Dual-mode immune structure based on titanium carbide SERS substrate and rare earth-doped sodium yttrium fluoride nanoparticles, preparation method therefor, and use thereof

Abstract

A dual-mode immune structure based on a titanium carbide SERS substrate and rare earth-doped sodium yttrium fluoride nanoparticles, a preparation method therefor, and the use thereof. The dual-mode immune structure comprises a titanium carbide / molybdenum disulfide composite immune substrate and a NaYF4:Yb, Er immune probe, the NaYF4:Yb, Er containing 78% of Y, 20% of Yb and 2% of Er. The preparation method comprises the following steps: (S10) using acid etching to prepare titanium carbide powder; (S20) preparing a titanium carbide / molybdenum disulfide composite immune substrate; and (S30) preparing a NaYF4:Yb, Er immune probe. The dual-mode immune structure is suitable for the use of prostate cancer detection, achieves high efficiency, high accuracy and high sensitivity in detection, and is suitable for clinical promotion and use.

Description

A dual-mode immune structure based on titanium carbide SERS substrate and rare earth doped sodium fluoroacetate nanoparticles and a preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of prostate cancer detection, and particularly relates to a dual-mode immune structure based on titanium carbide SERS substrate and rare earth doped sodium fluoroacetate nanoparticles and a preparation method and application thereof. BACKGROUND

[0002] In recent years, cancer has become an important factor of premature death and poses a serious threat to people's health. Prostate cancer is the most common malignant tumor and has been recognized as the third leading cause of male cancer-related death. Early detection and dynamic monitoring are crucial for effective treatment of most cancer patients, among which prostate specific antigen (PSA) has been widely recognized as a key biomarker. However, the current mainstream detection methods of PSA are relatively single, with long cycle, complicated operation steps, low detection accuracy and other problems. Compared with traditional detection methods, surface enhanced Raman scattering (SERS) as a highly efficient spectroscopic detection technology has extremely high detection sensitivity and molecular fingerprint recognition ability, and also has good application prospect in clinical detection. Upconversion luminescent material is a kind of luminescent material that can emit visible light under near-infrared light excitation, and is a luminescent material based on anti-Stokes principle. At present, upconversion luminescent material has a wide range of applications in biological fluorescence imaging, three-dimensional display and anti-counterfeiting. Upconversion luminescent material is usually composed of two parts: crystal matrix material and doped rare earth ions, among which fluoride and halide (halide oxide) have lower phonon energy and higher upconversion luminescence performance, and are the most common upconversion luminescent material matrix. At present, the preparation of rare earth doped fluoride upconversion nanoluminescent material has a relatively narrow absorption spectrum and emission spectrum, and is particularly suitable for specific marker testing in the medical field.

[0003] However, there is no report on the combination of titanium carbide SERS substrate and rare earth doped sodium fluoroacetate nanoparticles for dual-mode detection of cancer markers. In fact, the dual-mode detection technology based on upconversion luminescence and surface enhanced Raman scattering can realize the complementary advantages of the two detection methods and further improve the accuracy and sensitivity of cancer detection.

[0004] SUMMARY

[0005] An advantage of the present application is to provide a dual-mode immune structure based on titanium carbide SERS substrate and rare earth doped sodium fluoroacetate nanoparticles and a preparation method and application thereof, which can improve the efficiency and sensitivity of cancer detection and has important significance for early screening of cancer.

[0006] Another advantage of the present application is to provide a dual-mode immune structure based on titanium carbide SERS substrate and rare earth doped sodium fluoroethide nanoparticles and its preparation method and application.

[0007] Another advantage of the present application is to provide a dual-mode immune structure based on titanium carbide SERS substrate and rare earth doped sodium fluoroethide nanoparticles and its preparation method and application, compared with traditional fluorescent materials, upconversion materials can emit short-wave light under red or infrared light excitation as a fluorescent material, can convert low-energy long-wave light into high-energy short-wave light, and have a narrow absorption spectrum and emission spectrum, especially suitable for specific marker testing in the medical field.

[0008] Another advantage of the present application is to provide a dual-mode immune structure based on titanium carbide SERS substrate and rare earth doped sodium fluoroethide nanoparticles and its preparation method and application, SERS technology as another efficient spectral detection technology, has very high detection sensitivity and molecular fingerprint recognition ability, and has good application prospect in clinical detection, and has important significance for improving the accuracy and sensitivity of cancer detection.

[0009] Another advantage of the present application is to provide a dual-mode immune structure based on titanium carbide SERS substrate and rare earth doped sodium fluoroethide nanoparticles and its preparation method and application, the sandwich immune structure obtained after the above immune reaction is measured by a fluorescence spectrometer or a Raman spectrometer, and the concentration of the cancer marker antigen to be detected is calculated according to the linear relationship between the cancer marker antigen concentration and the fluorescence characteristic peak or the Raman characteristic peak intensity, and the result is more accurate.

[0010] Another advantage of the present application is to provide a dual-mode immune structure based on titanium carbide SERS substrate and rare earth doped sodium fluoroethide nanoparticles and its preparation method and application, the preparation method is simple, low in cost, convenient to use, and suitable for clinical popularization and application.

[0011] Another advantage of the present application is to provide a dual-mode immune structure based on titanium carbide SERS substrate and rare earth doped sodium fluoroethide nanoparticles and its preparation method and application, surface enhanced Raman scattering combined with the dual-mode structure in the present application improves the accuracy of the dual-mode detection technology of the present application.

[0012] According to one aspect of the present application, the present application provides a preparation method of a dual-mode immune structure based on titanium carbide SERS substrate and rare earth doped sodium fluoroethide nanoparticles, comprising the following steps:

[0013] (S10) preparing titanium carbide powder by acid etching method;

[0014] (S20) preparing titanium carbide / molybdenum disulfide composite immunization substrate; and

[0015] (S30) preparing NaYF4:Yb, Er immunization probe.

[0016] The step (S10) comprises the following steps: (S101) preparing hydrochloric acid solution containing lithium fluoride; (S102) adding titanium aluminum carbide powder into the above solution, and obtaining titanium carbide solution after reaction; (S103) centrifuging and washing the titanium carbide solution with dilute hydrochloric acid, and then washing with deionized water, and obtaining supernatant by ultrasonic treatment and centrifugation of the washed solution in ice bath; (S104) drying the supernatant to obtain titanium carbide (MXene) powder.

