CN114354574A - Multielement SERS biological detection method based on analog enzyme signal amplification - Google Patents
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Abstract
The invention relates to a multielement SERS biological detection method based on analog enzyme signal amplification in the technical field of biological material detection, and the detection method of the invention sequentially prepares SiO with different codes2And mixing the solid-phase immune substrate and the gold nanoparticles modified with different antibodies to prepare a sandwich immune sandwich structure microsphere mixed solution of the multi-component antigen marker, and performing surface enhanced Raman spectrum detection to perform a multi-element SERS biological detection method. In the detection method, the gold nanoparticles have excellent surface-enhanced Raman performance, and the inactive Raman reporter molecules can be oxidized into active Raman reporter molecules in the presence of hydrogen peroxide, so that the Raman signal of the surface-enhanced Raman probe is doubly enhanced, the detection sensitivity is further improved, the multi-element detection of different biomolecules is finally realized through the encoding/decoding of the silicon dioxide photonic crystal, and the high-sensitivity multi-element biological SERS detection is realized.
Description
Technical Field
The invention relates to the technical field of biological material detection, in particular to a multivariate SERS biological detection method based on analog enzyme signal amplification.
Background
Surface Enhanced Raman Scattering (SERS) is a powerful and highly sensitive analytical technique that can be used to perform detection without destroying the sample or adding reagents, and is now used in the fields of biochemistry, chemical molecule detection, drug analysis, biological detection, and the like. The multiplex immunoassay has a higher sample volume, less sample consumption, shorter detection time and lower cost than the conventional single molecule immunoassay. The multivariate bioanalysis method has wide application prospect in drug screening technology, gene function analysis and bioanalysis clinical diagnosis. However, the flow encoding carrier with single function has some defects, such as less encoding amount, difficult separation, low encoding stability and the like, so researchers can widen the application range of the flow encoding carrier by utilizing the properties of different materials. With the rapid development of nano science and material preparation technology, the SERS technology is also continuously advancing. However, the single enhancement effect of SERS substrate materials is no longer satisfied. Therefore, it becomes very important to prepare a SERS enhancing substrate which has high activity and can realize multiplex analysis.
Enzyme-linked immunosorbent assay (ELISA) is an analysis means with high sensitivity, simple operation and strong specificity, and is widely applied to the fields of early diagnosis of tumors, environmental analysis, food control and the like at present. However, the traditional enzyme-antibody conjugate can reduce the catalytic activity of enzyme labeling and the capture efficiency of the antibody to the antigen, so that the detection sensitivity of immunoassay is relatively low. In addition, the enzyme label, as a natural protein, is also less stable under harsh conditions. Therefore, the catalytic nano material or the composite material thereof is necessary to replace the problem of instability of natural enzymolysis.
The gold nanoparticles have extremely strong SERS performance and catalytic performance, and researches show that the gold nanoparticles are hidden days in artificial mimic enzymes, including mimic nucleases, esterase, glucose oxidase, peroxidase, superoxide dismutase and the like, and the gold nanoparticles are used for simulating the peroxidase to carry out integrin detection and the like. On the other hand, the local surface plasmon resonance characteristic enables the gold nanoparticles to have obvious electromagnetic field enhancement, and the SERS performance can be improved. Therefore, the combination of SERS activity and catalytic performance of gold nanoparticles will provide a special platform for the development of sensitive biological assays, which have been successfully used to develop active substrates for sensitive chemical and biomedical assays.
Recent studies show that the invention can be utilized to provide a multi-element biological SERS detection method based on analog enzyme signal amplification, which will promote a high-activity multi-component SERS biological analysis technology. The oxidation and reduction processes of the nitrophenol are monitored. Although plasma nanosensors for DNA detection have been successfully constructed using gold nanoparticles as oxidase mimics, the enzyme mimicking properties of gold nanoparticles and the combination of their SERS activity in biological detection remain to be explored.
Disclosure of Invention
Aiming at the problems of enzyme simulation characteristics of gold nanoparticles and SERS activity of the gold nanoparticles in the prior art in biological detection, the invention provides a multi-element SERS biological detection method based on simulated enzyme signal amplification, and realizes high-activity multi-component SERS biological detection.
