CN114136952B - Multi-element SERS biological detection method - Google Patents
Multi-element SERS biological detection method Download PDFInfo
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Abstract
The invention relates to a method for multi-element SERS biological detection, belonging to the technical field of biomedical research and clinical detection. According to the invention, hydrogel photonic crystals with different reflection peaks are respectively modified with different antibodies as substrates, and combined with silver nanoparticles with strong SERS signals to prepare the antibody/4-MBA/Ag nanoparticles as SERS immune probes, and finally the antibody/4-MBA/Ag nanoparticles are added into a multi-component antigen to be detected to form a sandwich composite structure of a solid-phase immune substrate, a corresponding antigen and the SERS immune probes. The kind of the antigen to be detected is determined by detecting the reflection peak position of the hydrogel photonic crystal encoded microsphere and a polarized light microscopic photograph, and finally, the simultaneous detection of various antigens is realized. The inverse opal hydrogel photonic crystal prepared by the invention has the advantages of stable coding, good biocompatibility, large specific surface area and the like, and the proposed multielement detection method has high sensitivity and small cross interference.
Description
Technical Field
The invention relates to the technical field of biomedical research and clinical detection, in particular to a method for performing multi-element SERS biological detection by utilizing hydrogel photonic crystal encoding microspheres with inverse opal structures.
Background
At present, the surface enhanced Raman spectrum has the advantages of high sensitivity, extremely high detection speed, low detection limit, no sample damage and the like, and is widely applied to the fields of environmental monitoring, biochemistry, imaging, chemical molecular detection and sensor application. In particular, in recent years, multiplex biological detection by SERS has become a hot spot and a difficult point of research.
In view of the current state of research, multicomponent bioassays principally achieve multicomponent detection through a variety of raman labeling modes and raman imaging. The multiple tag mode requires different tag molecules, which reduces the analytical throughput of the multi-component raman detection, while the raman imaging instrument is expensive, limiting its practical application. The photon crystal coding microsphere has the advantages of simple preparation, stable coding, simple decoding and the like, and can be used in the field of multi-element detection. At present, the multi-element biological analysis and detection based on photon crystal coding microspheres mainly adopts the analysis means of fluorescence spectrum. The absorption peak of the fluorescence spectrum is generally derived from the chemical structure of the molecule, so that the fluorescence spectrum is extremely easy to be limited by factors such as illumination, temperature, storage time and the like. Compared with fluorescence analysis, SERS has higher detection sensitivity and is very suitable for biological system research.
Traditional two-dimensional photonic crystal films, three-dimensional photonic crystal films and optical fibers can also be used as substrates for surface raman spectroscopy. However, the photonic crystal film is easy to fall off from the substrate, is difficult to prepare in a large area, has a limited detection range, and is most critical that the reproducibility of signals cannot be ensured. And the three-dimensional photonic crystal fiber cannot be widely applied due to complex manufacturing process and high cost. The silica and titania photonic crystals reported in recent years have advantages of stable encoding and simple decoding, but have low activity of immobilized probe molecules, slow binding reaction kinetics and unavoidable nonspecific surface adsorption. Hydrogel photonic crystals are considered ideal substrates, not only providing a biological environment similar to that of aqueous solutions, but also the unique three-dimensional structure of hydrogels can immobilize a large number of probe molecules, thereby improving the sensitivity of SERS detection. The coding/decoding characteristics of the photonic crystal are combined with the Raman signals, so that the multi-component detection of different biological molecules is realized, the stability and the accuracy of the coding can be ensured, the method is simple to operate, the distinction is obvious, and the cross interference can be ignored.