[0017] In the step (S101), 1-3 g of lithium fluoride is added into 20 mL of 9 mmol / mL hydrochloric acid, and stirring is continued at room temperature for 10 min; in the step (S102), 1-3 g of titanium aluminum carbide powder is slowly added into the solution containing lithium fluoride and hydrochloric acid while stirring, and the water bath is heated to 45°C, and the reaction is continued for 24-48 h to obtain titanium carbide solution; in the step (S103), the titanium carbide solution is centrifuged and washed with 1 mmol / mL dilute hydrochloric acid for three times, and then repeatedly washed with deionized water until the supernatant has a pH of 6-7.

[0018] The step (S20) comprises the following steps: (S201) dissolving ammonium molybdate and thiourea in deionized water, and then adding titanium carbide and ultrasonic dispersing to obtain a mixed solution; (S202) placing the mixed solution in a polytetrafluoroethylene-lined stainless steel autoclave, heating, cooling and centrifuging after reaction to obtain black precipitate; (S203) washing the black precipitate with ethanol and deionized water, and vacuum drying to obtain titanium carbide / molybdenum disulfide (MXene / MoS2) composite; (S204) adding the MXene / MoS2 composite in step (S203) into deionized water to prepare a solution, and dropping the solution on a silicon wafer, soaking the silicon wafer in DMF, and then washing with phosphate buffer solution; (S205) dropping the phosphate buffer solution containing NHS / EDC on the titanium carbide / molybdenum disulfide composite substrate; (S206) dropping the solution containing antibodies, soaking, and washing to remove excess unreacted antibodies to obtain MXene / MoS2 composite immunization substrate.

[0019] The step (S20) comprises the following steps: (S201) dissolving ammonium molybdate and thiourea in deionized water, and then adding titanium carbide to ultrasonically disperse into a mixed solution; (S202) placing the mixed solution in a polytetrafluoroethylene-lined stainless steel autoclave for heating, and then cooling and centrifuging after the reaction is completed to obtain a black precipitate; (S203) washing the black precipitate with ethanol and deionized water, and vacuum drying to obtain a titanium carbide / molybdenum disulfide (MXene / MoS2) composite; (S204) adding the MXene / MoS2 composite in the step (S203) into deionized water to prepare a solution, dropping the solution on a silicon wafer, immersing the silicon wafer in DMF, and then washing with a phosphate buffer solution; (S205) dropping a phosphate buffer solution of NHS / EDC on the titanium carbide / molybdenum disulfide composite substrate; (S206) dropping a solution containing an antibody, immersing, and washing to remove excess unreacted antibody to obtain a MXene / MoS2 composite immunization substrate.

[0020] In the step (S201), the ammonium molybdate is 15-20 mg, the thiourea is 30-50 mg, and the titanium carbide is 15-25 mg; in the step (S202), the mixed solution is heated to 200℃ in the polytetrafluoroethylene-lined stainless steel autoclave for 24 h, and then naturally cooled to room temperature after the reaction is completed; in the step (S203), the obtained black precipitate is washed with ethanol and deionized water for 4-6 times, and then vacuum dried at 60℃ for 12 h to obtain the titanium carbide / molybdenum disulfide (MXene / MoS2) composite.

[0021] In the step (S204), the MXene / MoS2 composite is added into deionized water at a mass-volume ratio of 1 mg:100 μL to prepare a solution, 10-30 μL of the solution is dropped on the silicon wafer, the silicon wafer is immersed in DMF for 2 h, and then washed with a phosphate buffer solution multiple times; in the step (S205), 1 mL of a phosphate buffer solution of NHS / EDC is dropped on the titanium carbide / molybdenum disulfide composite substrate, wherein the NHS / EDC is 1:1 and 10 mg / mL; in the step (S206), 10 μL of a solution containing a PSA antibody is dropped, immersed at room temperature for 2 h, and then washed to remove excess unreacted antibody and stored at 4℃ to obtain the MXene / MoS2 composite immunization substrate.

[0022] The step (S30) comprises the following steps: (S301) mixing and stirring the rare earth nitrate RE(NO3)3·6H2O with the aqueous citric acid solution; (S302) adding and stirring the sodium hydroxide solution, and then adding the sodium fluoride solution to obtain a colloidal suspension; (S303) transferring the colloidal suspension to a stainless steel reaction kettle with a polytetrafluoroethylene lining to react, cooling after the reaction is completed, and centrifuging to obtain a reaction product; (S304) washing the product with ethanol and deionized water, and drying; (S305) mixing and reacting the NaYF4:Yb,Er solution in the R6G solution; (S306) washing away the excess R6G, and then adding the NHS / EDC phosphate buffer solution, and incubating; (S307) after washing with the phosphate buffer solution, adding the solution containing the antibody, incubating, and washing to remove the excess unreacted antibody, to obtain the NaYF4:Yb,Er (78%Y, 20%Yb, 2%Er) immunoprobe.

[0023] In the step (S301), the rare earth nitrate is RE(NO3)3·6H2O (RE=78%Y, 20%Yb, 2%Er); in the step (S302), the obtained colloidal suspension is transferred to a stainless steel reaction kettle with a polytetrafluoroethylene lining after being continuously stirred for 30 min, and is reacted at 180°C for 12 h, and is naturally cooled to room temperature after the reaction is completed; in the step (S304), the reaction product is obtained by centrifuging and is repeatedly washed with ethanol and deionized water for 6 times, and the product is dried at 80°C for 12 h; in the step (S305), the NaYF4:Yb,Er solution is mixed and reacted in 5-10 mL of 1 mM R6G solution for 15 min, and the excess R6G is washed away; in the step (S306), the ratio of NHS / EDC is 1:1, and the concentration is 10 mg / mL.

[0024] In the step (S307), the solution containing the PSA antibody is added and incubated for 1 h, and the excess unreacted antibody is removed after washing, to obtain the NaYF4:Yb,Er immunoprobe.

[0025] Further comprising a step (S40) of assembling a cancer marker system: adding the phosphate buffer solution containing the to-be-detected antigen to the MXene / MoS2 composite immunization substrate dropwise, standing, allowing the immunoreaction between the antigen and the antibody, and washing to remove the excess unreacted to-be-detected antigen; then adding the NaYF4:Yb,Er immunoprobe to the MXene / MoS2 composite immunization substrate adsorbing the to-be-detected antigen, reacting at 37°C, and washing to remove the excess unreacted NaYF4:Yb,Er immunoprobe, to obtain the cancer marker detection system based on MXene / MoS2 and NaYF4:Yb,Er.

