CN115711994A - SERS (surface enhanced Raman Scattering) biosensor based on carbon-nitrogen four nanosheets and preparation method and application thereof - Google Patents
SERS (surface enhanced Raman Scattering) biosensor based on carbon-nitrogen four nanosheets and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of nano biology, and discloses a SERS (surface enhanced Raman Scattering) biosensor based on a carbon-nitrogen four nanosheet and a preparation method and application thereof; the SERS biosensor consists of an immunological probe, an immunological substrate and a glycoprotein 125 antigen; the immunoprobe is obtained by linking glycoprotein 125 antibody on magnetic nano molybdenum disulfide with functional markers; the immune substrate is obtained by linking glycoprotein 125 antibodies on the activated carbon-nitrogen-containing four nano-chips; and the immune probe and the glycoprotein 125 antibody on the immune substrate are specifically combined with the glycoprotein 125 antigen to form the SERS biosensor with a sandwich structure. The SERS immunosensor prepared by the method has good biocompatibility, the preparation process is simple, and double recycling of the substrate and the probe can be realized; and has excellent photocatalytic performance, and is beneficial to the rapid degradation of antigen, antibody and Raman molecules.
Description
Technical Field
The invention relates to the technical field of nano biology, in particular to a SERS (surface enhanced Raman scattering) biosensor based on carbon-nitrogen-rich nanosheets and a preparation method and application thereof.
Background
Early detection of cancer is key to improving cure rate. SERS has become one of the main strategies for detecting biomarkers compared to traditional detection techniques due to its unique fingerprint, non-destructive data acquisition, and sensitivity at the single molecule level.
Therefore, the prior art provides a SERS-based biosensor based on noble metal nanoparticles (Au, ag, etc.), which has high sensitivity and high flux, and SERS generally occurs on the surface of the noble metal nanoparticles, so that the SERS can directly detect an object to be detected, thereby drawing a great deal of attention.
However, the substrate and the probe of the SERS-based biosensor are both made of a large amount of noble metals, so that the preparation cost is high, and the noble metal immune substrate is generally required to be prepared into a noble metal layer (such as an Ag layer) by using processes such as electron beam, ion beam, photolithography and electrodeposition, the preparation process is complex and is not easy to operate, the controllability of the prepared substrate morphology is poor, the stability and the biocompatibility of the SERS-based biosensor are poor, and the detection effect of the SERS-based biosensor is influenced.
Therefore, the invention provides a SERS biosensor based on a carbon-nitrogen four nanosheet and a preparation method and application thereof.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a SERS biosensor based on a carbon-nitrogen-trinitrobenzene four-nanosheet and a preparation method and application thereof. The invention combines magnetic nano molybdenum disulfide and carbon three nitrogen four nano-sheets to manufacture an SERS biosensor for detecting tumor markers.
The invention discloses a SERS biosensor based on carbon-nitrogen-rich nanosheets, a preparation method and application thereof, which are realized by the following technical scheme:
the invention provides a SERS biosensor based on carbon-nitrogen-three nanosheets, which consists of an immunoprobe, an immunological substrate and glycoprotein 125 antigen;
the immunoprobe is obtained by linking a glycoprotein 125 antibody on functionalized marked magnetic nano molybdenum disulfide, and the diameter of the magnetic nano molybdenum disulfide is 100-1000 nm;
the immune substrate is obtained by linking the glycoprotein 125 antibody on a carbon three nitrogen four nanosheet, and the diameter of the carbon three nitrogen four nanosheet is 50-300 nm;
and the immune probe and the glycoprotein 125 antibody on the immune substrate are specifically combined with the glycoprotein 125 antigen to form the SERS biosensor with a sandwich structure.
Further, the functionalized magnetic nano molybdenum disulfide is obtained by the following steps:
adopting Raman molecules to mark magnetic nano molybdenum disulfide, namely obtaining the functionalized marked magnetic nano molybdenum disulfide;
the Raman molecule is any one of rhodamine 6G, methylene blue and crystal violet, and the dosage ratio of the Raman molecule to the magnetic nano molybdenum disulfide is 1-3mmol.
Further, the preparation method of the magnetic nano molybdenum disulfide comprises the following steps:
and uniformly dispersing molybdate, a sulfur source and ferroferric oxide in a solvent B, carrying out hydrothermal reaction at the temperature of 180-220 ℃ for 6-12 h, cooling to room temperature, washing and drying to obtain the magnetic nano molybdenum disulfide coated with the ferroferric oxide by molybdenum disulfide.
Further, the molybdate is sodium molybdate or ammonium molybdate;
the sulfur source is thioacetamide or thiourea;
the mol ratio of the molybdate to the sulfur source to the ferroferric oxide (232) is 1.5-2;
the dosage ratio of the molybdate to the solvent B is 0.15-0.2mol.
Further, the activated carbon three-nitrogen four-nanosheet is obtained through the following steps:
s1, adding carbon-nitrogen-IV powder into concentrated hydrochloric acid, performing ultrasonic treatment, and reacting at 70-90 ℃ for 8-20 h to obtain acidified carbon-nitrogen-IV;
wherein the dosage ratio of the carbon-nitrogen-containing four powder to the concentrated hydrochloric acid is 2-3mg;
s2, dispersing the acidified C, N and C in an aqueous solvent under the ultrasonic action, and then carrying out centrifugal treatment to obtain supernatant, namely a C, N and C nanosheet solution;
wherein the dosage ratio of the hydrosolvent to the carbon-nitrogen-tetrapowder is 1-2mg;
s3, uniformly dispersing the carbon-nitrogen-rich nanosheet solution and an imine compound in a phosphate buffer solution, and reacting at the temperature of 36-38 ℃ for 1-3 h to obtain an activated carbon-nitrogen-rich nanosheet solution;
wherein the imine compound is N-hydroxysuccinimide or 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide.
Furthermore, the dosage ratio of the glycoprotein 125 antibody to the activated carbon-trinitrogen tetrasheet solution is 0.002-0.006mg.