Disclosure of Invention
The invention aims to provide a method for performing multi-element SERS biological detection by utilizing hydrogel photonic crystal encoding microspheres with inverse opal structures, which can successfully realize simultaneous detection of multi-component tumor markers. The method is realized by the following technical scheme:
a method of multiplex SERS biological detection comprising the steps of:
s1, preparing inverse opal hydrogel photonic crystal encoded microspheres:
1) Soaking the silicon dioxide photonic crystal microspheres with different codes in a mixed solution of concentrated sulfuric acid and hydrogen peroxide, cleaning and drying in an inert environment to obtain an intermediate product 1;
2) Placing the intermediate product 1 in a hydrogel precursor solution containing PEGDA, acrylic acid, a photoinitiator and ultrapure water, standing for 4 hours, and then placing the mixture in an ultraviolet lamp for photo-curing for 10 minutes to obtain an intermediate product 2;
3) Etching the intermediate product 2 in hydrofluoric acid solution, and cleaning to obtain inverse opal hydrogel photonic crystal encoded microspheres;
s2, fixing an antibody:
1) Placing the inverse opal hydrogel photonic crystal encoded microspheres with the same kind of codes in a mixed solution of EDC and NHS for incubation to obtain an intermediate product 3;
2) Placing the intermediate product 3 into a buffer solution of an antibody for reaction to obtain inverse opal hydrogel photonic crystal encoded microspheres with the antibodies fixed on the surface;
s3, preparing a Raman signal amplification probe:
adding a buffer solution of an antibody into a buffer solution of a Raman probe molecule and a metal nanoparticle, and obtaining a Raman signal amplification probe of the antibody after reaction;
s4, multi-element SERS biological detection:
adding the inverse opal hydrogel photonic crystal encoding microsphere with the antibody fixed on the surface and the corresponding Raman signal amplification probe of the antibody into a biological mixed solution to be detected, reacting to form a product, detecting the Raman signal on the surface of the product, and determining the concentration of the biological molecules to be detected.
Further, the photoinitiator adopts HMPP, and the hydrogel precursor solution comprises the following components in percentage by mass: PEGDA40-90 wt%, acrylic acid 2-4 wt%, HMPP 1wt% and the balance ultrapure water. The hydrogel photonic crystal prepared within the range of the feeding mass ratio has good sphericity, high mechanical strength and stable reflection spectrum.
Further, in the metal nanoparticle buffer solution, the metal nanoparticles are at least one selected from gold nanoparticles and silver nanoparticles, and the silver nanoparticles are used for the metal nanoparticles, so that SERS signals of probe molecules can be greatly enhanced, and the detection sensitivity is improved.
Further, in the mixed solution of EDC and NHS, the mass ratio of EDC to NHS is 1-2:1. The effect of coating the hydrogel photonic crystal microsphere modified by the antibody in the range of the charging mass ratio is optimal.
Further, the biomolecule to be tested is selected from at least one of tumor markers CEA, AFP, CA, CA199, CA211 and CA 724.
Further, in S4, the reaction temperature is 25-37 ℃ and the reaction time is 25-60 minutes. The Raman signal of the tumor marker is best detected in a plurality of ways within the temperature and time range of the temperature bath reaction.
The beneficial effects of the invention are as follows:
1. the silica photonic crystal microsphere is used as a sacrificial template to prepare the inverse opal hydrogel photonic crystal encoding microsphere with a three-dimensional ordered structure, the template microsphere and the replicated hydrogel microsphere have a highly similar stable structure, the shape is regular and uniform, dense and ordered hole arrangement is realized, the structure can provide larger specific surface area for biological molecules, the biocompatibility is better than that of the silica photonic crystal microsphere, and the sensitivity of SERS detection can be improved.
2. The inverse opal hydrogel photonic crystal encoding microsphere is combined with a Raman signal to carry out multi-element SERS biological detection, the reflection peak position of the hydrogel photonic crystal and the characteristic color observed under a polarizing microscope are used for decoding the hydrogel photonic crystal, and the method has the advantages of small cross interference, high sensitivity, obvious distinction and simple operation. The invention has a large application background for detecting the multi-component tumor markers.
Drawings
FIGS. 1 (a) - (d) show SEM images of silica photonic crystal microsphere templates prepared in step S1 of example 1 and inverse opal hydrogel photonic crystal encoded microspheres replicated;
wherein FIG. 1 (a) is an overall view of a template microsphere;
FIG. 1 (b) is a template microsphere surface map;
FIG. 1 (c) is a surface view of a template microsphere filled with a hydrogel precursor solution;
FIG. 1 (d) is a surface structure diagram of inverse opal hydrogel photonic crystal encoded microspheres.