[0026] According to another aspect of the present application, the present application also provides a dual-mode immune structure based on a titanium carbide SERS substrate and rare earth-doped sodium fluoroethide nanoparticles, characterized in that it comprises a titanium carbide / molybdenum disulfide composite immune substrate and a NaYF4:Yb,Er immune probe, wherein the NaYF4:Yb,Er contains 78% Y, 20% Yb and 2% Er.

[0027] According to another aspect of the present application, the present application also provides an application of a dual-mode immune structure based on a titanium carbide SERS substrate and rare earth-doped sodium fluoroethide nanoparticles, wherein the dual-mode immune structure based on a titanium carbide SERS substrate and rare earth-doped sodium fluoroethide nanoparticles is suitable for application in detection of prostate cancer, colorectal cancer, ovarian cancer or pancreatic cancer.

[0028] The dual-mode immune structure based on a titanium carbide SERS substrate and rare earth-doped sodium fluoroethide nanoparticles comprises a titanium carbide / molybdenum disulfide composite immune substrate and a NaYF4:Yb,Er immune probe, wherein the NaYF4:Yb,Er contains 78% Y, 20% Yb and 2% Er, and the preparation method comprises the following steps: (S10) preparing a titanium carbide powder by using an acid etching method; (S20) preparing a titanium carbide / molybdenum disulfide composite immune substrate; and (S30) preparing a NaYF4:Yb,Er immune probe.

[0029] In the application process of the dual-mode immune structure based on a titanium carbide SERS substrate and rare earth-doped sodium fluoroethide nanoparticles, a phosphate buffer solution containing an antigen to be detected is added dropwise to the MXene / MoS2 composite immune substrate, which is placed at 37°C for 2h to allow an immune reaction between the antigen and the antibody, and the excess unreacted antigen to be detected is removed by washing; then 20-40μL of the NaYF4:Yb,Er immune probe is added dropwise to the MXene / MoS2 composite immune substrate adsorbed with the antigen to be detected, and the reaction is carried out at 37°C for 2h, and the excess unreacted NaYF4:Yb,Er immune probe is removed by washing, thereby obtaining a cancer marker detection system based on MXene / MoS2 and NaYF4:Yb,Er; the compound of the NaYF4:Yb,Er immune probe and the MXene / MoS2 immune substrate obtained after the immune reaction is measured by using a Raman spectrometer, and the concentration of the cancer marker antigen to be detected is calculated according to the linear relationship between the cancer marker antigen concentration and the Raman characteristic peak intensity. BRIEF DESCRIPTION OF DRAWINGS

[0030] FIG. 1 is a scanning electron microscope photo of the MXene / MoS2 composite immune substrate prepared in Example 1.

[0031] FIG. 2 is a scanning electron microscope photo of the MXene / MoS2 composite immune substrate prepared in Example 2.

[0032] Figure 3 is a scanning electron microscope photograph of the MXene / MoS2 composite immunobase prepared in Example 3.

[0033] Figure 4 is a scanning electron microscope photograph of the NaYF4:Yb,Er immunoprobes prepared in Example 1.

[0034] Figure 5 is a scanning electron microscope photograph of the NaYF4:Yb,Er immunoprobes prepared in Example 2.

[0035] Figure 6 is a scanning electron microscope photograph of the NaYF4:Yb,Er immunoprobes prepared in Example 3.

[0036] Figure 7 is a SERS spectrum of the MXene / MoS2-NaYF4:Yb,Er nanomaterial prepared in Example 1 for detecting different concentrations of prostate cancer.

[0037] Figure 8 is a SERS spectrum of the MXene / MoS2-NaYF4:Yb,Er nanomaterial prepared in Example 2 for detecting different concentrations of prostate cancer.

[0038] Figure 9 is a SERS spectrum of the MXene / MoS2-NaYF4:Yb,Er nanomaterial prepared in Example 3 for detecting different concentrations of prostate cancer.

[0039] Figure 10 is a upconversion spectrum of the MXene / MoS2-NaYF4:Yb,Er nanomaterial prepared in Example 1 for detecting different concentrations of prostate cancer.

[0040] Figure 11 is a upconversion spectrum of the MXene / MoS2-NaYF4:Yb,Er nanomaterial prepared in Example 2 for detecting different concentrations of prostate cancer.

[0041] Figure 12 is a upconversion spectrum of the MXene / MoS2-NaYF4:Yb,Er nanomaterial prepared in Example 3 for detecting different concentrations of prostate cancer. DETAILED DESCRIPTION

[0042] The following description is presented to enable any person skilled in the art to practice the application as claimed. The preferred embodiments disclosed herein are only examples of the application and alternative embodiments, modifications, improvements, equivalents, and the like can be made thereto without departing from the spirit and scope of the application as set forth in the following claims. The present application is defined by the claims.

[0043] Example 1

[0044] A preparation method of a dual-mode immune structure based on a titanium carbide SERS substrate and a rare earth doped sodium fluoroethide nanoparticle, comprising the following steps:

[0045] (1) Titanium carbide powder is prepared by acid etching method

[0046] 1g of lithium fluoride is added to 20mL of hydrochloric acid (9mmol / mL), and stirring is continued at room temperature for 10min, 1g of titanium aluminum carbide powder is slowly added to the solution containing lithium fluoride and hydrochloric acid while stirring, and water bath heating is performed to 45℃, and the reaction is continued for 24h to obtain a titanium carbide solution. The titanium carbide solution is washed with dilute hydrochloric acid (1mmol / mL) by centrifugation three times, and then washed repeatedly with deionized water until the supernatant has a pH of 6-7. The washed solution is ice-bathed and ultrasonicated for 1h, then centrifuged at 6000rpm for 10min to obtain the supernatant, and then the supernatant is dried at 60℃ for 12h to obtain titanium carbide (MXene) powder.

