CN112986561B - Multimode immune instant analysis excited by nano titanium carbide hybrid - Google Patents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a multimode immune instant analysis excited by a nano titanium carbide hybrid. The plasma nano hybrid Ti 3 C 2 The @ CuNCs has good photo-thermal conversion performance and can cause the SED temperature of the flexible conductive substrate to rise under the excitation of near infrared light. Further, Ti 3 C 2 The high catalyst activity of @ CuNCs can also catalyze the oxidation of a colorimetric reagent Lecuo-MB to generate blue methylene blue MB, so that the color of a substrate is changed from light blue to deep blue. Methylene blue MB also has excellent photo-thermal properties, Ti 3 C 2 The synergistic effect of @ CuNCs and MB photothermal effect further enhances light absorption and amplifies the temperature signal. Meanwhile, the continuously increased heat also accelerates the migration of charges inside the SED, so that the resistance change caused by temperature rise can be monitored in real time through a digital multimeter, and a temperature signal can be converted into an electric signal. The method realizes high sensitivity and multi-signal immunodetection of human anti-ASGPR, has great potential in clinical diagnosis of malignant diseases, and opens up a new way for point-of-care detection.
Description
Technical Field
The invention belongs to the technical field of novel functional materials and biological sensing detection, and particularly relates to a multi-mode immunoassay method for human anti-asialoglycoprotein receptor human anti-ASGPR detection by using a nano titanium carbide hybrid-based photo-thermal chemical sensor.
Background
The latent period of the etiology of many malignant diseases is long, the early diagnosis is difficult, the lack of effective preventive measures and other problems seriously threaten public health, and therefore, the design of a reliable early monitoring means is necessary for reducing the threat of malignant diseases to human health. The discovery of biomarkers in recent years provides important reference for early diagnosis of various malignant tumors. However, since the cost and complexity of biomarker detection are high, the need for professional operators and the like limit the popularization and promotion of ELISA in remote areas. The method is developed by people for the second time based on portable instruments and equipment, and is combined with ELISA (enzyme-linked immuno sorbent assay) to provide economic and effective daily health care services under the condition of limited resources.
Photothermal chemical detection uses light as an excitation signal and heat generated by light radiation as a detection signal. Higher sensitivity can be obtained by using different forms of energy changes caused by the local temperature rise of the substrate caused by the light radiation as detection signals. MXene is an emerging multifunctional two-dimensional photothermal material, and shows huge application potential in photothermal biosensing. Wherein, Ti 3 C 2 Tx MXene is the easiest to prepare and relatively inexpensive, its unique semiconductor and electromagnetic properties make Ti 3 C 2 Tx presents a plasma absorption peak and shows strong near infrared absorption and photo-thermal conversion efficiency under laser irradiation. Furthermore, studies have shown that the incorporation of metal plasmons can effectively improve the light-to-heat conversion efficiency, since the tunable Localized Surface Plasmon Resonance (LSPR) peak of the metal nanoparticles may effectively absorb NIR light and convert it to heat, which can be transferred to the ambient environment, causing a significant temperature rise.
In this experiment ionic nano-hybrid Ti 3 C 2 @ CuNCs has good photothermal conversion properties and is used as a signal reporter, and can cause the temperature of a flexible conductive Substrate (SED) to rise under the excitation of near infrared light. Further, Ti 3 C 2 The high catalyst activity of @ CuNCs can also catalyze the oxidation of a colorimetric reagent Lecuo-MB to generate blue MB, so that the color of a substrate is changed from light blue to deep blue. As an effective near-infrared dye, MB also has excellent photo-thermal properties, Ti 3 C 2 The stacking of @ CuNCs with MB photothermal effect further enhances light absorption and amplifies temperatureThe signal is raised in degree. Meanwhile, the continuously increased heat also accelerates the migration of charges inside the SED, so that the conductivity change caused by the temperature rise can be monitored in real time through a digital multimeter, and further, the temperature signal is successfully converted into an electric signal. The method realizes the multi-signal immunodetection with high sensitivity and high accuracy for human anti-asialoglycoprotein receptor human anti-ASGPR, the innovative multi-signal reading instant detection sensing platform has great potential in clinical diagnosis of malignant diseases, and a new way is opened for point-of-care detection.