The invention aims to realize the method, and the method is characterized by comprising the following steps of:
1) preparation of SiO2Solid-phase immunization substrate:
1.1) sampling SiO of different codes respectively2Photonic crystal encoded microspheres with addition of H2SO4And H2O2Soaking in the mixed solution for 10-16 h, filtering out microspheres, washing the silk yarns with ultrapure water, and then using N2Drying to obtain functional microspheres for modifying hydroxyl groups, adding a toluene solution with the mass concentration of 1% GPTMS into the functional microspheres, soaking for 10-16 h to silanize the microspheres, washing with toluene and absolute ethyl alcohol, and then washing with N2Blow drying to obtain activated SiO with different codes2Functional microspheres;
1.2) preparing each antibody according to a code: separately encoding each activated SiO2Adding a phosphate buffer solution of a corresponding antibody with the concentration of 0.5 mg/mL into the functional microspheres, placing the functional microspheres in a shaking table for 2 hours at 37 ℃, then standing the functional microspheres in a refrigerator at 4 ℃ for 10 to 16 hours, and finally washing the functional microspheres for 3 times by using the phosphate buffer solution; adding 1% bovine serum albumin, sealing for more than 2h at room temperature, cleaning the sealed microspheres with phosphate buffer solution, and storing to obtain Si with different codesO2A solid phase immunization substrate;
2) preparing gold nanoparticles for modifying antibodies: mixing a phosphate buffer solution with the antibody concentration of 0.5-2.5 mg/mL and a phosphate buffer solution with the concentration of 0.5-2.5 mg/mL of gold nanoparticles, placing the mixture in a shaking table at 37 ℃ for reaction for 2 hours, and then, standing the mixture in a refrigerator at 4 ℃ overnight; adding bovine serum albumin solution with the mass concentration of 1% and sealing for 2 hours at room temperature to obtain antibody-modified gold nanoparticles;
3) the multielement SERS biological detection method comprises the following steps: preparing n parts of a series of antigen mixed solutions with different concentrations by taking phosphate buffer solution as a solvent, and mixing each code SiO obtained in the step 1.2)2Respectively and simultaneously mixing the solid-phase immune substrate with An antigen mixed solution with the same concentration according to the proportion of 2-20 mg/mL to obtain a mixed solution A1, respectively sampling the antigen solution with each concentration to repeatedly prepare a mixed solution A2-An, respectively adding the step 2) to obtain the gold nanoparticles for modifying the antibody after the mixed solution is subjected to warm bath reaction to respectively form a sandwich immune sandwich structure microsphere mixed solution, washing for 3 times by using a phosphate buffer solution after the warm bath reaction, and adding H2O2And after the mixed solution of the gold nanoparticles and the TMB is subjected to molecular activation, the mixed solution is placed under a laser confocal Raman spectrometer, the light path is switched, and the SiO is amplified through double SERS signals of the gold nanoparticles2And detecting and decoding the Raman signal on the surface of the photonic crystal coding microsphere, determining the type of the biomolecule antigen to be detected through the coding information, and determining the concentration of the biomolecule antigen to be detected by using the detection Raman signal information.
Further, step 1.1) H2SO4And H2O2According to the volume ratio of 7: 3-5 mixing, wherein H2SO4The mass concentration of the SiO is 95-98 percent2Photonic crystal microspheres and H2SO4And H2O2The dosage proportion of the mixed solution is 5-20 mmg/mL; activated SiO2The dosage ratio of the functional microspheres to the phosphate buffer solution with the antibody concentration of 0.5 mg/mL is 30-50 mg/mL.
Further, in step 1.1), SiO2The photonic crystal encoded microspheres are prepared byThe preparation method comprises the following steps:
monodisperse SiO with the particle size range of 230-280 nm2Dispersing nano particles in deionized water, and collecting SiO in dimethyl silicone oil by using a co-flow type micro-fluidic device2Dripping, collecting the sedimentation layer after all the dripping subsides, standing in a 60 ℃ oven for 2-3 days, evaporating the water in the dripping to form SiO2Photonic crystal microspheres; then SiO2Washing the photonic crystal microspheres with n-hexane and ethanol in sequence, naturally drying the washed photonic crystal microspheres, and calcining the photonic crystal microspheres in a muffle furnace at a high temperature of 750 ℃ for 2 hours to obtain SiO with different particle size and different codes2The photonic crystal encodes microspheres.
Further, in the step 2), the particle size of the gold nanoparticles is 10-30 nm.
Further, in the step 2), the volume ratio of the antibody solution of the phosphate buffer solution to the phosphate buffer solution of the gold nanoparticles is 1: 1-3.
