhibit more excellent properties than single component nanomaterials. Therefore, the development of the composite material based on the long afterglow material and the plasma metal material has important significance and application prospect.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an enterotoxin spectral analysis method based on fluorescence and Raman double-signal enhancement. The method provided by the invention can effectively eliminate autofluorescence interference, improves the detection accuracy, and has high sensitivity and specificity.

The technical scheme of the invention is as follows:

a fluorescence Raman double spectrum analysis method for detecting enterotoxin specifically comprises the following steps: and (2) carrying out surface enterotoxin antibody modification on the long-afterglow nano particles to obtain ZGGO (chromium, Er, Yb/SEC-1) solution, mixing with Au NBPs @4-ATP/SEC-2 solution obtained by carrying out surface enterotoxin antibody modification on the gold cone nano particles, then adding enterotoxin SEC, uniformly mixing, and respectively measuring the fluorescence spectrum and the Raman spectrum of the solution.

The analysis method comprises the following specific steps:

(1) preparing long afterglow nano particle ZGGO, Cr, Er, Yb NPs and modifying surface SEC-1:

preparing long afterglow nano particle ZGGO, Cr, Er and Yb NPs:

mixing Cr (NO)₃)₂，Zn(NO₃)₂，Yb(NO₃)₂，Er(NO₃)₂With ammonium germanate solution to Ga (NO)₃)₂Uniformly mixing the solution to obtain a solution 1, then adding CTAB into the solution 1, adjusting the pH value of the solution to 7-9, performing hydrothermal reaction after uniform mixing, and heating at 110-130 ℃ for 14-16 h; cooling to room temperature, carrying out solid-liquid separation to obtain a precipitate, washing and drying to obtain the long-afterglow nano particles ZGGO, Cr, Er and Yb NPs;

② amination of long afterglow nano particle ZGGO, Cr, Er, Yb NPs: dispersing the obtained ZGGO, Cr, Er and Yb NPs into NaOH solution, uniformly mixing, carrying out solid-liquid separation to obtain precipitate, dispersing the precipitate into N, N-dimethylformamide reagent, adding 3-aminopropyltriethoxysilane APTES reagent, heating in 75-85 ℃ water bath, carrying out solid-liquid separation to obtain a solid phase after the reaction is finished, and dispersing the solid phase into water to obtain an aminated ZGGO, Cr, Er and Yb NPs solution;

③ Long persistence nanoparticle surface enterotoxin antibody (SEC-1) modification by glutaraldehyde and NaBH₄Adding the solution into an aminated ZGGO (chromium, iron and boron) NPs solution, stirring, carrying out solid-liquid separation, washing the precipitate, redissolving the precipitate by using a PBS (phosphate buffer solution), adding an SEC-1 solution, carrying out oscillation incubation, centrifuging, resuspending the product by using a BSA (bovine serum albumin) solution, storing at room temperature for 4-5 hours, collecting the precipitate after the reaction is finished, washing by using water, finally uniformly dispersing the precipitate in the PBS buffer solution again, and obtaining a ZGGO (chromium, iron and Yb/SCE-1 solution after the reaction is finished.

(2) Preparing a gold cone nano Au NBPs solution and modifying surface SEC-2:

① preparation of gold cone nano Au NBPs solution by adding HAuCl₄Cetyl trimethyl ammonium chloride CTAC, citric acid solution is addedNaBH₄Stirring the solution at room temperature for 1-3 min; heating the solution in 75-85 deg.C oil bath, stirring for 85-95min to obtain gold seed solution, adding CTAB, HAuCl₄，AgNO₃Adding the gold seed solution into a mixed solution of HCl and AA, and incubating for 1.8-2.2h to obtain a gold cone nano Au NBPs solution;

preparing Au NBPs @ 4-ATP: adding a 4-ATP solution into the gold cone nano Au NBPs solution, carrying out solid-liquid separation after the reaction is finished, taking a solid phase, washing with water, and dissolving the solid phase in water to obtain an Au NBPs @4-ATP solution;

③ modification of surface enterotoxin antibody SEC-2: adding enterotoxin antibody SEC-2 diluted by TBE buffer solution into AuNBPs @4-ATP solution for reaction, performing solid-liquid separation after the reaction is finished to obtain precipitate, using BSA solution to resuspend the precipitate, storing at room temperature for 2-3h, and blocking the binding site of unbound antibody; after the reaction is finished, centrifugally collecting precipitates, washing the precipitates with water, and finally re-dispersing the precipitates in a PBS buffer solution to obtain an Au NBPs @4-ATP/SEC-2 solution;

(3) constructing a fluorescence Raman double spectrum analysis method:

mixing the Au NBPs @4-ATP/SEC-2 solution with the prepared SEC standard solution, and incubating for 5-6h at room temperature; then adding ZGGO (chromium oxide), Cr, Er and Yb/SEC-1 solution, and incubating for 5-6h at room temperature; after the reaction is finished, a 250-one 260nm ultraviolet lamp is used for irradiation, the luminescence spectrum of each group of solution is tested, the logarithm of the SEC concentration is taken as the abscissa, and the luminescence recovery degree F-F of the probes ZGGO, Cr, Er, Yb/SEC-1 is detected₀A standard curve of ordinate; testing the Raman spectrum of each group of solution to obtain Raman signal change degree I-I by taking SEC concentration logarithm as abscissa and detecting probe Au NBPs @4-ATP/SEC-2₀A standard curve on the ordinate, in which F represents the fluorescence intensity in the presence of enterotoxin; i represents the Raman intensity in the presence of enterotoxin; f₀Represents the fluorescence intensity in the absence of enterotoxin; i is₀Representing the raman intensity in the absence of enterotoxin.