Disclosure of Invention
The invention aims to provide a multimode immune instant analysis method excited by a nano titanium carbide hybrid, which is used for instant detection of a biomarker.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
(1) preparation of SED/Ab modified substrate: 0.5 polyvinyl alcohol PVA solid was dissolved in 10 mL of ultrapure water, 2mL of glycerin was added and heated for 20 minutes with continuous stirring to finally obtain a homogeneous hydrosol. Then, the hydrosol was treated with 1 mL of 0.5 mol/L NaOH solution at 40 ℃ for 1 hour, and 1 mL of 1% by mass cyanuric chloride was added to the mixture to react for 1 hour. Then, an acetic acid solution with the mass fraction of 3% is dropped to neutralize the excess alkali. And then transferring the hydrosol to a 96-well plate with each well being 100 mu L, and cooling to room temperature to obtain the PVA hydrogel used as the SED (flexible temperature-sensitive conductive device). In order to further enlarge the adsorption volume, 30 μ L of 10% by mass HCl was added to the surface of the SED in a flexible temperature-sensitive conductive apparatus to etch for 10 minutes and washed several times with PH 7.4 phosphoric acid buffer solution. Then, 5. mu.L of an antibody against human asialoglycoprotein receptor human anti-ASGPR (Shanghai Jianglai Biotech Co., Ltd.) was added thereto at a concentration of 1.6U/mL, and the mixture was incubated at 4 ℃ for 40 min to wash out the unsuccessfully adsorbed antibody with a phosphate buffer solution of pH 7.4. Blocking the nonspecific sites with bovine serum albumin BSA solution with the concentration of 1.0 wt.% to obtain an SED/Ab modified substrate;
（2）SED/Ab/ Ag-Ti 3 C 2 preparation of @ CuNCs modified substrate: adding 250 mu L of Ti 3 C 2 The @ CuNCs complex was dispersed in 250. mu.L of 10mM 1-aminoethyl-3-methylimidazole bromideTreating Ti in ammonium solution by ultrasonic for 30min 3 C 2 The @ CuNCs complex is subjected to surface amination treatment. Then, 50. mu.L of human anti-asialoglycoprotein receptor human anti-ASGPR (Jianglai Biotech Co., Ltd.) at a concentration of 1.6U/mL and 10. mu.L of 25 wt% glutaraldehyde were added dropwise to the surface-aminated Ti 3 C 2 @ CuNCs solution, stirred at room temperature for 12 h. Finally, the mixture is centrifuged at 5000r/min for 10min, the supernatant is removed, the weakly bound molecules are separated and redispersed with 250 μ LpH 7.4.4 phosphoric acid buffer solution to obtain Ag-Ti 3 C 2 @ CuNCs probe; different concentrations of human anti-asialoglycoprotein receptor human anti-ASGPR standard solution (Shanghai Jianglai Biotech limited) and a labeled probe Ag-Ti 3 C 2 Dropping the mixed solution of @ CuNCs into the SED/Ab modified substrate prepared in the step 1), incubating for 40 min at 4 ℃, and combining the antibody fixed on the surface of the SED of the flexible temperature-sensitive conductive equipment by using a competitive reaction; washing the surface of the substrate with a phosphate buffer solution with pH 7.4 and naturally drying at room temperature to obtain SED/Ab/Ag-Ti 3 C 2 @ CuNCs modified substrates;
(3) detection of human anti-asialoglycoprotein receptor human anti-ASGPR: dripping 30 mu L of developer Leuco-MB on the SED/Ab/Ag-Ti prepared in the step 2) 3 C 2 The reaction is carried out for 90s on the substrate modified by @ CuNCs, so that the developer Leuco-MB is oxidized to generate methylene blue MB to obtain SED/Ab/Ag-Ti 3 C 2 Modification of substrate with @ CuNCs/MB, and observation of 1X 10 -6 U/mL-1×10 -2 Color change between U/mL; collecting SED/Ab/Ag-Ti at different concentrations through smart phone 3 C 2 @ CuNCs/MB modifies the photograph of the substrate and converts it into red, green and blue RGB images using the professional drawing software PowerPoint, calculates 1X 10 -6 U/mL–1×10 -2 Color values between U/mL. The color change may be defined by: color value = red value (R) + green value (G) × 256 + blue value (B) × 65536; using 808nm laser at optical power density of 5W cm -2 The following pairs SED/Ab/Ag-Ti 3 C 2 Irradiating the modified substrate of @ CuNCs/Leuco-MB for 1min, and measuring by 1 × 10 with electronic thermometer -8 U/mL–1×10 -3 Temperature change between U/mL; subjecting SED/Ab/Ag-Ti 3 C 2 The @ CuNCs/Leuco-MB modified substrate is connected with a digital multimeter, and 808nm laser is used for measuring the optical power density at 5W cm -2 For SED/Ab/Ag-Ti 3 C 2 Irradiating the substrate modified with @ CuNCs/Leuco-MB for 1min, and recording 1 × 10 -8 U/mL–1×10 -2 Resistance change between U/mL; drawing a working curve by recording different temperature changes, resistance changes and color value changes generated before and after 808nm laser radiation; and replacing the human asialoglycoprotein receptor (asialoglycoprotein receptor) anti-ASGPR standard solution with the sample solution to be detected for detection, and checking the detection result through a working curve.