The second purpose of the present invention is to provide a method for preparing the SERS biosensor, comprising the following steps:
step 1, preparing an immunoprobe:
adding a glycoprotein 125 antibody into the functionalized and marked magnetic nano molybdenum disulfide, and then carrying out incubation treatment A, so as to link the glycoprotein 125 antibody on the Raman molecule marked magnetic nano molybdenum disulfide, and obtain the immune probe;
step 2, preparing an immune substrate:
activating the carbon-nitrogen-III nanosheets, adding a glycoprotein 125 antibody, and performing incubation treatment B to link the glycoprotein 125 antibody on the activated carbon-nitrogen-III nanosheets to obtain an immune substrate;
step 3, preparing the SERS biosensor with the sandwich structure:
dispersing an immune substrate in a phosphate buffer solution A to form an immune substrate solution, and then adding a glycoprotein 125 antigen for incubation treatment C to obtain a mixed solution A;
and dispersing the immune probe in a phosphate buffer solution B to form an immune probe solution, and then adding the immune probe solution into the mixed solution A for incubation treatment D to obtain the SERS biosensor.
Further, the mass ratio of the glycoprotein 125 antibody to the functionalized magnetic nano molybdenum disulfide is 2-6;
the dosage ratio of the immunological substrate to the phosphate buffer solution A is 1-2mg;
the dosage ratio of the immunoprobe to the phosphate buffer solution B is 10mg;
the dosage ratio of the glycoprotein 125 antigen to the immune substrate solution is 0.002-0.006mg;
the dosage ratio of the immune probe solution to the mixed solution A is 1-1.5mL;
the concentration of the glycoprotein 125 antigen is 80-120 IU/mL.
Further, the treatment temperature of the incubation treatment A is 3-5 ℃, and the treatment time is 1.5-2.5 h;
the incubation treatment B is carried out at the treatment temperature of 3-5 ℃ for 1.5-2.5 h;
the incubation treatment C is carried out at the treatment temperature of 36-38 ℃ for 1-3 h;
the incubation temperature of the incubation treatment D is 36-38 ℃, and the incubation time is 1-3 h.
The third purpose of the invention is to provide an application of the SERS biosensor in tumor marker detection.
Compared with the prior art, the invention has the following beneficial effects:
the SERS biosensor consists of an immunological probe, an immunological substrate and a glycoprotein 125 antigen, wherein the immunological probe is obtained by linking a functional-labeled magnetic nano molybdenum disulfide with a glycoprotein 125 antibody, the immunological substrate is obtained by linking the glycoprotein 125 antibody on an activated carbon-three-nitrogen four-nano chip, the preparation process is simple and easy to operate, and the activated g-C is 3 N 4 The nanosheet has larger surface area and more active electrons, shows improved Raman scattering performance, has a cross energy band structure which is matched with molybdenum disulfide very well, has a synergistic chemical enhancement effect, greatly promotes the chemical enhancement effect, causes more charge transfer processes to increase the polarizability, obviously amplifies a Raman signal of a labeled molecule, and further can cause excellent SERS activity, so that the SERS immunosensor disclosed by the invention has good biocompatibility and the detection efficiency is greatly improved.
According to the invention, a small amount of magnetic particle ferroferric oxide is introduced in the preparation process of molybdenum disulfide, so that the ferroferric oxide is wrapped in the molybdenum disulfide, and further, the stacking and self-assembly of molybdenum disulfide nanosheets into flower shapes are promoted, higher specific surface area and active sites are exposed, and the molecular adsorption is facilitated. In the SERS immunosensor prepared by the invention, the synergistic chemical enhancement effect of the carbon three-nitrogen four-nano sheet and the magnetic nano molybdenum disulfide causes more charge transfer processes to increase the polarizability and improve SERS signals, so that excellent SERS activity can be caused.
The SERS immunosensor prepared by the method has good biocompatibility, and the introduction of magnetic ferroferric oxide realizes the dual-cycle utilization of the substrate and the probe. Meanwhile, by virtue of the high carrier mobility of the two semiconductors, the immunosensor also has excellent photocatalytic performance, and is beneficial to rapid degradation of antigens, antibodies and Raman molecules.
Drawings
FIG. 1 is a scanning electron micrograph of an immunoprobe prepared in example 1;
FIG. 2 is a scanning electron micrograph of an immunoprobe prepared in example 2;
FIG. 3 is a scanning electron micrograph of an immunoprobe prepared in example 3;
FIG. 4 is a scanning electron micrograph of the immune substrate prepared in example 1;
FIG. 5 is a scanning electron micrograph of the SERS biosensor prepared in example 1;
FIG. 6 shows the Raman detection results of rhodamine 6G by the immunological probe and the immunological substrate prepared in example 1;
FIG. 7 shows the Raman detection result of rhodamine 6G by the immunological probe prepared in example 2;
FIG. 8 shows the Raman detection results of the immuno probe prepared in example 3 for rhodamine 6G;
fig. 9 shows the raman detection result of the SERS biosensor prepared in example 1 on glycoprotein 125 in serum.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention.
The invention provides a SERS biosensor based on carbon-nitrogen-three nanosheets, which consists of an immunological probe, an immunological substrate and a glycoprotein 125 antigen, and the preparation method comprises the following steps:
step 1, preparing an immunoprobe:
1.1 preparation of magnetic nanometer molybdenum disulfide
It should be noted that, in the present invention, magnetic flower-like nano molybdenum disulfide is preferably used as the magnetic nano molybdenum disulfide, and optionally, the magnetic flower-like nano molybdenum disulfide is prepared by the following steps:
1.1.1 preparation of ferroferric oxide:
uniformly dispersing an iron source, a stabilizer and a surfactant in a solvent A in sequence, and carrying out hydrothermal reaction for 10 hours at the temperature of 200 ℃ to obtain ferroferric oxide small particles;
the present invention is not limited to a specific type of the iron source as long as Fe can be supplied, and may be, for example, any one selected from the group consisting of ferrous sulfate heptahydrate, ferric dichloride tetrahydrate, and ferric chloride hexahydrate.
The present invention is not limited to a specific type of the stabilizer as long as it can prevent agglomeration among particles, and may be, for example, any one selected from sodium acetate, sodium citrate and sodium acetate.
The present invention is not limited to a specific type of surfactant as long as agglomeration between particles can be prevented.