Further, the dosage ratio of the bovine serum albumin solution to the sealed mixed solution in the steps 1) and 2) is 1-3: 1.
Further, the concentration ratio of the gold nanoparticles for modifying the antibody added in the step 3) to the phosphate buffer solution is 0.5-2.5 mmol/L, and the mixing ratio of the gold nanoparticles to the mixed solution A is as follows: 1:1
And further. In the step 3), the temperature of the medium-temperature bath reaction is 25-37 ℃, the time of the medium-temperature bath is 30-60 min, and the activation time is 5-30 min.
Preferably, in step 3), the antigen of the biomolecule to be detected is CEA, AFP, IgG, CA125, CA199, CA211 and/or CA 724.
Preferably, step 3), H2O2And the concentration of the Raman reporter molecule in the mixed solution of the Raman reporter molecule and the TMB is 10- 4moL/L, and the volume ratio of the microsphere mixed solution relative to the sandwich immune sandwich structure is 1: 1.
The invention has the beneficial effects that: the hydrogen peroxide of the gold nano-particles simulates the enzyme performance and SiO2The coding characteristics of the photonic crystal are combined, and meanwhile SERS detection is carried out on the multi-element biomolecules. Modifying antibodies to have coding propertiesOf (2) functionalized SiO2The sandwich immune structure is prepared by adding gold nanoparticles modified with different antibodies and antigens to be detected on the surface of the photonic crystal microsphere. On one hand, the gold nanoparticles have SERS performance; on the other hand in the presence of H2O2Under the condition, the gold nanoparticles oxidize the inactive Raman reporter molecules into active Raman reporter molecules, so that the effect of Raman dual signal enhancement is achieved, and the Raman detection sensitivity is further improved. Finally, SiO is reused2And decoding the reflection peak of the photonic crystal and the polarization microscope photo to realize the multielement SERS biological detection. The method is simple to operate, has good anti-interference performance, and keeps higher consistency with the detection result of an actual sample.
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FIG. 1 shows that the SiO particles obtained in step 1 of example 1 have a particle size of 230nm and 280nm2The scanning electron micrograph, the corresponding reflection spectrogram and the microscope picture of the coding solid-phase immune substrate; wherein 1a is SiO with the grain diameter of 230nm2CEA antibody immune substrate of coded photonic crystal, 1b is SiO with particle size of about 28 nm2AFP antibody immune substrates encoding photonic crystals.
FIG. 2 is a TEM image of gold nanoparticles of step (2) of example 1.
FIG. 3 is a TMB Raman spectrum of the gold nanoparticles of step (2) in example 1 with hydrogen peroxide mimicking enzyme performance; wherein 3a is a SERS Raman spectrogram of TMB; 3b is SERS Raman spectrogram of gold nanoparticles and TMB; 3c Picture gold nanoparticles + TMB + H2O2SERS spectrum of (a).
FIG. 4 is a SERS Raman spectrum and a fitted curve graph of the SERS intensity variation versus antigen concentration for each concentration of 2 different tumor markers in example step (4).
Wherein FIGS. 4a and 4c are SiO2The photonic crystal encoded microspheres detect SERS Raman spectrograms of CEA (a) and AFP (c) with different concentrations; the b and d figures are at 1605 cm-1The peak is a fitting curve graph of SERS intensity change of CEA and AFP obtained by reference peak.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in detail below with reference to examples.
Example 1
(1) Preparation of SiO2Photonic crystal encoded microspheres:
monodisperse SiO2Dispersing nano particles in deionized water, and collecting SiO in dimethyl silicone oil by using a co-flow type micro-fluidic device2Liquid drops, collecting and standing in a 60 ℃ oven for 2-3 days after all the liquid drops are settled, and evaporating water in the liquid drops to form SiO2Photonic crystal microspheres. Take out SiO2Cleaning the photonic crystal microspheres, naturally drying the microspheres, and calcining the microspheres in a muffle furnace at a high temperature of 750 ℃ for 2 hours to obtain SiO2The photonic crystal coding microspheres in the embodiment respectively obtain SiO with two codes with the grain sizes of 230nm and 280nm2The photonic crystal encodes microspheres.