FIG. 2 (a) shows a polarized photomicrograph of two encoded inverse opal hydrogel photonic crystal encoded microspheres prepared in step S1 of example 1;
FIG. 2 (b) shows the reflection peak positions of two encoded inverse opal hydrogel photonic crystal encoded microspheres prepared in step S1 of example 1.
FIG. 3 is a SERS Raman spectrum for detecting CEA at different concentrations.
FIG. 4 is a schematic diagram of 1587 and 1587cm -1 The peak at this point is a standard plot of the concentration-dependent CEA SERS intensity change obtained from the reference peak.
FIG. 5 is a SERS Raman spectrum for detecting AFP at different concentrations.
FIG. 6 is a schematic diagram of 1587cm -1 The peak at this point is a standard plot of the concentration-dependent SERS intensity variation of AFP obtained from the reference peak.
Description of the embodiments
In order to more clearly illustrate the technical aspects of the present invention, the following examples are set forth, but the present invention is not limited thereto.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; the reagents, materials, etc. used in the examples described below are commercially available unless otherwise specified.
Example 1
S1, preparing inverse opal hydrogel photonic crystal encoded microspheres:
1) Taking two coded silicon dioxide photonic crystals with reflection peak positions of 530nm and 650nm respectively, soaking the two coded silicon dioxide photonic crystals in a mixed solution of concentrated sulfuric acid and hydrogen peroxide in a volume ratio of 7:3 for 12 h, hydroxylating the surfaces of the microspheres, repeatedly cleaning the microspheres with ultrapure water, and drying with nitrogen;
2) Placing the obtained photonic crystal in a hydrogel precursor solution for reaction 4h, and exposing the photonic crystal and the hydrogel precursor solution under an ultraviolet lamp for 10 minutes simultaneously to solidify the hydrogel precursor solution when the photonic crystal changes from milky to transparent;
wherein the hydrogel precursor solution comprises the following components in percentage by mass: 40 wt% PEGDA (polyethylene glycol diacrylate), 2wt% parts acrylic acid, 1wt% HMPP ((2-hydroxy-2-methyl-1-phenylpropion) and the balance ultrapure water;
3) The photonic crystal is put into hydrofluoric acid with the concentration of 1wt percent to be etched for 2 h, and the inverse opal hydrogel photonic crystal encoded microsphere with two different encodings can be obtained.
S2, fixing an antibody:
1) Respectively taking 0.0075 and g of the prepared two inverse opal hydrogel photonic crystal encoding microspheres, placing the microspheres in a mixed solution of EDC and NHS with the mass ratio of 2:1, incubating for 20 minutes at room temperature, activating carboxyl groups on the surface of the hydrogel, and washing the surface of the hydrogel with a phosphate buffer solution;
2) The obtained hydrogel photonic crystal microsphere with the reflection peak position of 530nm is placed in phosphate buffer solution (200 mu L,0.5 mg/mL) of CEA coating antibody, the hydrogel photonic crystal microsphere with the reflection peak position of 650nm is placed in phosphate buffer solution (200 mu L,0.5 mg/mL) of AFP coating antibody, after triggering reaction for 2 hours in a shaking table at 37 ℃, the temperature is 4 ℃ overnight, and the coating antibody Ab1 is modified on the surface of the hydrogel photonic crystal microsphere. The next day, after the microspheres are taken out and washed by PBS phosphate buffer solution, 100 mu L of 1wt percent bovine serum albumin is respectively added into the two buffer solutions, the microspheres are sealed for 2 hours at room temperature, and the sealed microspheres are washed by PBS phosphate buffer solution for later use.