The specific type of the solution A of the present invention is only required to form a uniform solution or suspension with the iron source, the stabilizer and the surfactant.
The present invention is not limited to the manner of dispersing the iron source, the stabilizer and the surfactant in the solvent a, as long as the iron source, the stabilizer and the surfactant can be uniformly dispersed in the solvent a.
1.1.2 hydrothermal synthesis:
uniformly dispersing molybdate, a sulfur source and ferroferric oxide small particles in a solvent B, carrying out hydrothermal reaction for 6-12 h at the temperature of 180-220 ℃, cooling to room temperature, washing and drying to obtain magnetic nano molybdenum disulfide with ferroferric oxide coated by molybdenum disulfide;
the present invention is not limited to a particular type of molybdate as long as it can provide molybdenum ions, such as sodium molybdate or ammonium molybdate.
The invention is not limited to a particular type of sulfur source as long as it is capable of providing S, such as may be selected from thioacetamide or thiourea.
The invention is not limited to a particular type of solution B, as long as it forms a homogeneous solution or suspension with the molybdate, the sulfur source, and the ferroferric oxide.
The invention does not limit the dispersion mode of the molybdate, the sulfur source and the ferroferric oxide in the solvent B, as long as the molybdate, the sulfur source and the ferroferric oxide can be uniformly dispersed in the solvent B.
The present invention does not limit the specific manner of the washing treatment as long as impurities (e.g., unreacted substances) on the surface of the product can be removed.
The present invention is not limited to a specific manner of the drying treatment as long as the solvent remaining on the surface of the product can be removed.
1.2 labeling of magnetic nanometer molybdenum disulfide
In order to facilitate marking, the magnetic nano molybdenum disulfide is prepared into solution and then marked: the obtained magnetic nano molybdenum disulfide powder is uniformly dissolved in deionized water under ultrasonic wave to prepare a magnetic nano molybdenum disulfide solution with the concentration of 5-15 mg/mL.
Mixing Raman molecules with a magnetic nano molybdenum disulfide solution, then carrying out culture treatment A, then adding a phosphate buffer saline solution of an imine compound, carrying out culture treatment B, and after carrying out magnetic separation treatment, uniformly dispersing the imine molecules in the phosphate buffer saline solution to obtain a magnetic nano molybdenum disulfide solution marked by rhodamine 6G;
it should be noted that the invention does not limit the specific type of raman molecule, as long as the magnetic nano molybdenum disulfide probe can be labeled, such as: the Raman molecule can be selected from any one of rhodamine 6G, methylene blue and crystal violet, and other Raman molecules with larger scattering cross sections can also be selected.
The invention does not limit the specific mode of the Raman molecular marking magnetic nano molybdenum disulfide as long as the marking can be realized.
In order to facilitate the subsequent treatment of the marked magnetic nano molybdenum disulfide, the method also comprises the step of carrying out magnetic separation and cleaning for 3 times before carrying out the culture treatment B so as to remove the unbound Raman molecules in the culture treatment A. In the magnetic separation and cleaning process, ferroferric oxide endows the molybdenum disulfide nanoflowers with good magnetism, the magnetic nano molybdenum disulfide can be firmly adsorbed on the wall of a test tube by a magnet, the solution in the test tube is washed out, deionized water is added, and the operations are repeated.
The invention does not limit the specific magnetic separation mode after the culture treatment B, as long as the Raman molecules which are not adsorbed on the magnetic nano molybdenum disulfide can be removed. Optionally, in this embodiment, a magnetic adsorption manner is adopted to magnetically separate the raman molecules on the magnetic nano molybdenum disulfide, so as to obtain the magnetically treated mixture a.
The invention does not limit the specific way of culturing the molybdenum dioxide nanoflower, and only needs to realize the marking of the magnetic molybdenum dioxide nanoflower by the Raman molecules for subsequent SERS detection. Preferably, the present invention uses a phosphate buffered saline solution of N-hydroxysuccinimide as a phosphate buffered saline solution of the imine compound, the phosphate buffered saline solution having a salt balance, an adjustable pH buffer, and a suitable pH buffer.
1.3 preparation of immunoprobes
Adding a glycoprotein 125 antibody into the labeled magnetic nano molybdenum disulfide solution of the Raman molecule, performing incubation treatment A, then adding a bovine serum albumin sealing solution, uniformly mixing, and performing culture treatment C to obtain an immunoprobe;
it should be noted that the invention does not limit the specific way of the incubation treatment a, as long as the antibody can be linked to the magnetic nano-molybdenum disulfide for the subsequent immune reaction.
In order to improve the blocking effect of the bovine serum albumin blocking solution on the non-specific adsorption sites on the probe, before the culture treatment C, the magnetic nano molybdenum disulfide solution after the incubation treatment A is subjected to magnetic separation treatment to remove redundant glycoprotein 125 antibodies, so as to obtain a mixed material B.
The present invention is not limited to a specific mode of the culture treatment C, as long as it can block the nonspecific adsorption sites on the immunoprobe.
1.4 preparation of an immunoprobe solution
Dispersing the obtained immune probe in phosphate buffer saline solution to obtain immune probe solution;
it should be noted that, in order to improve the detection effect of the probe, the invention washes the immunoprobe through phosphate buffer solution of N-hydroxysuccinimide before dispersing the immunoprobe in phosphate buffer solution, magnetically collects the molybdenum disulfide nanoflower through a magnet, cleans the collected molybdenum disulfide nanoflower for many times to remove redundant bovine serum albumin, and then dissolves the cleaned bovine serum albumin in the phosphate buffer solution to obtain the immunoprobe solution.
It should be further noted that, the functions of the immunoprobe solution prepared by phosphate buffered saline solution in the present invention are as follows: the function of dissolving the protective reagent is achieved; the reason is that phosphate buffer has a salt balance, a suitable pH buffering action that can be adjusted. Generally, other materials cannot be used for replacement, for example, distilled water does not have salt balance effect and can destroy the structure and biological characteristics of biological protein; the physiological saline has no effect of adjusting pH, and the complete and active substance can not be ensured to participate in biological reaction under the optimal condition, so the phosphate buffer solution is preferred.