(2) Preparation of the solid phase immune substrates for different codes: a plurality of 0.0075 g of the two encoded SiO samples were taken2H with the mass fraction of 98 percent is added into the photonic crystal microspheres2SO4Solution and H2O21mL (wherein, H)2SO4/H2O2Volume ratio of 7:3), soaking for 12h, washing with ultrapure water for 3 times, and then using N2Blow-drying to obtain the SiO modified with hydroxyl groups2Photonic crystal microspheres; then 1mL of 1% GPTMS toluene solution is added into each part, the mixture is soaked for 12 hours to be silanized, and then the microspheres are washed three times by toluene and absolute ethyl alcohol respectively and then are washed by N2Blow-drying to respectively obtain the functional SiO modified with the activated epoxy group2Photonic crystal microspheres; finally sampling 230nm of functionalized SiO in each part2200 mu L of phosphate buffer solution (0.5 mg/mL) of CEA coating antibody is respectively added into the photonic crystal microspheres, and 280nm of functionalized SiO for each sampling2Respectively adding 200 mu L phosphate buffer solution (0.5 mg/mL) of AFP coating antibody into the photonic crystal microspheres, placing the photonic crystal microspheres in a shaking table, reacting for 2h at 37 ℃, standing for 12h in a refrigerator at 4 ℃, and respectively modifying two types of coating antibodies on SiO with functionalized different codes2The surface of the photonic crystal microsphere; taking out the microsphere sample from the refrigerator, washing the microsphere sample with phosphate buffer solution, adding 100 mu L of 1% bovine serum albumin, and sealing at room temperature for 2h to obtain SiO of solid-phase immune substrates with different codes2And (4) respectively cleaning the microspheres with phosphate buffer solution for later use. Two coded solid-phase immune-based SiO of this step2The scanning electron micrograph, the corresponding reflection spectrum and the microscope of the microsphere are shown in FIG. 1, wherein the left image of FIG. 1a is SiO with a particle size of 230nm2Electron microscope scanning image of CEA antibody immune base of coded photonic crystal, the curve of right reflection spectrum is the central green photograph under the polarizing microscope when the reflection peak position is about 510 nm, FIG. 1b is SiO with particle size of 280nm2The curve of the reflection spectrum of the AFP antibody immune base of the coded photonic crystal is a photograph with red center under a polarizing microscope when the position of a reflection peak is about 600 nm.
(3) Preparing gold nanoparticles for modifying different antibodies: respectively sampling 200 mu L of phosphate buffer solution of CEA-labeling antibody with the concentration of 0.5 mg/mL and 200 mu L of phosphate buffer solution of AFP-labeling antibody with the concentration of 0.5 mg/mL, respectively adding 200 mu L of prepared gold nanoparticle phosphate buffer solution (0.5 mg/mL) with the particle size of 10-30 nm into each solution, placing the solutions in a shaking table, reacting for 2 hours at 37 ℃, standing for 12 hours in a refrigerator environment at 4 ℃, then taking out a standing sample, centrifuging to remove unreacted antibody, adding 100 mu L of 1% bovine serum albumin, sealing for 2 hours at room temperature, and respectively obtaining two kinds of gold nanoparticles modifying different antibodies. As shown in FIG. 2, a TEM image of the gold nanoparticles of this example is shown.
FIG. 3 is a Raman spectrum of the hydrogen peroxide mimic enzyme performance of the gold nanoparticles in this example.
Wherein curve a is a concentration of 10-4Raman signal curve spectrum of the TMB solution of mol/L, curve b is Raman signal curve spectrum of the gold nano-particle added into the TMB solution, curve c is continuous H addition2O2The Raman signal curve atlas shows that the Raman signal generated by the independent TMB is very weak in the TMB and the gold nano-particles from the curves a, b and c of the pictureThe Raman signal generated by the sub-mixed solution is enhanced to a certain extent, because the gold nanoparticles have SERS performance, the Raman signal of TMB can be enhanced; at H2O2Under the existing condition, the gold nanoparticles can oxidize the inactive TMB into the active TMB, thereby greatly enhancing the Raman signal of the TMB. In addition, when the gold nanoparticles and TMB are separately mixed in the test, the color of the mixed solution is pink; once H is added2O2Then, the gold nanoparticles exert the hydrogen peroxide mimic enzyme performance, and the color is changed into purple.