S3, preparing a Raman signal amplification probe:
taking a phosphate buffer solution (0.5 mg/mL) of Raman probe molecules 4-MBA (100 [ mu ] L,2.6 mg/mL) and 200 [ mu ] L of silver nano particles, and adding the solution into the phosphate buffer solution (200 [ mu ] L,0.5 mg/mL) of CEA-labeling antibody (M5B); and then taking a phosphate buffer solution (0.5 mg/mL) of Raman probe molecules 4-MBA (100 mu L,2.6 mg/mL) and 200 mu L of silver nano particles, adding the Raman probe molecules into the phosphate buffer solution (200 mu L,0.5 mg/mL) of AFP labeling antibody (9K 5), standing at 4 ℃ overnight after triggering reaction, centrifuging and washing to remove unreacted 4-MBA and antibody, respectively adding 100 mu L of 1wt% bovine serum albumin, sealing for 2 hours at room temperature, centrifuging to remove redundant bovine serum albumin, and redispersing the bovine serum albumin in the phosphate buffer solution of 1 mL to obtain two Raman signal amplification probes (antibody/4-MBA/Ag nano particles).
S4, a multi-element SERS biological detection method comprises the following steps:
the concentration gradient was set to 1X 10 3 -1×10 -9 ng/mL of CEA and AFP antigen mixed solution, placing two different coded anti-opal hydrogel photon crystal coded microspheres immobilized with antibodies into the mixed solution, carrying out warm water bath reaction for 50 minutes at 37 ℃, and washing the microspheres by using PBS phosphate buffer solution. Adding corresponding Raman signal amplification probes (antibodies/4-MBA/Ag nanoparticles), carrying out warm water bath reaction for 1 hour at 37 ℃, flushing with PBST flushing liquid, finally forming a product which has a sandwich composite structure of a solid-phase immune substrate-antigen-Raman signal amplification probe, determining the type of an antigen to be detected by measuring the reflection peak position of the encoded microsphere and the characteristic color observed under a polarizing microscope, detecting the Raman signal by using a laser confocal Raman spectrometer, and determining the concentration of the biomolecule to be detected.
FIGS. 1 (a) - (d) show SEM images of silica photonic crystal microsphere templates prepared in step S1 of example 1 and inverse opal hydrogel photonic crystal encoded microspheres replicated. FIG. 1 (a) is an overall view of a template microsphere, showing that the silica photonic crystal microsphere has a regular spherical shape; FIG. 1 (b) is a surface view of template microspheres showing that the microspheres are densely packed in a hexagonal packed structure, and the microspheres are regular in arrangement and large in specific surface area; FIG. 1 (c) is a surface view of a template microsphere filled with a hydrogel precursor solution, showing that the hydrogel precursor solution has penetrated into the interior pores of the silica photonic crystal microsphere; FIG. 1 (d) is a surface structure diagram of inverse opal hydrogel photonic crystal-encoded microspheres, which shows that the resulting inverse opal hydrogel photonic crystal-encoded microspheres exhibit a highly ordered arrangement of pores and are uniform in size.
Example 2
S1, preparing inverse opal hydrogel photonic crystal encoded microspheres:
1) Taking two coded silicon dioxide photonic crystals with reflection peak positions of 530nm and 650nm respectively, soaking the two coded silicon dioxide photonic crystals in mixed solution of concentrated sulfuric acid and hydrogen peroxide in a volume ratio of 7:3 for 10 hours to hydroxylate the surfaces of the microspheres, repeatedly cleaning the microspheres with ultrapure water, and drying with nitrogen;
2) Placing the obtained photonic crystal in a hydrogel precursor solution (the hydrogel precursor solution comprises 90wt% of PEGDA, 2wt% of acrylic acid, 1wt% of HMPP and the balance of ultrapure water according to mass fraction) for reaction for 4 hours, and exposing the photonic crystal and the hydrogel precursor solution under an ultraviolet lamp for 10 minutes until the photonic crystal is changed from milky to transparent color, so that the hydrogel precursor solution is solidified;
3) And (3) putting the photonic crystal into hydrofluoric acid with the concentration of 2wt% to etch for 2 hours, so as to obtain the inverse opal hydrogel photonic crystal encoded microsphere with two different encodings.