Step 2, preparing an immune substrate
2.1 preparation of carbon-three-nitrogen-four-nanosheets
The present invention does not limit the specific preparation method of the carbon trinitrogen tetrasheet, as long as the carbon trinitrogen tetrasheet with a diameter of 50-300 nm can be obtained. Optionally, the carbon-nitrogen nanosheet is obtained by the following steps:
according to the weight ratio of carbon-nitrogen-tetranitrate powder and concentrated hydrochloric acid 2-3 mg: 1-2 mL, adding carbon-nitrogen-containing four powder into concentrated hydrochloric acid, and carrying out ultrasonic treatment for 1-3 h under the ultrasonic power of 800W. Then, the mixed solution reacts for 8 to 20 hours at a temperature of between 70 and 90 ℃. And then washing the mixture by using a large amount of deionized water until the washing liquid is neutral to remove redundant hydrochloric acid to obtain acidified carbon, nitrogen and nitrogen four. Dispersing the acidified C, N and D into deionized water under the ultrasonic action (the ultrasonic power is 800W, and the ultrasonic time is 5-10 min) according to the dosage ratio of 1-2mg of the hydrosolvent to 1-1.5 mL of the C, N and D powder. Centrifuging the dispersed carbon three-nitrogen four at the rotating speed of 2500-3500 r/min for 5-20 min, sinking the un-peeled massive carbon three-nitrogen four at the bottom of the wall of the test tube, and leaving the protonated carbon three-nitrogen four-nano sheet in the supernatant, wherein the supernatant is the solution for obtaining the carbon three-nitrogen four-nano sheet.
2.2 preparation of immune-based solutions
2.2.1 activated carbon, three, four:
uniformly dispersing the carbon three nitrogen four nanosheet solution and an imine compound in a phosphate buffer solution, and reacting at the temperature of 36-38 ℃ for 1-3 h to activate the carbon three nitrogen four nanosheets, so that the coupling efficiency of the protein is improved, and the activated carbon three nitrogen four nanosheet solution is obtained;
it should be noted that the present invention does not limit the specific dispersion manner of the carbon three nitrogen four nanosheet solution and the imine compound in the phosphate buffer solution, as long as the activation of the carbon three nitrogen four nanosheet by the imine compound can be achieved to improve the coupling efficiency of the protein.
2.2.2 formation of immune substrate:
mixing the glycoprotein 125 antibody with the activated carbon-three-nitrogen four-nanosheet solution, performing incubation treatment B, and performing solid-liquid separation to obtain a solid component, namely the immune substrate;
it should be noted that the invention is not limited to the specific manner of incubation treatment B in step 2.2.2, as long as it is possible to achieve linking of the antibody to the carbonitridine substrate for subsequent immunological reactions.
In order to improve the processing effect of the bovine serum albumin, the invention also carries out centrifugal treatment on the mixed material C before the bovine serum albumin is added into the mixed material C so as to remove the redundant glycoprotein 125 antibody in the mixed material C.
The invention does not limit the concrete mode of solid-liquid separation, and optionally, the embodiment adopts a centrifugal mode to separate, and the product after the hatching treatment B is centrifugally washed twice to remove the excessive bovine serum albumin, so as to obtain the immune substrate.
2.2.3 preparation of immune-based solutions
Dissolving the immune substrate in phosphate buffer salt solution under the action of ultrasound to obtain carbon-nitrogen-tetraimmune substrate solution;
the ultrasonic power of the ultrasonic action is 800W, and the ultrasonic time is 5-10 min.
Step 3, preparing SERS biosensor with sandwich structure
Adding glycoprotein 125 antigen into the carbon-nitrogen-four immune substrate solution, and performing incubation treatment C to obtain a mixed solution A;
adding the immunological probe into the mixed solution A, mixing, performing incubation treatment D, and combining the immunological substrate and the immunological probe through an antigen and an antibody to form a sandwich structure to obtain the SERS biosensor;
it should be noted that the present invention does not limit the specific manner of incubation treatment C, as long as the antigen can be linked to the carbon-nitrogen-four substrate for subsequent immunoreaction.
In order to avoid the influence of the uncontained antigen in the solution on subsequent treatment, phosphate buffer solution is adopted to carry out centrifugal washing treatment on the solid components obtained in the incubation treatment C (the solid components sink on the wall of the test tube, and the unconjugated antigen is sucked away in the solution) for multiple times so as to remove the uncaptured antigen.
The present invention does not limit the specific manner of incubation treatment D, as long as it is possible to achieve linking of the antigen on the carbon-three-nitrogen-four substrate for subsequent immunoreaction.
It should be noted that the SERS biosensor with sandwich structure prepared in this example is stored in 1mL of phosphate buffer solution for subsequent SERS detection. After the SERS biosensor with the sandwich structure prepared in this embodiment performs SERS detection, the immune structure is catalyzed by a visible light simulator, and raman molecules, antigens, and antibodies can be degraded based on the good photocatalytic activity of the molybdenum disulfide nanoflowers and the carbon trinitrogen nanosheets. And after the catalysis is finished, separating and collecting the immunoprobe and the immunopotentiator by adopting a magnet, and continuously carrying out subsequent tumor marker detection. Repeating the steps, and realizing recyclable immunodetection.
Example 1
The embodiment provides a SERS biosensor based on a carbon-nitrogen-III nanosheet, and the preparation method of the SERS biosensor based on a carbon-nitrogen-III nanosheet of the embodiment is as follows:
step 1, preparing an immunoprobe:
1.1 preparation of magnetic Nano molybdenum disulfide
1.1.1 preparation of ferroferric oxide:
in this example, ferric chloride hexahydrate was used as the iron source; sodium acetate is used as a stabilizer; polyethylene glycol is used as a surfactant; glycol is taken as a solvent A;
adding 1.35g of ferric chloride hexahydrate into a solvent A, then performing ultrasonic treatment for 10min under the ultrasonic power of 800W to dissolve the ferric chloride hexahydrate, sequentially adding 3.6g of sodium acetate and 1g of polyethylene glycol, performing ultrasonic treatment for 10min under the ultrasonic power of 800W, then stirring for 30min at the stirring speed of 300-500 r/min to uniformly disperse an iron source, the sodium acetate and the polyethylene glycol into ethylene glycol, and performing hydrothermal reaction for 10h at the temperature of 200 ℃ to obtain the ferroferric oxide small particles.