(4) SERS detection of multi-component tumor markers (biomolecules or antigens): respectively preparing 1mL of a series of antigen concentrations of 1 × 10 with phosphate buffer solution as solvent-3ng/mL、1×10-2 ng/mL、1×10-1 ng/mL、1ng/mL、10 ng/mL、1×102 ng/mL、1×103ng/mL of a mixed solution of CEA and AFP antigen, wherein the mass ratio of the CEA to the AFP antigen in the mixed solution of each concentration is 1: 1; SiO two different coded antibodies prepared in the step 2)2Simultaneously placing photonic crystal microspheres (the sampling amount of each microsphere is 0.0075 g) in a mixed solution with each antigen concentration, carrying out warm bath reaction on the mixed solutions at 37 ℃ for 1 h, and cleaning the microspheres with a phosphate buffer solution for later use; adding the two gold nanoparticles modified with the corresponding antibodies prepared in the step (3) into each mixed solution respectively to obtain a microsphere mixed solution with a sandwich immune sandwich structure, carrying out warm bath reaction at 37 ℃ for 60min, washing for 3 times by using a phosphate buffer solution, and then dropwise adding 1mL of H2O2And (TMB concentration of 10)-4mol/L) is activated for 30 min at 37 ℃, and then SERS detection is carried out immediately, the SERS detection spectra corresponding to antigens with different concentrations are shown in figures 4a and 4c, and figures 4b and 4d are shown in 1605 cm corresponding to different antigen concentrations-1The peak is a standard curve graph of the CEA and AFP concentration and SERS intensity change obtained by reference peaks. As can be seen from the figure, the detectable antigen concentration range of the method is wide, and the linear fitting relation is good.
Tumor markers containing unknown concentrations of at least one of the two antigens were performed according to the methods of this exampleThe SERS detection of the object directly adopts the products prepared in the processes (1), (2) and (3) to prepare the microsphere mixed solution with a sandwich structure from the unknown tumor marker antigen solution according to the process of the embodiment (4), and the mixture is subjected to warm bath, washing and dropwise adding H2O2And TMB (concentration of 10)-4mol/L) mixed solution, activating for 30 min at 37 ℃, then performing SERS detection and decoding immediately, determining the type of the tumor marker antigen according to decoding information, and determining the concentration of the antigen according to the intensity of a Raman signal detected by SERS, thereby realizing the detection of multiple biological molecule antigens with unknown concentration, namely the tumor marker.
Example 2:
(1) preparation of SiO2Photonic crystal encoded microspheres:
as in the preparation method of example 1, two kinds of encoded SiO with particle sizes of 230nm and 280nm were prepared respectively2The photonic crystal encodes microspheres.
(2) Preparation of solid-phase immune substrate: a plurality of 0.0075 g of the two encoded SiO samples were taken2H with the mass fraction of 98 percent is added into the photonic crystal microspheres2SO4And H2O21mL of the mixed solution (wherein H2SO4And H2O2The volume ratio is 7:5), soaking for 10h, then washing with ultrapure water for three times, and then using N2Blowing dry and modifying the SiO with hydroxyl groups2Photonic crystal microspheres. Adding 1mL of 1% GPTMS toluene solution, soaking for 12h to silanize, washing with toluene and anhydrous ethanol for three times, and adding N2Drying and modifying the activated epoxy group. Finally 230nm functionalized SiO2200 mu L of phosphate buffer solution (0.5 mg/mL) of CA125 coating antibody is respectively added into the photonic crystal microspheres, and the mixture is functionalized into SiO at 2800nm2Respectively adding 200 mu L of phosphate buffer solution (0.5 mg/mL) of CA199 coating antibody into the photonic crystal microspheres, placing the photonic crystal microspheres in a shaking table, reacting for 2h at 37 ℃, standing overnight at 4 ℃, and respectively modifying the coating antibody on different codes of functionalized SiO2The surface of the photonic crystal microsphere; taking out the microspheres, washing with phosphate buffer solution, and adding 100 μ L of 1% bovine serumSealing the protein at room temperature for 2h, and cleaning the sealed microspheres with phosphate buffer solution for later use.