S2, fixing an antibody:
1) Taking 0.0075g of each of the prepared two inverse opal hydrogel photonic crystal encoding microspheres, placing the microspheres in a mixed solution of EDC and NHS with a mass ratio of 2:1, incubating for 20 minutes at room temperature, activating carboxyl groups on the surface of the hydrogel, and then washing the microspheres with a phosphate buffer solution;
2) The obtained hydrogel photonic crystal microsphere with the reflection peak of 530nm is placed in phosphate buffer solution (200 mu L,0.5 mg/mL) of CEA coating antibody, the hydrogel photonic crystal microsphere with the reflection peak of 650nm is placed in phosphate buffer solution (200 mu L,0.5 mg/mL) of CA125 coating antibody, after triggering reaction for 2 hours in a shaking table at 37 ℃, overnight at 4 ℃, and the coating antibody Ab1 is modified on the surface of the hydrogel photonic crystal microsphere. The next day, after the microspheres are taken out and washed by PBS phosphate buffer solution, 100 mu L of 1wt percent bovine serum albumin is respectively added into the two buffer solutions, the microspheres are sealed for 2 hours at room temperature, and the sealed microspheres are washed by PBS phosphate buffer solution for later use.
S3, preparing a Raman signal amplification probe:
adding a phosphate buffer solution (0.5 mg/mL) of Raman probe molecules 4-MBA (100 [ mu ] L,2.6 mg/mL) and 200 [ mu ] L of silver nano particles into a phosphate buffer solution (200 [ mu ] L,0.5 mg/mL) of CEA-labeling antibody; adding a phosphate buffer solution (0.5 mg/mL) of Raman probe molecules 4-MBA (100 mu L,2.6 mg/mL) and 200 mu L of silver nano particles into a phosphate buffer solution (200 mu L,0.5 mg/mL) of CA125 labeling antibody, standing overnight at 4 ℃ after triggering reaction, centrifuging and washing to remove unreacted 4-MBA and antibody, respectively adding 100 mu L of 1wt% bovine serum albumin, sealing for 2 hours at room temperature, centrifuging to remove redundant bovine serum albumin, and re-dispersing in the phosphate buffer solution of 1 mL to obtain two Raman signal amplification probes (4-MBA/antibody/Ag nano particles).
S4, a multi-element SERS biological detection method comprises the following steps:
the concentration gradient was set to 1X 10 3 -1×10 -9 ng/mL of CEA and CA125 antigen mixed solution, placing two different coded inverse opal hydrogel photon crystal coded microspheres immobilized with antibodies into the mixed solution, carrying out warm water bath reaction for 60 minutes at 25 ℃, and washing the microspheres by using PBS phosphate buffer solution. Adding corresponding Raman signal amplification probes (4-MBA/antibody/Ag nano particles), carrying out a warm water bath reaction at 37 ℃ for 1 hour, flushing with PBST flushing liquid to finally form a product, wherein the product has a sandwich composite structure of a solid-phase immune substrate-antigen-Raman signal amplification probe, determining the type of an antigen to be detected by measuring the reflection peak position of an encoded microsphere and the characteristic color observed under a polarizing microscope, detecting the Raman signal of the antigen by using a laser confocal Raman spectrometer, and determining the antigen to be detectedConcentration of the molecule.
Test case
For the relevant test effect of the multi-element SERS biological detection method using the inverse opal structured hydrogel photonic crystal encoded microspheres in example 1 of the present invention, the following analysis was performed.
FIGS. 2 (a) - (b) are reflection peak positions of two encoded inverse opal hydrogel photonic crystal encoded microspheres prepared in step S1 of example 1, wherein the inset shows characteristic colors shown under a polarizing microscope. FIG. 2 (a) shows an inverse opal hydrogel photonic crystal encoded microsphere prepared by using a silicon dioxide photonic crystal with a particle size of 190 nm as a template, wherein the reflection peak position of the photonic crystal is 530nm, and the characteristic color is green; fig. 2 (b) shows an inverse opal hydrogel photonic crystal encoded microsphere prepared by using a silica photonic crystal with a particle size of 320 nm as a template, wherein the reflection peak position of the photonic crystal is 650nm, and the characteristic color is red. The difference between the two is obvious, and the detection result is accurate. According to the reflection peak position and the characteristic color displayed under a polarizing microscope, the encoding and decoding functions of the inverse opal hydrogel photonic crystal encoding microsphere can be successfully realized.