1.1.2 hydrothermal synthesis:
in the embodiment, ammonium molybdate is used as molybdate, thiourea is used as a sulfur source, and deionized water is used as a solvent B;
in addition, in the embodiment, the dispersion treatment is performed in an ultrasonic manner, 0.35g of ammonium molybdate, 0.76g of thiourea and 15.7mg of the small ferroferric oxide particles are added into 10mL of deionized water, and ultrasonic treatment is performed for 10min under the ultrasonic power or power of 800W, so that molybdate, a sulfur source and the small ferroferric oxide particles are uniformly dispersed in the solvent B; then carrying out hydrothermal reaction for 10h at the temperature of 200 ℃, cooling to room temperature, and then washing for 3 times by using absolute ethyl alcohol and deionized water in sequence to remove impurities on the surface of the product; and then drying at 60 ℃ for 6-12 h to remove residual solvent on the surface of the product, thus obtaining the magnetic nano molybdenum disulfide.
1.2 labeling of magnetic Nano molybdenum disulfide
In the embodiment, the concentration of the prepared magnetic nano molybdenum disulfide solution is 10mg/mL; rhodamine 6G is used as a Raman molecule; in the embodiment, 200 μ L of rhodamine 6G with a concentration of 10mmol/L and 1mL of magnetic nano molybdenum disulfide solution with a concentration of 10mg/mL are weighed, labeling is performed in an ultrasonic mode, the ultrasonic power is 800W, the ultrasonic time is 3min, and then the mixture is treated at room temperature for 12h to complete the culture treatment A, so that the rhodamine 6G molecule and the magnetic nano molybdenum disulfide are combined, and the labeled magnetic nano molybdenum disulfide is obtained.
And (3) carrying out magnetic separation and washing on the labeled magnetic nano molybdenum disulfide for 3 times to remove the unbound rhodamine 6G molecules in the culture treatment A process. In the magnetic separation and cleaning process, ferroferric oxide endows the molybdenum disulfide nanoflowers with good magnetism, the magnetic nano molybdenum disulfide can be firmly adsorbed on the wall of a test tube by a magnet so as to carry out magnetic separation on rhodamine 6G molecules on the magnetic nano molybdenum disulfide, a solution in the test tube is washed out, deionized water is added, and the operation is repeated for 3 times to obtain the magnetic labeled nano molybdenum disulfide after magnetic treatment, namely the mixed material A. Since the rhodamine 6G solution is red, the wash is considered complete when the solution in the cuvette is not transparent.
Then, a phosphate buffered saline solution of N-hydroxysuccinimide having a salt balance, an adjustable pH buffer proper, was used as a phosphate buffered saline solution of the imine compound. Weighing 1mL of N-hydroxysuccinimide with the concentration of 0.02mol/L, adding the obtained mixed material A, uniformly dispersing, and then culturing at the culture temperature of 37 ℃ for 1h to finish the culture treatment B so as to improve the coupling efficiency of the protein and obtain the rhodamine 6G labeled magnetic nano molybdenum disulfide solution.
1.3 preparation of immunoprobes
In the embodiment, 20 μ L of glycoprotein 125 antibody with the concentration of 0.2mg/mL is weighed and added into the rhodamine 6G labeled magnetic nano molybdenum disulfide solution, the mixture is uniformly mixed and reacts at the temperature of 4 ℃ for 2 hours, and the incubation treatment A is completed, so that the antibody is linked with the magnetic nano molybdenum disulfide; then carrying out magnetic separation treatment to remove redundant glycoprotein 125 antibodies to obtain a mixed material B; and then, weighing 10 mu L of bovine serum albumin blocking solution with the mass concentration of 3% and adding the bovine serum albumin blocking solution into the mixed material B, uniformly mixing, reacting for 1h at the temperature of 37 ℃, and finishing the culture treatment C so as to block the non-specific adsorption sites on the immunoprobe and obtain the immunoprobe.
1.4 preparation of an immunoprobe solution
In this embodiment, the immunoprobe is washed by a phosphate buffer solution of N-hydroxysuccinimide at a concentration of 0.02mol/L, and the molybdenum disulfide nanoflower is magnetically collected by a magnet and washed for 4 times to remove excess bovine serum albumin, and then the bovine serum albumin is dissolved in 1mL of phosphate buffer solution to obtain an immunoprobe solution, so as to improve the detection effect of the probe.
In this example, the use of phosphate buffered saline solution for the preparation of the immunoprobe solution has the following effects: the function of dissolving the protective reagent is realized; the reason is that phosphate buffer has a salt balance, a suitable pH buffering action that can be adjusted. 1. Other materials cannot be used for replacement, for example, distilled water does not have salt balance effect and can destroy the structure and biological characteristics of the biological protein; the physiological saline has no effect of adjusting pH, and the complete and active substance can not be ensured to participate in biological reaction under the optimal condition, so the phosphate buffer solution is preferred.
Step 2, preparing an immune substrate
2.1 preparation of carbon-three-nitrogen-four-nanosheets
The invention adopts the following steps to obtain the carbon three nitrogen four nano-sheet:
25mg of carbon-nitrogen-tetrapowder is added into 15mL of concentrated hydrochloric acid and ultrasonic sound is carried out for 2h under the ultrasonic power of 800W. Then, the mixed solution was transferred to a 25mL reaction vessel and reacted at 80 ℃ for 12 hours. Then, the mixture is washed with a large amount of deionized water until the washing solution is neutral, so as to remove the redundant hydrochloric acid. The acidified C, N, C, N and C are dispersed into 5mL of deionized water under the ultrasonic action (the ultrasonic power is 800W, and the ultrasonic time is 5-10 min). Centrifuging the dispersed carbon-nitrogen-III for 10min at the rotating speed of 3000r/min, sinking the un-peeled large carbon-nitrogen-III at the bottom of the wall of the test tube, leaving the protonated carbon-nitrogen-III-IV nanosheet in a supernatant, wherein the supernatant is the solution for obtaining the carbon-nitrogen-III-IV nanosheet, and obtaining the carbon-nitrogen-III-IV nanosheet with the diameter of 50-300 nm.