The above Ti 3 C 2 The @ CuNCs complex is prepared by the following method:
250 μ L of 5mg/mL Ti 3 C 2 MXene nanoplatelets were dissolved in N, N-dimethylformamide and 100uL of 100mM CuCl was injected 2 And 20uL of 50mM ascorbic acid AA, stirred well. Subsequently, 20 μ L of 0.25mg/mL copper nanocluster CuNCs was added to the mixture and refluxed at 100 ℃ for 12h until the mixture color changed from gray to brown. Then, the obtained mixture was centrifuged at 5000r/min for 10min, and the precipitate was collected and washed with deionized water to remove free ions. Dissolving the precipitate with 250 μ L deionized water to obtain Ti 3 C 2 @ CuNCs plasma nano hybrid.
The Ti 3 C 2 The MXene nanosheet material is prepared by the following method:
50mg of Ti 3 AlC 2 MXene powder (Shanghai Michelin reagent Co., Ltd.) was dissolved in 3mL of 50 wt% hydrofluoric acid HF and stirred at room temperature for 48 hours. The resulting solution was centrifuged at 10000 r/min for 10 minutes to remove the supernatant and harvest the product, which was then sonicated and washed with 0.5M NaOH to neutralize excess acid. The collected precipitate was then washed several times with deionized water until the solution became neutral. Drying the product at 70 ℃ overnight to obtain Ti 3 C 2 MXene nano-sheet.
The copper nanocluster Cu NCs material is prepared by the following method:
will 100mg of yeast extract dissolved in 8mL of ultrapure water and admixed with 2mL of 100mM CuCl 2 Mixing the solutions at room temperature, and stirring for 2-3 min; refluxing the mixture at 100 deg.C for 12h until the solution changes color from light blue to dark green; and finally, centrifuging the obtained sample for 10min under 10000rmp, and taking supernatant to obtain a purified copper nanocluster Cu NCs solution.
The Leuco-MB color developing agent is prepared by the following method:
mixing 5 mM of methylene blue MB and 50mM of ascorbic acid AA according to the volume ratio of 1:1, shaking for 20min until the color of the solution is changed from dark blue to light blue, and obtaining the Leuco-MB color developing agent
A multimode immune instant analysis sensor excited by nano titanium carbide hybrid is characterized in that SED/Ab/Ag-Ti 3 C 2 The substrate of @ CuNCs is prepared by the following method: 1) preparation of SED/Ab modified substrate: 0.5 polyvinyl alcohol PVA solid was dissolved in 10 mL of ultrapure water, 2mL of glycerin was added and heated for 20 minutes with continuous stirring to finally obtain a homogeneous hydrosol. Then, the hydrosol was treated with 1 mL of 0.5 mol/L NaOH solution at 40 ℃ for 1 hour, and 1 mL of 1% by mass cyanuric chloride was added to the mixture to react for 1 hour. Then, an acetic acid solution with a mass fraction of 3% was added dropwise to neutralize the excess alkali. Subsequently, the hydrosol is transferred to a 96-well plate with 100 μ L of each well, and is cooled to room temperature to obtain the PVA hydrogel which is used as the SED (temperature sensitive conductive device). To further expand the adsorption volume, 30 μ L of 10% HCl by mass was added to the SED surface and etched for 10 minutes and rinsed several times with pH 7.4 phosphate buffer. Then, 5. mu.L of a human anti-asialoglycoprotein receptor human anti-ASGPR antibody (Shanghai Jianglai Biotech Co., Ltd.) was added thereto at a concentration of 1.6U/mL, and the mixture was incubated at 4 ℃ for 40 min to wash out unsuccessfully adsorbed antibody with a phosphate buffer solution of pH 7.4. Blocking the non-specific sites with 1.0 wt.