[0047] (2) Preparation of titanium carbide / molybdenum disulfide composite immune substrate

[0048] A. 15mg of ammonium molybdate and 30mg of thiourea are dissolved in 4mL of deionized water, and then slowly added to a 4mL solution containing 15mg of titanium carbide, and ultrasonically dispersed for 10min. The mixed solution is placed in a polytetrafluoroethylene-lined stainless steel autoclave, heated to 200℃, and kept for 24h. After the reaction is completed, it is naturally cooled to room temperature, centrifuged, and the black precipitate obtained is washed with ethanol and deionized water 4-6 times, and then vacuum dried at 60℃ for 12h to obtain a titanium carbide / molybdenum disulfide (MXene / MoS2) composite.

[0049] B. The MXene / MoS2 composite in step (2) A is added to deionized water in a mass-volume ratio of 1mg:100μL to prepare a solution, 10μL of the solution is added to a silicon wafer, the silicon wafer is soaked in DMF for 2h, then washed with phosphate buffer solution several times, then 1mL of NHS / EDC (1:1, 10mg / mL) phosphate buffer is added to the titanium carbide / molybdenum disulfide composite substrate. Then, 10μL of a solution containing PSA antibody is added, soaked at room temperature for 2h, and the excess unreacted antibody is removed by washing and stored at 4℃ to obtain a MXene / MoS2 composite immune substrate.

[0050] (3) Preparation of NaYF4:Yb,Er(78%Y,20%Yb,2%Er) immune probe

[0051] A. First, 1 mmol of rare earth nitrate RE(NO3)3·6H2O (RE = 78% Y, 20% Yb, 2% Er) was mixed with 10 mL of citric acid aqueous solution (0.4 mmol / mL) and stirred for 1 h. Then, 0.2 mL of sodium hydroxide solution (5 mmol / mL) was added and stirred for 15 min. Next, 8 mL of sodium fluoride solution (1 mmol / mL) was added to obtain a colloidal suspension. The resulting colloidal suspension was stirred for another 30 min and then transferred to a 40 mL stainless steel reactor with a polytetrafluoroethylene liner. The reaction was carried out at 180 °C for 12 h. After the reaction was completed, the mixture was allowed to cool naturally to room temperature. The reactants were centrifuged to obtain the product and washed repeatedly with ethanol and deionized water 6 times. The product was then dried at 80 °C for 12 h.

[0052] B. The NaYF4:Yb,Er solution was mixed in 5 mL of R6G solution (1 mmol / mL) and reacted for 15 min. Excess R6G was washed away. Then, 1 mL of NHS / EDC (1:1, 10 mg / mL) phosphate buffer solution was added and incubated at 37 °C for 1 h. After washing with phosphate buffer, a solution containing PSA antibody was added and incubated for 1 h. After washing away excess unreacted antibody, the NaYF4:Yb,Er immunoprobe was obtained.

[0053] (4) Assembly of cancer biomarker detection system

[0054] A phosphate buffer solution containing the antigen to be tested was added dropwise to the MXene / MoS2 composite immunomodulator and incubated at 37°C for 2 hours to allow for a full immune reaction between the antigen and antibody. Excess unreacted antigen was then washed away. Next, 20 μL of NaYF4:Yb,Er immunoprobe was added dropwise to the MXene / MoS2 composite immunomodulator containing the antigen to be tested and reacted at 37°C for 2 hours. Excess unreacted NaYF4:Yb,Er immunoprobe was then washed away, resulting in a cancer biomarker detection system based on MXene / MoS2 and NaYF4:Yb,Er. Raman spectroscopy was used to measure the spectra of the NaYF4:Yb,Er immunoprobe and the MXene / MoS2 immunomodulator complex obtained after the above immune reaction. The concentration of the cancer biomarker antigen to be tested was calculated based on the linear relationship between the concentration of the cancer biomarker antigen and the intensity of the Raman characteristic peak.

[0055] Figure 1 shows a scanning electron microscope image of the MXene / MoS2 composite prepared in this embodiment. As can be seen from Figure 1, the MXene sheets are encapsulated in a mesh-like structure by the MoS2, forming a composite nanostructure.

[0056] Figure 4 shows the NaYF4:Yb,Er upconversion luminescent nanoparticles prepared in this embodiment. As can be seen from Figure 4, the upconversion luminescent nanoparticles exhibit a spherical shape.

[0057] Figure 7 is a Raman spectrum obtained by Raman detection of the substrate after immunoreaction with NaYF4:Yb,Er upconversion luminescent nanoparticle immunoprobes and MXene / MoS2 composite SERS immunosubstrates and different concentrations of the antigen to be detected (concentrations of 10 -2 mg / mL to 10 -6 mg / mL). As can be seen from the figure, as the concentration of the antigen to be detected decreases, the Raman characteristic spectrum intensity of the labeled molecules gradually decreases, and until the concentration of the antigen to be detected decreases to 10 -6 mg / mL, the Raman characteristic peak of the labeled molecules is still very obvious relative to the background signal, and this concentration is the detection limit of the antigen to be detected in the present scheme.

[0058] Figure 10 is an upconversion luminescence spectrum obtained by luminescence detection of the substrate after immunoreaction with NaYF4:Yb,Er upconversion luminescent nanoparticle immunoprobes and MXene / MoS2 composite SERS immunosubstrates and different concentrations of the antigen to be detected (concentrations of 10 -2 mg / mL to 10 -6 mg / mL). As can be seen from the figure, as the concentration of the antigen to be detected decreases, the upconversion luminescence spectrum intensity gradually decreases, and until the concentration of the antigen to be detected decreases to 10 -6 mg / mL, the upconversion luminescence spectrum peak is still very obvious relative to the background signal, and this concentration is the detection limit of the antigen to be detected in the present scheme.

[0059] Example 2

[0060] A preparation method of a dual-mode immunological structure based on a titanium carbide SERS substrate and rare earth doped sodium fluoroethide nanoparticles, comprising the following steps:

[0061] (1) Titanium carbide powder is prepared by acid etching

[0062] 2 g of lithium fluoride is added to 20 mL of hydrochloric acid (9 mmol / mL), continuously stirred at room temperature for 10 min, and 2 g of titanium aluminum carbide powder is slowly added to the solution containing lithium fluoride and hydrochloric acid while stirring, heated to 45°C in a water bath, and continuously reacted for 36 h to obtain a titanium carbide solution. The titanium carbide solution is washed three times by centrifugation with dilute hydrochloric acid (1 mmol / mL), and then repeatedly washed with deionized water until the supernatant PH is 6-7. The washed solution is ultrasonically treated in an ice bath for 1 h, and then centrifuged at 6000 rpm for 10 min to obtain the supernatant. The supernatant is dried at 60°C for 12 h to obtain titanium carbide (MXene) powder.