2.2 preparation of immune-based solutions
2.2.1 activated carbon, three, four:
dispersing by adopting an ultrasonic mode, taking N-hydroxysuccinimide phosphate buffer salt solution as phosphate buffer salt solution of an imine compound, weighing 1mL of N-hydroxysuccinimide phosphate buffer salt solution with the concentration of 0.02mol/L, adding the carbon-nitrogen-tetrad solution obtained by the step 2.1, performing ultrasonic treatment for 3min under the ultrasonic power of 800W to uniformly disperse, and reacting for 1h at the temperature of 37 ℃ to activate the carbon-nitrogen-tetrad nanosheet, further improving the coupling efficiency of protein, and obtaining the activated carbon-nitrogen-tetrad nanosheet solution.
2.2.2 formation of immune substrate:
weighing 20 mu L of glycoprotein 125 antibody with the concentration of 0.2mg/mL, adding the glycoprotein 125 antibody into the 1mL of activated carbon-nitrogen-tetrananosheet solution, uniformly mixing, and culturing at the culture temperature of 4 ℃ for 2h to obtain a mixed material C; centrifuging the mixed material C at 8000r/min for 10min to remove excessive glycoprotein 125 antibody; then adding 10 mu L of bovine serum albumin phosphate buffer solution with the mass concentration of 3%, and then incubating for 1h at room temperature to realize that the antibody is linked on the carbon-nitrogen-carbon substrate to complete incubation treatment B; and then, separating the product after the incubation treatment B in a centrifugal mode, and removing excessive bovine serum albumin by centrifugally washing the product after the incubation treatment B twice to obtain the immune substrate with the concentration of 1-2 mg/mL.
2.2.3 preparation of immune-based solutions
And (3) carrying out ultrasonic treatment on the immune substrate for 5-10 min under the ultrasonic power of 800W to dissolve the immune substrate in 1mL of phosphate buffer solution to obtain the carbon-nitrogen-four immune substrate solution.
Step 3, preparing SERS biosensor with sandwich structure
Weighing 10-30 mu L of glycoprotein 125 antigen with the initial concentration of 100IU/mL, adding the glycoprotein 125 antigen into 1mL of the carbon-three-nitrogen-four immune substrate solution, and incubating at the incubation temperature of 37 ℃ for 2h to link the glycoprotein 125 antigen on the carbon-three-nitrogen-four substrate so as to realize incubation treatment C; subsequently, the solid components obtained in the incubation treatment C (the solid components sink on the test tube wall, and the unlinked antigens are sucked away in the solution) were centrifuged at 8000r/min for 10min by using a phosphate buffer solution to remove the uncaptured antigens, obtaining a mixed solution a; then, 0.5-1.5 mL of the above immunoprobe solution is weighed and added into the above mixed solution A, and cultured at a culture temperature of 37 ℃ for 2h, and an incubation treatment D is completed, so as to form the SERS biosensor with a sandwich structure based on the specific immunoreaction between the antigen and the antibody.
It should be noted that the SERS biosensor with sandwich structure prepared in this example is stored in 1mL of phosphate buffer solution for subsequent SERS detection. After SERS detection is performed on the SERS biosensor with the sandwich structure, the immune structure is catalyzed by a visible light simulator, and Raman molecules, antigens and antibodies can be degraded based on good photocatalytic activity of the molybdenum disulfide nanoflowers and the carbon-nitrogen-carbon nanosheets. And after the catalysis is finished, separating and collecting the immunoprobe and the immunological substrate by adopting a magnet, and continuously detecting the subsequent tumor marker. The above steps are repeated to realize the circulating immunoassay.
Example 2
The present embodiment provides a SERS biosensor based on carbon-nitrogen nanosheets, and the preparation method thereof differs from embodiment 1 only in that:
in step 1 of this example, the dosage of the ferroferric oxide particles is 62.8mg.
Example 3
The present embodiment provides a SERS biosensor based on carbon-nitrogen nanosheets, and the preparation method thereof differs from embodiment 1 only in that:
in step 1 of this example, the dosage of the ferroferric oxide particles is 125.6mg.
Example 4
The present embodiment provides a SERS biosensor based on carbon-nitrogen nanosheets, and the preparation method thereof differs from embodiment 1 only in that:
in step 1.1.1 of this example: ferrous sulfate heptahydrate is used as an iron source, and sodium citrate is used as a stabilizer;
in step 1.1.2 of this example:
sodium molybdate is used as molybdate, thioacetamide is used as a sulfur source, and the molar ratio of the molybdate to the sulfur source and ferroferric oxide is 1.5;
the temperature of the hydrothermal reaction is 180 ℃, and the reaction time is 12h;
in step 1.2 of this example:
methylene blue is used as a Raman molecule, and the dosage ratio of the Raman molecule to the magnetic nano molybdenum disulfide is 10g;
the treatment temperature of the culture treatment A is 20 ℃, and the treatment time is 14h;
the treatment temperature of the culture treatment B was 36 ℃ and the treatment time was 2 hours.
In step 1.3 of this example:
the mass ratio of the glycoprotein 125 antibody to the rhodamine 6G marked magnetic nano molybdenum disulfide is 2;
the incubation treatment A is carried out at the treatment temperature of 3 ℃ for 2.5h;
the treatment temperature of the culture treatment C was 36 ℃ and the treatment time was 2 hours.
In step 1.4 of this example:
the dosage ratio of the molybdenum disulfide nanoflower immunoprobe to the phosphate buffer solution is 10mg.
In step 2.1 of this example:
the dosage ratio of the carbon-nitrogen-containing four powder to the concentrated hydrochloric acid is 2mg, 1mL, and the reaction is carried out for 20 hours at the acidification temperature of 70 ℃;
the dosage ratio of the hydrosolvent to the carbon-nitrogen-four powder is 1-2mg.