% bovine serum albumin BSA solution to obtain SED/Ab modified substrate, 2) adding 250. mu.L Ti 3 C 2 The @ CuNCs complex was dispersed in 250. mu.L of 10mM 1-aminoethyl-3-methylimidazolium bromide solution and sonicated for 30min for Ti 3 C 2 The @ CuNCs complex is subjected to surface amination treatment. Then, 50. mu.L of human anti-asialoglycoprotein receptor at a concentration of 1.6U/mLhuman anti-ASGPR (Shanghai Jianglai Biotech Co., Ltd.) and 10. mu.L of 25 wt% glutaraldehyde were dropped to the surface aminated Ti 3 C 2 @ CuNCs solution, and stirred at room temperature for 12 h. Finally, the mixture is centrifuged at 5000r/min for 10min, the supernatant is removed, the weakly bound molecules are separated and redispersed with 250 μ LpH 7.4.4 phosphoric acid buffer solution to obtain Ag-Ti 3 C 2 @ CuNCs probe; different concentrations of human anti-asialoglycoprotein receptor human anti-ASGPR standard solution and labeled probe Ag-Ti 3 C 2 Dropping the mixed solution of @ CuNCs into the SED/Ab modified substrate prepared in the step 1), incubating for 40 min at 4 ℃, and binding the antibody fixed on the SED surface by using a competitive reaction; washing the surface of the substrate with a phosphate buffer solution with pH 7.4 and naturally drying at room temperature to obtain SED/Ab/Ag-Ti 3 C 2 @ CuNCs modified substrate;
a multimode immune instant analysis method excited by a nano titanium carbide hybrid is characterized by comprising the following steps: 1) 30 μ L of Leuco-MB was dropped on the SED/Ab/Ag-Ti 3 C 2 The reaction is carried out for 90s on the substrate modified by @ CuNCs, so that the developer Leuco-MB is oxidized to generate methylene blue MB to obtain SED/Ab/Ag-Ti 3 C 2 Modifying a substrate with @ CuNCs/MB, and using 808nm laser to perform SED/Ab/Ag-Ti 3 C 2 Irradiating a @ CuNCs/MB modified substrate; 2) measurement of human anti-asialoglycoprotein receptor human anti-ASGPR: photos of SED under different concentrations are collected through a smart phone, converted into red, green and blue RGB images through professional drawing software PowerPoint, and 1 x 10 is calculated -6 U/mL–1×10 -2 Color values between U/mL. The color change may be defined by: color value = red value (R) + green value (G) × 256 + blue value (B) × 65536; using 808nm laser to SED/Ab/Ag-Ti 3 C 2 @ CuNCs/MB modified substrate radiation for 1min (optical power density 5W cm) -2 ) 1X 10 measured with an electronic thermometer -8 U/mL–1×10 -3 Temperature change between U/mL; subjecting SED/Ab/Ag-Ti 3 C 2 The @ CuNCs/MB modified substrate is connected with a digital multimeter, and SED/Ab/Ag-Ti is subjected to laser alignment at 808nm 3 C 2 Irradiation of substrate with @ CuNCs/MB modification 1min (optical power density 5W cm) -2 ) Recording 1X 10 by portable fluorescence spectrometer -8 U/mL–1×10 -2 Resistance change between U/mL; drawing a working curve by recording different temperature changes, resistance changes and color value changes generated before and after 808nm laser radiation; replacing human anti-asialoglycoprotein receptor human anti-ASGPR standard solution with to-be-detected sample solution for detection, and checking the detection result through a working curve
The invention has the following remarkable advantages:
(1)Ti 3 C 2 the synergistic effect between MXene and copper nanoclusters CuNCs can obviously improve Ti 3 C 2 The photo-thermal property of the @ CuNCs nano hybrid causes the temperature of a reaction interface to be sharply increased under the excitation of near infrared light.
(2)Ti 3 C 2 The @ CuNCs nano hybrid has good catalytic activity, can catalyze the Lecuo-MB solution to change color and regenerate blue methylene blue MB, and further amplifies a thermal signal by the photothermal effect of the methylene blue MB under the irradiation of near infrared.