[0063] (2) Preparation of titanium carbide / molybdenum disulfide composite immunosubstrate

[0064] A. 17 mg ammonium molybdate, 40 mg thiourea were dissolved in 4 mL deionized water, and then slowly added to a 4 mL solution containing 20 mg titanium carbide, and ultrasonically dispersed for 10 min. The mixed solution was placed in a stainless steel autoclave lined with polytetrafluoroethylene, heated to 200°C, and maintained for 24 h. After the reaction was completed, it was naturally cooled to room temperature, centrifuged, and the black precipitate was washed with ethanol and deionized water 4-6 times. After vacuum drying at 60°C for 12 h, a titanium carbide / molybdenum disulfide (MXene / MoS2) composite was obtained.

[0065] B. The MXene / MoS2 composite in step (2) A was added to deionized water at a mass-volume ratio of 1 mg: 100 μL to prepare a solution. 20 μL of the solution was dropped onto a silicon wafer, which was immersed in DMF for 2 h, then washed with a phosphate buffer solution several times. Then, 1 mL of a phosphate buffer solution containing NHS / EDC (1:1, 10 mg / mL) was added to the titanium carbide / molybdenum disulfide composite substrate. Then, 10 μL of a solution containing PSA antibodies was added, and soaked at room temperature for 2 h. After washing to remove excess unreacted antibodies, the MXene / MoS2 composite immunization substrate was stored at 4°C.

[0066] (3) Preparation of NaYF4:Yb,Er (78% Y, 20% Yb, 2% Er) immunization probes

[0067] A. First, 2 mmol of rare earth nitrate RE(NO3)3·6H2O (RE = 78% Y, 20% Yb, 2% Er) was mixed with 10 mL of an aqueous citric acid solution (0.4 mmol / mL) and stirred for 1 h. Then, 0.2-0.4 mL of a sodium hydroxide solution (5 mmol / mL) was added and stirred for 15 min. Then, 9 mL of a sodium fluoride solution (1 mmol / mL) was added to obtain a colloidal suspension. The colloidal suspension was further stirred for 30 min and then transferred to a 40 mL stainless steel reactor with a polytetrafluoroethylene lining. The reaction was carried out at 180°C for 12 h. After the reaction was completed, it was naturally cooled to room temperature. The reaction product was obtained by centrifugation and washed repeatedly with ethanol and deionized water 6 times. The product was dried at 80°C for 12 h.

[0068] B. The NaYF4:Yb,Er solution was mixed with 7 mL of an R6G solution (1 mM) and reacted for 15 min. The excess R6G was washed away. Then, 1 mL of a phosphate buffer solution containing NHS / EDC (1:1, 10 mg / mL) was added, and incubated at 37°C for 1 h. Then, it was washed with a phosphate buffer solution, and then a solution containing PSA antibodies was added, and incubated for 1 h. After washing to remove excess unreacted antibodies, the NaYF4:Yb,Er immunization probe was obtained.

[0069] (4) Cancer marker detection system assembly

[0070] The phosphate buffer solution containing the antigen to be tested is added dropwise to the MXene / MoS2 composite immunization substrate, and it is placed at 37°C for 2h, so that the immune reaction between the antigen and the antibody is fully carried out, and the excess unreacted antigen to be tested is removed by washing; 30μL of NaYF4:Yb,Er immunization probe is added dropwise to the MXene / MoS2 composite immunization substrate adsorbed with the antigen to be tested, and reacted at 37°C for 2h, and the excess unreacted NaYF4:Yb,Er immunization probe is removed by washing, to obtain a cancer marker detection system based on MXene / MoS2 and NaYF4:Yb,Er. The complex of the NaYF4:Yb,Er immunization probe and the MXene / MoS2 immunization substrate obtained after the above immune reaction is measured by a Raman spectrometer, and the concentration of the antigen to be tested is calculated according to the linear relationship between the concentration of the cancer marker antigen and the intensity of the Raman characteristic peak.

[0071] Figure 2 shows the scanning electron microscope photos of the MXene / MoS2 composite prepared in this embodiment. As can be seen from Figure 2, the MoS2 in a net shape wraps the MXene sheet, forming a composite nanostructure.

[0072] Figure 5 shows the NaYF4:Yb,Er upconversion luminescent nanoparticles prepared in this embodiment. As can be seen from Figure 5, the upconversion luminescent nanoparticles have a spherical shape.

[0073] Figure 8 is the Raman spectrum of the substrate after the immune reaction of the NaYF4:Yb,Er upconversion luminescent nanoparticle immunization probe prepared in this embodiment and the MXene / MoS2 composite SERS immunization substrate with different concentrations of antigen to be tested (the concentration is 10 -2 mg / mL to 10 -6 mg / mL). As can be seen from the figure, as the concentration of the antigen to be tested decreases, the Raman characteristic spectrum intensity of the labeled molecules gradually decreases, and when the concentration of the antigen to be tested decreases to 10 -6 mg / mL, the Raman characteristic peak of the labeled molecules is still very obvious relative to the background signal, and this concentration is the detection limit of the antigen to be tested in this scheme.

[0074] Figure 11 is the Raman spectrum of the substrate after the immune reaction of the NaYF4:Yb,Er upconversion luminescent nanoparticle immunization probe prepared in this embodiment and the MXene / MoS2 composite SERS immunization substrate with different concentrations of antigen to be tested (the concentration is 10 -2 mg / mL to 10 -6The upconversion spectrum of the substrate after the immunoreaction was detected by luminescence detection. As can be seen from the figure, as the concentration of the antigen to be detected decreases, the upconversion luminescence spectrum gradually decreases, and when the concentration of the antigen to be detected decreases to 10 -6 mg / mL, the upconversion luminescence spectrum peak is still obvious relative to the background signal, and this concentration is the detection limit of the antigen to be detected in the present scheme.