In step 2.2.1 of this example:
the temperature of the activation treatment is 36 ℃, and the treatment time is 2h;
in step 2.2.2 of this example:
the dosage ratio of the glycoprotein 125 antibody to the activated carbon-nitrogen four-nanosheet solution is 0.0026mg;
the incubation treatment B is carried out at the treatment temperature of 3 ℃ for 2.5h;
in step 2.2.3 of this example:
the dosage ratio of the immune substrate to the phosphate buffer solution is 1mg;
in step 3 of this embodiment:
the concentration of the glycoprotein 125 antigen is 80IU/mL, and the dosage ratio of the glycoprotein 125 antigen to the immune substrate solution is 0.002mg;
the incubation treatment C is carried out at the treatment temperature of 36 ℃ for 3h;
the dosage ratio of the immune probe solution to the mixed solution A is 1.5mL;
the incubation temperature of the incubation treatment D is 36 ℃, and the incubation time is 3h;
and the phosphate buffer solution used in each step is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide phosphate buffer solution with the concentration of 0.07 mol/L.
Example 5
The present embodiment provides a SERS biosensor based on carbon-nitrogen nanosheets, and the preparation method thereof differs from embodiment 1 only in that:
in step 1.1.1 of this example: ferric chloride tetrahydrate is used as an iron source, and sodium acetate is used as a stabilizer.
In step 1.1.2 of this example: taking sodium molybdate as molybdate, wherein the mol ratio of the molybdate to the sulfur source and ferroferric oxide is 2; the temperature of the hydrothermal reaction is 220 ℃, and the reaction time is 8h;
in step 1.2 of this example:
taking crystal violet as a Raman molecule, wherein the dosage ratio of the Raman molecule to the magnetic nano molybdenum disulfide is 3mmol;
the treatment temperature of the culture treatment A is 25 ℃, and the treatment time is 8h;
the treatment temperature of the culture treatment B was 38 ℃ and the treatment time was 0.5 hour.
In step 1.3 of this example:
the mass ratio of the glycoprotein 125 antibody to the rhodamine 6G marked magnetic nano molybdenum disulfide is 6, 1000, namely 30 microliter of the glycoprotein 125 antibody with the concentration of 0.2mg/mL is added into 1mL of the rhodamine 6G marked magnetic nano molybdenum disulfide solution;
the incubation treatment A is carried out at the treatment temperature of 5 ℃ for 1.5h;
the treatment temperature of the culture treatment C was 38 ℃ and the treatment time was 0.5 hour.
In step 1.4 of this example:
the dosage ratio of the molybdenum disulfide nanoflower immunoprobe to the phosphate buffer solution is 10mg.
In step 2.1 of this example:
the dosage ratio of the carbon-nitrogen-containing four powder to the concentrated hydrochloric acid is 3mg, namely 2mL, and the reaction is carried out for 8 hours at the acidification temperature of 90 ℃;
the dosage ratio of the water solvent to the carbon-nitrogen-tetrapowder is 2mg.
In step 2.2.1 of this example:
the temperature of the activation treatment is 38 ℃, and the treatment time is 0.5h;
in step 2.2.2 of this example:
the dosage ratio of the glycoprotein 125 antibody to the activated carbon-nitrogen four-nanosheet solution is 0.006mg;
the incubation treatment B is carried out at the treatment temperature of 5 ℃ for 1.5h;
in step 2.2.3 of this example:
the dosage ratio of the immune substrate to the phosphate buffer solution is 2mg;
in step 3 of this embodiment:
the concentration of the glycoprotein 125 antigen is 120IU/mL, and the dosage ratio of the glycoprotein 125 antigen to the immune substrate solution is 0.006mg;
the incubation treatment C is carried out at the treatment temperature of 38 ℃ for 1h;
the dosage ratio of the immunoprobe solution to the mixed solution A is 1.3mL;
the incubation temperature of the incubation treatment D is 38 ℃, and the incubation time is 1h;
and the phosphate buffered saline solution used in each step was 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide phosphate buffered saline solution at a concentration of 0.09 mol/L.
Test section
(I) scanning electron microscope testing
Scanning electron microscope tests were performed on the immunoprobes prepared in example 1, example 2 and example 3, respectively, and the immuno-substrate and SERS biosensor prepared in example 1, and the results are shown in fig. 1-5, respectively.
Fig. 1 is a scanning electron microscope photograph of the immunoprobe prepared in example 1, and it can be seen that, in the immunoprobe prepared in example 1, molybdenum disulfide nanosheets grow on the surface of the ferroferric oxide particles. Because less ferroferric oxide particles are added, a lot of molybdenum disulfide exists independently.
Fig. 2 is a scanning electron micrograph of the immunoprobe prepared in example 2, and it can be seen that more molybdenum disulfide nanoflowers were formed in the immunoprobe prepared in example 2 as the added amount of the ferroferric oxide particles was increased.
Fig. 3 is a scanning electron microscope photograph of the immunoprobe prepared in example 3, and it can be seen that, in the immunoprobe prepared in example 3, as the number of the ferroferric oxide particles continues to increase, the ferroferric oxide particles are embedded in molybdenum disulfide nanosheets.
Fig. 4 is a scanning electron micrograph of the immuno-substrate prepared in example 1, and it can be seen that the immuno-substrate prepared in example 1 has a smaller and larger surface area after the carbonitridation.
Fig. 5 is a scanning electron microscope photograph of the SERS biosensor prepared in example 1, and it can be seen that, in the SERS biosensor prepared in example 1, due to the specific reaction between glycoprotein 125 antigen and antibody, the magnetic nano molybdenum disulfide and carbon-nitrogen nanosheet are tightly bound together.
(II) Raman test
In the invention, the immunological probes and the immunological substrates prepared in the embodiments 1, 2 and 3 and the SERS biosensor prepared in the embodiment 1 are subjected to Raman detection of rhodamine 6G under 532nm laser, and the tests are respectively shown in FIGS. 6 to 9.
FIG. 6 shows the Raman detection results of the immuno-probe and the immuno-substrate prepared in example 1 on rhodamine 6G, and it can be seen that the immuno-probe and the immuno-substrate prepared in example 1 both have a good Raman enhancement effect on rhodamine 6G, which is 1360cm -1 The raman signal intensity at position 6913 and 5041 respectively.
FIG. 7 shows the Raman detection result of the immuno probe prepared in example 2 on rhodamine 6G, and it can be seen that the immuno probe prepared in example 2 has a good Raman enhancement effect on rhodamine 6G, which is 1360cm -1 The raman signal intensity reached 8246.