(3) The flexible conductive electronic device SED based on the polyelectrolyte hydrogel can convert the temperature change on a reaction interface into an electric signal, thereby realizing the ultra-sensitive and multi-mode analysis on a target object.
Drawings
FIG. 1 is a schematic diagram of a preparation process of a human anti-asialoglycoprotein receptor human anti-ASGPR multi-mode chemical sensor.
FIG. 2A is a graph of the change in fluorescence intensity after laser irradiation of substrates modified with different concentrations of human anti-asialoglycoprotein receptor human anti-ASGPR.
FIG. 2B is a graph of the linear relationship between the resistance change of the sensing substrate under laser irradiation and the concentration of human anti-asialoglycoprotein receptor human anti-ASGPR standard solution.
FIG. 3A is a graph of the color change after addition of Lecuo-MB solution to substrates modified with the different concentrated human anti-asialoglycoprotein receptor human anti-ASGPR.
FIG. 3B is a linear relationship of color change versus concentration of human anti-asialoglycoprotein receptor human anti-ASGPR standard solution.
Detailed Description
The present invention is further illustrated by the following examples, but the scope of the present invention is not limited to the following examples.
Example 1
A multimode immune instant analysis method excited by nano titanium carbide hybrid (as shown in figure 1):
1) dissolving 0.5 of polyvinyl alcohol (PVA) solid in 10 mL of ultrapure water, adding 2mL of glycerol, and heating for 20 minutes under continuous stirring to finally obtain uniform hydrosol;
2) then, the hydrosol was treated with 1 mL of 0.5 mol/L NaOH solution at 40 ℃ for 1 hour, and 1 mL of 1% by mass cyanuric chloride (dissolved in 1, 4-dioxane) was added and reacted for 1 hour. Then, dripping 3 percent of acetic acid solution to neutralize redundant alkali;
3) then transferring the hydrosol to a 96-pore plate with each pore being 100 mu L, and cooling to room temperature to obtain polyvinyl alcohol hydrogel used as a flexible temperature-sensitive conductive device SED;
4) adding 30 mu L of HCl with the mass fraction of 10% to the surface of the SED of the flexible temperature-sensitive conductive equipment, etching for 10 minutes, and washing for several times by using a phosphoric acid buffer solution with the pH of 7.4;
5) the unsuccessfully adsorbed antibody was washed away by adding 5. mu.L of a human anti-asialoglycoprotein receptor human anti-ASGPR antibody (Shanghai Jianglai Biotech Co., Ltd.) and incubating at 4 ℃ for 40 min with a phosphate buffer solution of pH 7.4. Blocking the nonspecific sites with bovine serum albumin BSA solution with the concentration of 1.0 wt.% to obtain an SED/Ab modified substrate;
6）SED/Ab/ Ag-Ti 3 C 2 preparation of a @ CuNC modified substrate: adding 250 mu L of Ti 3 C 2 The @ CuNCs complex was dispersed in 250. mu.L of 10mg/mL 1-aminoethyl-3-methylimidazolammonium bromide solution (manufactured by the institute for chemical and physical, Inc., Lanzhou, Zhongkojie) and sonicated for 30min for Ti 3 C 2 The @ CuNCs complex is subjected to surface amination treatment. Then, 50. mu.L of human asialoglycoprotein receptor human anti-ASGPR (Shanghai Jianglai Biotech Co., Ltd.) and 1 were added at a concentration of 1.6U/mL0 mu L of 25 wt% glutaraldehyde is dropped into Ti aminated on the surface 3 C 2 @ CuNCs solution, and stirred at room temperature for 12 h. Finally, the mixture is centrifuged at 5000r/min for 10min, the supernatant is removed, the weakly bound molecules are separated and redispersed with 250 μ LpH 7.4.4 phosphoric acid buffer solution to obtain Ag-Ti 3 C 2 @ CuNCs probe; different concentrations of human anti-asialoglycoprotein receptor human anti-ASGPR standard solution (Shanghai Jianglai Biotech limited) and a labeled probe Ag-Ti 3 C 2 Dropping the mixed solution of @ CuNCs into the SED/Ab modified substrate prepared in the step 1), incubating for 40 min at 4 ℃, and binding the antibody fixed on the SED surface by using a competitive reaction; washing the surface of the substrate with a phosphate buffer solution with pH 7.4 and naturally drying at room temperature to obtain SED/Ab/Ag-Ti 3 C 2 @ CuNCs modifies the substrate.