[0075] Example 3

[0076] A preparation method of a dual-mode immune structure based on a titanium carbide SERS substrate and rare earth doped sodium fluoroacetate nanoparticles, comprising the following steps:

[0077] (1) Titanium carbide powder is prepared by acid etching

[0078] 3g of lithium fluoride is added to 20mL of hydrochloric acid (9mmol / mL), and stirring is continued at room temperature for 10min. 3g of titanium aluminum carbide powder is slowly added to the solution containing lithium fluoride and hydrochloric acid while stirring, and the solution is heated to 45℃ in a water bath and reacted for 48h to obtain a titanium carbide solution. The titanium carbide solution is washed with dilute hydrochloric acid (1mmol / mL) by centrifugation three times, and then washed with deionized water until the supernatant has a pH of 6-7. The washed solution is ultrasonically treated in an ice bath for 1h, and then centrifuged at 6000rpm for 10min to obtain the supernatant. The supernatant is then dried at 60℃ for 12h to obtain titanium carbide (MXene) powder.

[0079] (2) Preparation of titanium carbide / molybdenum disulfide composite immune substrate

[0080] A. 20mg of ammonium molybdate and 50mg of thiourea are dissolved in 4mL of deionized water, and then slowly added to a 4mL solution containing 25mg of titanium carbide. The mixture is ultrasonically dispersed for 10min, and then placed in a polytetrafluoroethylene-lined stainless steel autoclave and heated to 200℃ for 24h. After the reaction is completed, the mixture is naturally cooled to room temperature, centrifuged, and the black precipitate obtained is washed with ethanol and deionized water 4-6 times. The mixture is then vacuum dried at 60℃ for 12h to obtain a titanium carbide / molybdenum disulfide (MXene / MoS2) composite.

[0081] B. The MXene / MoS2 composite in step (2) A was added in deionized water at a mass volume ratio of 1 mg: 100 μL to make a solution, 10-30 μL of the solution was dropped on the silicon wafer, the silicon wafer was soaked in DMF for 2 h, then washed with phosphate buffer solution for several times, then 1 mL of NHS / EDC (1:1, 10 mg / mL) phosphate buffer solution was dropped on the titanium carbide / molybdenum disulfide composite substrate. Then, 10 μL of the solution containing PSA antibody was dropped, soaked at room temperature for 2 h, and the excess unreacted antibody was removed by washing and stored at 4°C to obtain the MXene / MoS2 composite immunosubstrate.

[0082] (3) Preparation of NaYF4:Yb,Er (78% Y, 20% Yb, 2% Er) immunoprobes

[0083] A. First, 3 mmol of rare earth nitrate RE(NO3)3·6H2O (RE = 78% Y, 20% Yb, 2% Er) was mixed with 10 mL of aqueous citric acid solution (0.4 mmol / mL) and stirred for 1 h, then 0.4 mL of sodium hydroxide solution (5 mmol / mL) was added and stirred for 15 min, then 10 mL of sodium fluoride solution (1 mmol / mL) was added to obtain a colloidal suspension. The colloidal suspension was further stirred for 30 min and then transferred to a 40 mL stainless steel reactor with a polytetrafluoroethylene liner. The reaction was carried out at 180°C for 12 h, and after the reaction was completed, it was naturally cooled to room temperature. The reaction was centrifuged and washed with ethanol and deionized water for 6 times, and the product was dried at 80°C for 12 h.

[0084] B. The NaYF4:Yb,Er solution was mixed with 5-10 mL of R6G solution (1 mM) for 15 min, and the excess R6G was washed away. Then, 1 mL of NHS / EDC (1:1, 10 mg / mL) phosphate buffer solution was added, incubated at 37°C for 1 h, washed with phosphate buffer solution, then a solution containing PSA antibody was added, incubated for 1 h, and the excess unreacted antibody was removed by washing to obtain the NaYF4:Yb,Er immunoprobes.

[0085] (4) Assembly of cancer marker detection system

[0086] The phosphate buffer solution containing the antigen to be detected is added dropwise to the MXene / MoS2 composite immunization substrate, and it is placed at 37°C for 2h, so that the immune reaction between the antigen and the antibody is fully carried out, and the excess unreacted antigen to be detected is removed by washing; 40μL of NaYF4:Yb,Er immune probe is added dropwise to the MXene / MoS2 composite immunization substrate adsorbed with the antigen to be detected, and reacted at 37°C for 2h, and the excess unreacted NaYF4:Yb,Er immune probe is removed by washing, thereby obtaining a cancer marker detection system based on MXene / MoS2 and NaYF4:Yb,Er. The complex of the NaYF4:Yb,Er immune probe and the MXene / MoS2 immunization substrate obtained after the above immune reaction is measured by a Raman spectrometer, and the concentration of the cancer marker antigen to be detected is calculated according to the linear relationship between the concentration of the cancer marker antigen and the intensity of the Raman characteristic peak.

[0087] Figure 3 shows the scanning electron microscope photos of the MXene / MoS2 composite prepared in the embodiment. As can be seen from Figure 3, the MoS2 in a net shape wraps the MXene sheet, forming a composite nanostructure.

[0088] Figure 6 shows the NaYF4:Yb,Er upconversion luminescent nanoparticles prepared in the embodiment. As can be seen from Figure 6, the upconversion luminescent nanoparticles have a spherical shape.

[0089] Figure 9 is a Raman spectrum obtained by Raman detection of the substrate after immune reaction of the NaYF4:Yb,Er upconversion luminescent nanoparticle immune probe and the MXene / MoS2 composite SERS immunization substrate prepared in the embodiment with different concentrations of antigen to be detected (the concentration is 10 -2 mg / mL to 10 -6 mg / mL). As can be seen from the figure, as the concentration of the antigen to be detected decreases, the Raman characteristic spectrum intensity of the labeled molecule gradually decreases, and when the concentration of the antigen to be detected decreases to 10 -6 mg / mL, the Raman characteristic peak of the labeled molecule is still obvious relative to the background signal, and this concentration is the detection limit of the antigen to be detected in the present scheme.

[0090] Figure 12 is an upconversion spectrum obtained by luminescence detection of the substrate after immune reaction of the NaYF4:Yb,Er upconversion luminescent nanoparticle immune probe and the MXene / MoS2 composite SERS immunization substrate prepared in the embodiment with different concentrations of antigen to be detected (the concentration is 10 -2 mg / mL to 10 -6 mg / mL). As can be seen from the figure, as the concentration of the antigen to be detected decreases, the upconversion luminescence spectrum intensity gradually decreases, and when the concentration of the antigen to be detected decreases to 10-6 When the concentration of the antigen is 0.1 mg / mL, the up-conversion luminescence spectrum peak is still obvious relative to the background signal, and this concentration is the detection limit of the present application for the antigen to be detected.