(3) Preparing gold nanoparticles for modifying antibodies: respectively sampling 200 mu L of phosphate buffer solution of antibody for CA125 labeling with the concentration of 2.5mg/mL and 200 mu L of phosphate buffer solution (0.5 mg/mL) of antibody for CA199 labeling with the concentration of 2.5mg/mL, respectively adding 400 mu L of prepared gold nanoparticle phosphate buffer solution (2.5 mg/mL) into each solution, placing the solutions in a shaking table, reacting for 2 hours at 37 ℃, standing overnight at 4 ℃, centrifuging the next day to remove unreacted antibody, and adding 100 mu L of 1% bovine serum albumin for sealing for 2 hours at room temperature;
(4) SERS detection of multi-component tumor markers: a series of different concentrations (1X 10) with a volume of 1mL are prepared-4~1×102ng/mL) mixed solution of CA125 and CA199 antigens, (wherein the mass ratio of the CA125 to the CA199 antigens in the mixed solution of each concentration is 1: 1) two different encoded antibody-immobilized SiO prepared in (2)20.0075 g of each photonic crystal microsphere is placed in the mixed antigen solution, the mixture is subjected to warm bath reaction for 50 min at the temperature of 30 ℃, and the microspheres are washed by phosphate buffer washing liquid for later use. Adding the gold nanoparticles modified with the corresponding antibody prepared in the step (3) to obtain a microsphere mixed solution with a sandwich structure, carrying out warm bath reaction for 50 min at 30 ℃, washing for 3 times by using a phosphate buffer solution, and dropwise adding 1mL of H2O2Mixed solution with TMB (wherein the concentration of TMB is 10)-4mol/L) and activating for 25 min at 37 ℃, and then performing SERS detection to obtain a SERS detection spectrum similar to that of figure 4 and a standard curve graph of antigen concentration and SERS intensity change, and performing detection and decoding of CA125 and CA199 markers with unknown concentrations.
The present invention is not limited to the modification of the antibody and the detection of the antibody described in the above examples, and is also not limited to the simultaneous detection of the concentrations of two marker molecules. For example, in the step (1), three or more kinds of SiO having different particle diameters can be prepared2Photon crystal coding microspheres, wherein in the step (2), each coding microsphere is modified with an antibody, in the step (3), gold nanoparticles corresponding to the antibodies are respectively modified, and then the preparation is carried outAnd (4) performing SERS detection and decoding on the corresponding multi-component sandwich immune sandwich microspheres in the step (4) to detect the corresponding multi-component marker molecule concentration.
Claims (10)
1. A multivariate SERS biological detection method based on analog enzyme signal amplification is characterized by comprising the following steps:
1) preparation of SiO2Solid-phase immunization substrate:
1.1) sampling SiO of different codes respectively2Photonic crystal encoded microspheres with addition of H2SO4And H2O2Soaking in the mixed solution for 10-16 h, filtering out microspheres, washing the silk yarns with ultrapure water, and then using N2Drying to obtain functional microspheres for modifying hydroxyl groups, adding a toluene solution with the mass concentration of 1% GPTMS into the functional microspheres, soaking for 10-16 h to silanize the microspheres, washing with toluene and absolute ethyl alcohol, and then washing with N2Blow drying to obtain activated SiO with different codes2Functional microspheres;
1.2) preparing each antibody according to a code: separately encoding each activated SiO2Adding a phosphate buffer solution of a corresponding antibody with the concentration of 0.5 mg/mL into the functional microspheres, placing the functional microspheres in a shaking table for 2 hours at 37 ℃, then standing the functional microspheres in a refrigerator at 4 ℃ for 10 to 16 hours, and finally washing the functional microspheres for 3 times by using the phosphate buffer solution; adding 1% bovine serum albumin, sealing for more than 2h at room temperature, cleaning the sealed microspheres with phosphate buffer solution, and storing to obtain SiO with different codes2A solid phase immunization substrate;
2) preparing gold nanoparticles for modifying antibodies: mixing a phosphate buffer solution with the antibody concentration of 0.5-2.5 mg/mL and a phosphate buffer solution with the concentration of 0.5-2.5 mg/mL of gold nanoparticles, placing the mixture in a shaking table at 37 ℃ for reaction for 2 hours, and then, standing the mixture in a refrigerator at 4 ℃ overnight; adding bovine serum albumin solution with the mass concentration of 1% and sealing for 2 hours at room temperature to obtain antibody-modified gold nanoparticles;
3) the multielement SERS biological detection method comprises the following steps: respectively preparing a series of n parts of antigen mixed solution with different concentrations by using phosphate buffer solution as a solvent, and carrying out the step 1.2)Each of the obtained encoded SiO2Respectively and simultaneously mixing the solid-phase immune substrate with An antigen mixed solution with the same concentration according to the proportion of 2-20 mg/mL to obtain a mixed solution A1, respectively sampling the antigen solution with each concentration to repeatedly prepare a mixed solution A2-An, respectively adding the step 2) to obtain antibody-modified gold nanoparticles after the mixed solution is subjected to warm bath reaction to respectively form a sandwich immune sandwich structure microsphere mixed solution, washing for 3 times by using a phosphate buffer solution after the warm bath reaction, and adding H2O2After the mixed solution of the gold nanoparticles and the TMB is subjected to molecular activation, the mixed solution is placed under a laser confocal Raman spectrometer, the light path is switched, and the SiO is amplified through double SERS signals of the gold nanoparticles2And detecting and decoding the Raman signal on the surface of the photonic crystal coding microsphere, determining the type of the biomolecule antigen to be detected through the coding information, and determining the concentration of the biomolecule antigen to be detected by using the detection Raman signal information.