FIGS. 3-6 are SERS plots of the multiplex SERS bioassay performed on the final product of example 1. The two different coded inverse opal hydrogel photonic crystal coding microspheres immobilized with the antibody in the figures 2 (a) - (b) are respectively placed in CEA and AFP mixed antigen solution, then antibody/4-MBA/Ag nano particles are added as Raman signal amplification probes, after antigen-antibody specific binding, the inverse opal hydrogel photonic crystal coding microspheres are immediately placed under a Raman confocal spectrometer, and Raman signals are detected.
3-6, FIG. 3 is a SERS chart for detecting CEA at different concentrations, the detection range is from 103-10-9ng/mL, FIG. 4 is a standard graph of concentration-dependent SERS intensity variation obtained by taking a peak at 1587cm-1 as a reference peak, and the obtained data are shown in Table 1; FIG. 5 is a SERS plot of AFP at various concentrations ranging from 103-10-7ng/mL, FIG. 6 is a standard plot of concentration dependent SERS intensity changes with a peak at 1587cm-1 as a reference peak, and the data obtained are shown in Table 2.
TABLE 1 detection of SERS intensity variation for different CEA concentrations
TABLE 2 detection of SERS intensity variation for different concentrations of AFP
From fig. 6, it can be seen that the detection limit range is wider and the linear fitting relation is good when the method is used for multi-element SERS detection.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (5)
1. A method for multiplex SERS biological detection, comprising the steps of:
s1, preparing inverse opal hydrogel photonic crystal encoded microspheres:
1) Soaking the silicon dioxide photonic crystal microspheres with different codes in a mixed solution of concentrated sulfuric acid and hydrogen peroxide, cleaning and drying in an inert environment to obtain an intermediate product 1;
2) Placing the intermediate product 1 in a hydrogel precursor solution containing PEGDA, acrylic acid, a photoinitiator and ultrapure water, standing for 4 hours, and then placing the mixture in an ultraviolet lamp for photo-curing for 10 minutes to obtain an intermediate product 2;
3) Etching the intermediate product 2 in hydrofluoric acid solution, and cleaning to obtain inverse opal hydrogel photonic crystal encoded microspheres;
s2, fixing an antibody:
1) Placing the inverse opal hydrogel photonic crystal encoded microspheres with the same kind of codes in a mixed solution of EDC and NHS for incubation to obtain an intermediate product 3;
2) Placing the intermediate product 3 into a buffer solution of an antibody for reaction to obtain inverse opal hydrogel photonic crystal encoded microspheres with the antibodies fixed on the surface;
s3, preparing a Raman signal amplification probe:
adding a buffer solution of an antibody into a buffer solution of a Raman probe molecule and a metal nanoparticle, and obtaining a Raman signal amplification probe of the antibody after reaction;
s4, multi-element SERS biological detection:
adding the inverse opal hydrogel photonic crystal encoding microsphere with the antibody fixed on the surface and the corresponding Raman signal amplification probe of the antibody into a biological mixed solution to be detected, reacting to form a product, detecting the Raman signal on the surface of the product, and determining the concentration of the biological molecules to be detected.
2. The method of multiplex SERS biological detection according to claim 1, wherein the photoinitiator is HMPP, and the hydrogel precursor solution comprises the following components in mass fraction: PEGDA40-90 wt%, acrylic acid 2-4 wt%, HMPP 1wt% and the balance ultrapure water.
3. The method of multiplex SERS biological detection according to claim 1, wherein the metal nanoparticles are in a buffer solution of metal nanoparticles, and the metal nanoparticles are at least one selected from gold nanoparticles and silver nanoparticles.
4. The method of multiplex SERS biological detection according to claim 1, wherein the mass ratio of EDC to NHS in the mixed solution of EDC and NHS is 1-2:1.
5. The method of multiplex SERS biological detection according to claim 1, wherein in S4, the reaction is carried out at a temperature of 25 to 37 ℃ for a reaction time of 25 to 60 minutes.
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