Preferably, the Cr (NO) in step (1) ①₃)₂、Zn(NO₃)₂、Yb(NO₃)₂、Er(NO₃)₂Ammonium germanate and Ga (NO)₃)₂The molar ratio of 0.004-0.006:1.2-1.4:0.02-0.03:0.002-0.003:0.2-0.3: 1.4-1.6; CTAB is used in an amount of 15-17 mg.

Preferably, the mass ratio of ZGGO to Cr, Er, Yb NPs to solid NaOH in the step (1) is 8-12:0.48-1.2, and the volume ratio of the N, N-dimethylformamide solvent to the APTES solution is 3-5: 0.02-0.08.

Preferably, the mass concentration of the glutaraldehyde in the step (1) ③ is 20-25%, and the NaBH is₄The concentration of the solution is 24-26mM, the concentration of the BSA solution is 8-10mg/mL, the concentration of the SEC-1 solution is 10-14 mu M, and the aminated ZGGO is Cr, Er, Yb NPs solution, glutaraldehyde and NaBH₄The volume ratio of the solution, the SEC-1 solution and the BSA solution is 1-2:1-2:2-4:5-6: 8-10.

Preferably, HAuCl used in the preparation of the gold seed solution as described in step (2) ①₄CTAC, citric acid solution and NaBH₄In a molar ratio of 2.2 to 2.5: 495-505: 45-55: 6.1-6.5; CTAB and HAuCl in preparation of Au NBPs₄、AgNO₃The molar ratio of HCl to AA is 8-12:0.04-0.06:0.0008-0.0012:1-3: 0.07-0.09.

Preferably, the molar ratio of Au NBPs to 4-ATP in the step (2) is 1: 50-1: 60.

preferably, the concentration of the SEC-2 solution in the third step (2) is 10-14 mu M; the molar ratio of the SEC-2 solution to the Au NBPs @4-ATP solution is 1:100-1: 110.

Preferably, the concentration of the PBS buffer solution in the steps (1), (2) and (3) is 8-12mM, and the pH is 7.2-7.6; TBE buffer concentration is 10-15. mu.M.

The beneficial technical effects of the invention are as follows:

SEC refers to enterotoxin antigen, SEC-1 and SEC-2 are primary antibody and secondary antibody of enterotoxin, wherein SEC-2 represents capture antibody, SEC-1 represents recognition antibody, and the antibodies are paired and recognized in the preparation process of the antibodies, so that a double-antibody sandwich structure is formed for assembly and detection.

The invention adopts the long afterglow nano particles ZGGO Cr, Er and Yb NPs with red emission as the luminescent material, and SEC-1 is modified on the surfaces of the ZGGO Cr, Er and Yb NPs in a mode of forming covalent bonds between aldehyde groups and amino groups to prepare the detection probe capable of specifically identifying enterotoxin. The surface of the gold cone is modified with a secondary antibody SEC-2 of a Raman beacon and enterotoxin, which is recorded as Au NBPs @ 4-ATP/SEC-2. In the presence of SEC, ZGGO-Cr, Er, Yb NPs form assemblies with Au NBPs @4-ATP due to specific recognition by antigen-antibody. The rigid structure of the antibody provides a proper distance between the gold cone and ZGGO, Cr, Er and Yb NPs, and the fluorescence of the ZGGO, Cr, Er and Yb NPs is enhanced; the charge transfer exists between ZGGO Cr, Er, Yb NPs and Au NBPs @4-ATP, the SERS signal is enhanced, and meanwhile, the electromagnetic field enhancement caused by the gold cone promotes the radiation attenuation rate of the fluorescent substance, so that the fluorescence is enhanced. The method can effectively eliminate autofluorescence interference, improves detection accuracy, and has high sensitivity and specificity.

The fluorescence Raman double spectrum analysis method for detecting SEC provided by the invention has very high sensitivity and specificity, in-situ excitation is not needed in the detection method, the background interference of autofluorescence in a detection environment can be eliminated, and the detection sensitivity is greatly improved; the long afterglow material can enhance the SERS enhancement effect of Au NBPs @4-ATP, Au NBPs @4-ATP can effectively enhance the fluorescence of long afterglow, the detection result of SEC can be reflected by fluorescence spectrum and Raman spectrum at the same time, and the long afterglow material can enhance the synergistic effect between Au NBPs @4-ATP and Au NBPs @4-ATP to further improve the detection accuracy.

Drawings

FIG. 1 is a TEM image of the long-afterglow nanoparticles ZGGO, Cr, Er and Yb NPs prepared in example 2;

FIG. 2 is a TEM image of Au NBPs of gold cone nanoparticles prepared in example 2;

FIG. 3 is a schematic diagram of the principle of a fluorescence Raman aptamer sensor for detecting SEC in example 2;

FIG. 4 is a fluorescence spectrum, a Raman spectrum and a calibration curve of the fluorescence Raman sensor in the presence of different concentrations of SEC in example 2.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings and examples.