2. The multivariate SERS bioassay method based on mimetic enzyme signal amplification as claimed in claim 1, wherein H in step 1.1)2SO4And H2O2According to the volume ratio of 7: 3-5 mixing, wherein H2SO4The mass concentration of the SiO is 95-98 percent2Photonic crystal microspheres and H2SO4And H2O2The dosage proportion of the mixed solution is 5-20 mmg/mL; activated SiO2The dosage ratio of the functional microspheres to the phosphate buffer solution with the antibody concentration of 0.5 mg/mL is 30-50 mg/mL.
3. The multivariate SERS bioassay method based on mimic enzyme signal amplification as claimed in claim 1, wherein in step 1.1), SiO is used2The photonic crystal coding microsphere is prepared by the following method:
monodisperse SiO with the particle size range of 230-280 nm2Dispersing nano particles in deionized water, and collecting SiO in dimethyl silicone oil by using a co-flow type micro-fluidic device2Dripping, collecting the sedimentation layer after all the dripping subsides, standing in a 60 deg.C oven for 2-3 days, and steamingThe water in the liquid drops is released to form SiO2Photonic crystal microspheres;
then SiO2Washing the photonic crystal microspheres with n-hexane and ethanol in sequence, naturally drying the washed photonic crystal microspheres, and calcining the photonic crystal microspheres in a muffle furnace at a high temperature of 750 ℃ for 2 hours to obtain SiO with different particle size and different codes2The photonic crystal encodes microspheres.
4. The multivariate SERS biological detection method based on mimetic enzyme signal amplification as recited in claim 1, wherein in the step 2), the particle size of the gold nanoparticles is 10-30 nm.
5. The multivariate SERS biological detection method based on mimetic enzyme signal amplification as recited in claim 1, wherein in the step 2), the volume ratio of the antibody solution of the phosphate buffer solution to the phosphate buffer solution of the gold nanoparticles is 1: 1-3.
6. The multivariate SERS biological detection method based on mimetic enzyme signal amplification as recited in claim 1, wherein the ratio of the bovine serum albumin solution to the sealed mixed solution in step 1) and step 2) is 1-3: 1.
7. The multivariate SERS biological detection method based on mimetic enzyme signal amplification as recited in claim 1, wherein the concentration ratio of the gold nanoparticles of the modified antibody added in step 3) to the phosphate buffer is 0.5-2.5 mmol/L, and the volume mixing ratio of the gold nanoparticles to the mixed solution A is: 1: 1.
8. the multivariate SERS biological detection method based on simulated enzyme signal amplification as recited in claim 1, wherein in the step 3), the temperature of the warm bath reaction is 25-37 ℃, the time of the warm bath is 30-60 min, and the activation time is 5-30 min.
9. The multivariate SERS bioassay method based on mimetic enzyme signal amplification as recited in claim 1, wherein in step 3), the biomolecule antigen to be detected is CEA, AFP, IgG, CA125, CA199, CA211, and/or CA 724.
10. The multivariate SERS bioassay method based on mimetic enzyme signal amplification as claimed in claim 1, wherein in step 3), H)2O2And the concentration of the Raman reporter molecule in the mixed solution of the Raman reporter molecule and the TMB is 10-4moL/L, and the volume ratio of the microsphere mixed solution relative to the sandwich immune sandwich structure is 1: 1.
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| CN115097038A (en) * | 2022-06-22 | 2022-09-23 | 山东国仓健生物科技有限公司 | Screening and identifying method and application of soybean phytophthora root rot-resistant related metabolites |
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