FIG. 8 shows the Raman detection result of the immuno-probe prepared in example 3 on rhodamine 6G, and it can be seen that the immuno-probe prepared in example 3 has an SERS enhancement effect on rhodamine 6G, which is 1360cm -1 The raman signal intensity reached 6959.
Fig. 9 shows the raman detection result of the SERS biosensor prepared in example 1 on the glycoprotein 125 in serum, and it can be seen that the SERS biosensor prepared in example 1 can realize ultra-sensitive detection of the glycoprotein 125 in serum.
It is to be understood that the above-described embodiments are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Claims (10)
1. The SERS biosensor based on the carbon-nitrogen-carbon four nanosheets is characterized by consisting of an immunological probe, an immunological substrate and a glycoprotein 125 antigen;
the immunoprobe is obtained by linking glycoprotein 125 antibody on functionalized marked magnetic nano molybdenum disulfide, and the diameter of the magnetic nano molybdenum disulfide is 100-1000 nm;
the immune substrate is obtained by linking the glycoprotein 125 antibody on an activated carbon three nitrogen four nanosheet, and the diameter of the carbon three nitrogen four nanosheet is 50-300 nm;
and the immune probe and the glycoprotein 125 antibody on the immune substrate are specifically combined with the glycoprotein 125 antigen to form the SERS biosensor with a sandwich structure.
2. The SERS biosensor of claim 1, wherein the functionalized labeled magnetic nano molybdenum disulfide is obtained by:
marking magnetic nano molybdenum disulfide by adopting Raman molecules to obtain the magnetic nano molybdenum disulfide marked by functionalization;
the Raman molecule is any one of rhodamine 6G, methylene blue and crystal violet, and the dosage ratio of the Raman molecule to the magnetic nano molybdenum disulfide is 1-3mmol.
3. The SERS biosensor of claim 1, wherein the magnetic nano molybdenum disulfide is prepared by the following method:
and uniformly dispersing molybdate, a sulfur source and ferroferric oxide in a solvent B, carrying out hydrothermal reaction at the temperature of 180-220 ℃ for 6-12 h, cooling to room temperature, washing and drying to obtain the magnetic nano molybdenum disulfide with ferroferric oxide coated by molybdenum disulfide.
4. The SERS biosensor of claim 3, wherein the molybdate is sodium molybdate or ammonium molybdate;
the sulfur source is thioacetamide or thiourea;
the molar ratio of the molybdate to the sulfur source to the ferroferric oxide is (1.5-2);
the dosage ratio of the molybdate to the solvent B is 0.15-0.2mol.
5. The SERS biosensor of claim 1, wherein the activated carbon trion-nitrogen tetrananosheets are obtained by:
s1, adding carbon-nitrogen-IV powder into concentrated hydrochloric acid, performing ultrasonic treatment, and reacting at 70-90 ℃ for 8-20 h to obtain acidified carbon-nitrogen-IV;
wherein the dosage ratio of the carbon-nitrogen-containing four powder to the concentrated hydrochloric acid is 2-3mg;
s2, dispersing the acidified C, N and C in a water solvent under the ultrasonic action, and then carrying out centrifugal treatment to obtain supernatant, namely a C, N and C nanosheet solution;
wherein the dosage ratio of the hydrosolvent to the carbon-nitrogen-tetrapowder is 1-2mg;
s3, uniformly dispersing the carbon-nitrogen-rich nanosheet solution and an imine compound in a phosphate buffer solution, and reacting at the temperature of 36-38 ℃ for 1-3 h to obtain an activated carbon-nitrogen-rich nanosheet solution;
wherein the imine compound is N-hydroxysuccinimide or 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide.
6. The SERS biosensor as recited in claim 5, wherein the ratio of the amount of the glycoprotein 125 antibody to the activated carbon-trinitrogen tetrasheet solution is 0.002-0.006mg.
7. A method for preparing the SERS biosensor according to any one of claims 1 to 6, comprising the steps of:
step 1, preparing an immunoprobe:
adding a glycoprotein 125 antibody into the functionalized and marked magnetic nano molybdenum disulfide, and then carrying out incubation treatment A to link the glycoprotein 125 antibody on the functionalized and marked magnetic nano molybdenum disulfide to obtain the immunoprobe;
step 2, preparing an immune substrate:
adding a glycoprotein 125 antibody into the activated carbon-three-nitrogen four-nano sheet for incubation treatment B, so as to link the glycoprotein 125 antibody on the activated carbon-three-nitrogen four-nano sheet, thereby obtaining an immune substrate;
step 3, preparing the SERS biosensor with the sandwich structure:
dispersing an immune substrate in a phosphate buffer solution A to form an immune substrate solution, and then adding a glycoprotein 125 antigen to perform incubation treatment C to obtain a mixed solution A;
and dispersing the immune probe in a phosphate buffer solution B to form an immune probe solution, and then adding the immune probe solution into the mixed solution A for incubation treatment D to obtain the SERS biosensor.
8. The preparation method of claim 1, wherein the mass ratio of the glycoprotein 125 antibody to the functionalized and labeled magnetic nano molybdenum disulfide is 2-6;
the dosage ratio of the immunological substrate to the phosphate buffer solution A is 1-2mg;
the dosage ratio of the immunoprobe to the phosphate buffer solution B is 10mg;
the dosage ratio of the glycoprotein 125 antigen to the immune substrate solution is 0.002-0.006mg;
the dosage ratio of the immunoprobe solution to the mixed solution A is 1-1.5mL;
the concentration of the glycoprotein 125 antigen is 80-120 IU/mL.
9. The method according to claim 1, wherein the incubation treatment A is carried out at a temperature of 3 to 5 ℃ for 1.5 to 2.5 hours;
the incubation treatment B is carried out at the treatment temperature of 3-5 ℃ for 1.5-2.5 h;
the incubation treatment C is carried out at the treatment temperature of 36-38 ℃ for 1-3 h;
the incubation temperature of the incubation treatment D is 36-38 ℃, and the incubation time is 1-3 h.
10. The use of the SERS biosensor as claimed in any one of claims 1-6 in the preparation of a test agent for detecting ovarian tumor markers.
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