[0091] As can be seen from the above examples and the drawings, the preparation method of the dual-mode immune structure based on the titanium carbide SERS substrate and the rare earth doped sodium fluoroacetate nanoparticles provided by the present application has high detection sensitivity and high detection efficiency in application, can be applied to screening of cancer, is crucial for understanding the course of cancer, and is suitable for clinical popularization and application.

[0092] And according to the different antigens, the dual-mode immune structure based on the titanium carbide SERS substrate and the rare earth doped sodium fluoroacetate nanoparticles provided by the present application is not only suitable for application in detection of prostate cancer, but also can be applied to detection of colorectal cancer, ovarian cancer or pancreatic cancer and other cancers.

[0093] Those skilled in the art will understand that the embodiments of the present application shown in the above description and the drawings are only examples and do not limit the present application. The purpose of the present application has been completely and effectively achieved. The function and structural principle of the present application has been shown and explained in the embodiments, and the embodiments of the present application can have any modification or change without departing from the principle.

Claims

1. A method for the preparation of a dual-mode immunostucture based on a titanium carbide SERS substrate and rare-earth doped sodium fluoroethide nanoparticles, characterized by, Comprise the following steps: (S10) adopt acid etching method to prepare titanium carbide powder; (S20) preparation of titanium carbide / molybdenum disulfide composite immune substrate; And (S30) preparation of NaYF4:Yb, Er immune probe.

2. The method for preparing a dual-mode immunological structure based on a titanium carbide SERS substrate and rare-earth-doped sodium fluoroethide nanoparticles according to claim 1, wherein the step (S10) comprises the following steps: (S101) preparation of hydrochloric acid solution containing lithium fluoride; (S102) add titanium carbide aluminum powder to the above solution, and obtain titanium carbide solution after reaction; (S103) centrifugal washing of titanium carbide solution with dilute hydrochloric acid, then washing with deionized water, ice bath ultrasonic cleaning of the washed solution, and centrifugal separation to obtain supernatant; (S104) drying of the supernatant to obtain titanium carbide (MXene) powder.

3. The preparation method of the double-mode immune structure based on titanium carbide SERS substrate and rare earth doped sodium fluoroethide nanoparticles according to claim 2, wherein in the step (S101), 1-3 g of lithium fluoride is added to 20 mL of 9 mmol / mL hydrochloric acid, and stirring is continued at room temperature for 10 min; in the step (S102), 1-3 g of titanium carbide aluminum powder is slowly added to the solution containing lithium fluoride and hydrochloric acid while stirring, and water bath heating is carried out to 45℃, and the reaction is continued for 24-48 h to obtain titanium carbide solution; in the step (S103), the titanium carbide solution is centrifugally washed three times with 1 mmol / mL dilute hydrochloric acid, and then repeatedly washed with deionized water until the supernatant has a pH of 6-7.

4. The method for preparing a dual-mode immunostucture based on titanium carbide SERS substrate and rare earth doped sodium fluoroethylate nanoparticle according to claim 1, wherein the step (S20) comprises the following steps: (S201) take ammonium molybdate, thiourea and dissolve in deionized water, then add titanium carbide and ultrasonic dispersion to form a mixed solution; (S202) place the mixed solution in a polytetrafluoroethylene-lined stainless steel autoclave, heat, cool and centrifuge after the reaction is completed, and obtain black precipitate; (S203) wash the black precipitate with ethanol and deionized water, and vacuum dry to obtain titanium carbide / molybdenum disulfide (MXene / MoS2) composite; (S204) add the MXene / MoS2 composite in the step (S203) to deionized water to form a solution, drop the solution on a silicon wafer, immerse the silicon wafer in DMF, and then wash with a phosphate buffer solution; (S205) drop the phosphate buffer solution containing NHS / EDC on the titanium carbide / molybdenum disulfide composite substrate; (S206) drop the solution containing antibodies, soak, and wash to remove excess unreacted antibodies to obtain a MXene / MoS2 composite immune substrate.

5. The method for preparing a dual-mode immunostucture based on titanium carbide SERS substrate and rare-earth doped sodium fluoroethide nanoparticles according to claim 2, wherein the step (S20) comprises the following steps: (S201) take ammonium molybdate, thiourea and dissolve in deionized water, then add titanium carbide and ultrasonic dispersion to form a mixed solution; (S202) place the mixed solution in a polytetrafluoroethylene-lined stainless steel autoclave, heat, cool and centrifuge after the reaction is completed, and obtain black precipitate; (S203) wash the black precipitate with ethanol and deionized water, and vacuum dry to obtain titanium carbide / molybdenum disulfide (MXene / MoS2) composite; (S204) add the MXene / MoS2 composite in the step (S203) to deionized water to form a solution, drop the solution on a silicon wafer, immerse the silicon wafer in DMF, and then wash with a phosphate buffer solution; (S205) NHS / EDC phosphate buffer solution was added dropwise on the titanium carbide / molybdenum disulfide composite substrate; (S206) the solution containing the antibody was added dropwise, soaked, and cleaned to remove the excess unreacted antibody, to obtain the MXene / MoS2 composite immunosubstrate.

6. The preparation method of the dual-mode immunological structure based on the titanium carbide SERS substrate and the rare earth doped sodium fluoroethide nanoparticles according to claim 4, wherein in the step (S201), the ammonium molybdate is 15-20 mg, the thiourea is 30-50 mg, and the titanium carbide is 15-25 mg; in the step (S202), the mixed solution is heated to 200°C in a polytetrafluoroethylene-lined stainless steel autoclave for 24 h, and then naturally cooled to room temperature after the reaction is completed; in the step (S203), the obtained black precipitate is cleaned with ethanol and deionized water for 4-6 times, and then vacuum dried at 60°C for 12 h to obtain the titanium carbide / molybdenum disulfide (MXene / MoS2) composite.

7. The preparation method of the dual-mode immunological structure based on the titanium carbide SERS substrate and the rare earth doped sodium fluoroethide nanoparticles according to claim 6, wherein in the step (S204), the MXene / MoS2 composite is added in deionized water at a mass-volume ratio of 1 mg:100 μL to prepare a solution, 10-30 μL of the solution is added dropwise on a silicon wafer, the silicon wafer is soaked in DMF for 2 h, and then washed with a phosphate buffer solution multiple times; in the step (S205), 1 mL of the NHS / EDC phosphate buffer solution is added dropwise on the titanium carbide / molybdenum disulfide composite substrate, wherein the NHS / EDC is 1:1, and the concentration is 10 mg / mL; in the step (S206), 10 μL of the solution containing the PSA antibody is added dropwise, soaked at room temperature for 2 h, cleaned to remove the excess unreacted antibody, and stored at 4°C to obtain the MXene / MoS2 composite immunosubstrate.

8. The method for the preparation of a dual-mode immunological structure based on a titanium carbide SERS substrate and rare-earth doped sodium fluoroethide nanoparticles according to any one of claims 1 to 7, wherein said step (S30) comprises the following steps: (S301) The rare earth nitrate RE(NO3)3·6H2O is mixed with the aqueous citric acid solution and stirred; (S302) the sodium hydroxide solution is added and stirred, and then the sodium fluoride solution is added to obtain a colloidal suspension; (S303) the colloidal suspension is transferred to a stainless steel reaction kettle with a polytetrafluoroethylene liner for reaction, and then cooled and centrifuged after the reaction is completed to obtain the reaction product; (S304) the product is cleaned with ethanol and deionized water, and dried; (S305) the NaYF4:Yb,Er solution is mixed with the R6G solution for reaction; (S306) the excess R6G is washed away, and then the NHS / EDC phosphate buffer solution is added and incubated; (S307) after washing with the phosphate buffer solution, the solution containing the antibody is added and incubated, and then cleaned to remove the excess unreacted antibody to obtain the NaYF4:Yb,Er (78% Y, 20% Yb, 2% Er) immunological probe.

9. The method of claim 8, wherein in the step (S301), the rare earth nitrate is RE(N03)3-6H20 (RE = 78% Y, 20% Yb, 2% Er); in the step (S302), the obtained colloidal suspension is transferred to a stainless steel reactor with a polytetrafluoroethylene liner after being continuously stirred for 30 min, and is reacted at 180°C for 12 h, and is naturally cooled to room temperature after the reaction is completed; in the step (S304), the reaction product is obtained by centrifugation and is repeatedly washed with ethanol and deionized water for 6 times, and the product is dried at 80°C for 12 h; in the step (S305), the NaYF4:Yb, Er solution is mixed with 5-10 mL of a 1 mmol / mL R6G solution for reaction for 15 min, and the excess R6G is washed away; in the step (S306), the ratio of NHS / EDC is 1:1, and the concentration is 10 mg / mL; in the step (S307), the solution containing the PSA antibody is added, incubated for 1 h, and the excess unreacted antibody is removed by washing to obtain the NaYF4:Yb, Er immunoprobe.

10. The method of claim 8, further comprising a step (S40) of assembling a cancer marker system: a phosphate buffer solution containing an antigen to be detected is added dropwise to the MXene / MoS2 composite immunobase, and is allowed to stand to allow an immune reaction between the antigen and the antibody, and the excess unreacted antigen to be detected is removed by washing; and then the NaYF4:Yb, Er immunoprobe is added dropwise to the MXene / MoS2 composite immunobase on which the antigen to be detected is adsorbed, and is reacted at 37°C, and the excess unreacted NaYF4:Yb, Er immunoprobe is removed by washing to obtain a cancer marker detection system based on MXene / MoS2 and NaYF4:Yb, Er.

11. A dual-mode immunoscaffold based on titanium carbide SERS substrate and rare earth doped sodium fluoroethide nanoparticles, characterized in that, including: a titanium carbide / molybdenum disulfide composite immunobase and a NaYF4:Yb, Er immunoprobe, wherein the NaYF4:Yb, Er contains 78% Y, 20% Yb, and 2% Er.

12. The dual-mode immunostucture based on a titanium carbide SERS substrate and rare earth-doped sodium fluoroethide nanoparticles of claim 11, wherein the dual-mode immunostucture based on a titanium carbide SERS substrate and rare earth-doped sodium fluoroethide nanoparticles is prepared by the method of any one of claims 1 to 10.

13. Use of a dual-mode immunostucture based on a titanium carbide SERS substrate and rare-earth doped sodium fluoroethide nanoparticles, characterized in that, The dual-mode immunostucture based on a titanium carbide SERS substrate and rare earth-doped sodium fluoroethide nanoparticles is suitable for application in the detection of prostate cancer, colorectal cancer, ovarian cancer, or pancreatic cancer.

14. Use of a dual-mode immunological structure based on a titanium carbide SERS substrate and rare-earth doped sodium fluoroethylate nanoparticles according to claim 13, wherein the dual-mode immunological structure based on a titanium carbide SERS substrate and rare-earth doped sodium fluoroethylate nanoparticles comprises a titanium carbide / molybdenum disulfide composite immunological substrate and NaYF4:Yb,Er immunological probes, wherein 78% Y, 20% Yb, 2% Er are comprised in NaYF4:Yb,Er, the method for its preparation comprising the following steps: (S10) titanium carbide powder is prepared by an acid etching method; (S20) a titanium carbide / molybdenum disulfide composite immunobase is prepared; and (S30) a NaYF4:Yb, Er immunoprobe is prepared.

15. The dual-mode immunostucture based on titanium carbide SERS substrate and rare earth doped sodium fluoroethylate nanoparticles according to claim 14, wherein in the application process, phosphate buffer solution containing the antigen to be tested is added dropwise to the MXene / MoS2 composite immunosubstrate, which is placed at 37°C for 2h, so that the antigen and antibody are allowed to react, and the excess unreacted antigen to be tested is removed by washing; 20-40 μL of NaYF4:Yb,Er immune probe is then added dropwise to the MXene / MoS2 composite immunosubstrate adsorbed with the antigen to be tested, and reacted at 37°C for 2h, and the excess unreacted NaYF4:Yb,Er immune probe is removed by washing, thereby obtaining a cancer marker detection system based on MXene / MoS2 and NaYF4:Yb,Er; the compound of the NaYF4:Yb,Er immune probe and the MXene / MoS2 immunosubstrate obtained after the above immunoreaction is measured by a Raman spectrometer, and the concentration of the cancer marker antigen to be tested is calculated according to the linear relationship between the concentration of the cancer marker antigen and the intensity of the Raman characteristic peak.

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