The above Ti 3 C 2 The @ CuNCs complex is prepared by the following method: 250 μ L of Ti with a concentration of 5mg/mL 3 C 2 MXene nanoplatelets were dissolved in N, N-dimethylformamide and 100uL of 100mM CuCl was injected 2 And 20uL of ascorbic acid (vitamin C, abbreviated as AA) with a concentration of 50mM, sufficiently stirred; subsequently, 20 μ L of 0.25mg/mL of the above copper nanocluster CuNCs was added to the mixture and refluxed at 100 ℃ for 12h until the mixture color changed from gray to brown; then, centrifuging the obtained mixture for 10min at 5000r/min, collecting the precipitate, and washing away free ions by using deionized water; dissolving the precipitate with 250 μ L deionized water to obtain Ti 3 C 2 @ CuNCs plasma nano hybrid;
the Ti 3 C 2 The MXene nanosheet material is prepared by the following method:
50mg of Ti 3 AlC 2 MXene (Shanghai Michelin reagent Co., Ltd.) powder was dissolved in 3mL of 50 wt% hydrofluoric acid and stirred at room temperature for 48 hours; centrifuging the resulting solution at 10000 r/min for 10 minutes to remove supernatant and harvest the product, and washing with 0.5M NaOH after sonication to neutralize excess acid; then, washing the collected precipitate with deionized water for multiple times until the solution becomes neutral; drying the product at 70 ℃ overnight to obtain Ti 3 C 2 MXene nano-sheet.
The copper nanocluster Cu NCs material is prepared by the following method:
100 mg of yeast extract was dissolved in 8mL of ultrapure water and mixed with 2mL of 100mM CuCl 2 Mixing the solutions at room temperature, and stirring for 2-3 min; refluxing the mixture at 100 deg.C for 12h until the solution changes color from light blue to dark green; and finally, centrifuging the obtained sample for 10min under 10000rmp, and taking supernatant to obtain a purified copper nanocluster Cu NCs solution.
The Leuco-MB developer is prepared by the following method:
mixing 5 mM of methylene blue MB and 50mM of ascorbic acid AA according to the volume ratio of 1:1, and shaking for 20min until the color of the solution is changed from dark blue to light blue, thus obtaining the Leuco-MB color developing agent.
Example 2
A multimode immune instant analysis method excited by a nano titanium carbide hybrid comprises the following steps:
1) dropping 30 μ L of Leuco-MB developer on SED/Ab/Ag-Ti 3 C 2 Reaction on a @ CuNC modified substrate for 90s to obtain SED/Ab/Ag-Ti 3 C 2 Modifying a substrate with @ CuNCs/MB, and using 808nm laser to perform SED/Ab/Ag-Ti 3 C 2 Irradiating a @ CuNCs/MB modified substrate;
2) measurement of human anti-asialoglycoprotein receptor human anti-ASGPR: collecting SED/Ab/Ag-Ti at different concentrations through smart phone 3 C 2 @ CuNCs/MB modifies the photograph of the substrate and converts it into red, green and blue RGB images using the professional drawing software PowerPoint, calculates 1X 10 -6 U/mL–1×10 -2 Color values between U/mL. The color change may be defined by: color value = red value (R) + green value (G) × 256 + blue value (B) × 65536; using 808nm laser to SED/Ab/Ag-Ti 3 C 2 The substrate modified by @ CuNCs/Leuco-MB is radiated for 1min (the optical power density is 5W cm) -2 ) 1X 10 measured with an electronic thermometer -8 U/mL–1×10 -3 Temperature change between U/mL; subjecting SED/Ab/Ag-Ti 3 C 2 The @ CuNCs/MB modified substrate is connected with a digital multimeter,using 808nm laser to SED/Ab/Ag-Ti 3 C 2 @ CuNCs/MB modified substrate radiation for 1min (optical power density 5W cm) -2 ) Recording 1X 10 by digital multimeter -8 U/mL–1×10 -2 Resistance change between U/mL; drawing a working curve by recording different temperature changes, resistance changes and color value changes generated before and after 808nm laser radiation;
3) and replacing the human asialoglycoprotein receptor (asialoglycoprotein receptor) anti-ASGPR standard solution with the sample solution to be detected for detection, and checking the detection result through a working curve.
Claims (4)
1. A non-disease diagnosis multimode immune instant analysis method excited by nano titanium carbide hybrid is characterized by comprising the following steps:
(1) preparation of SED/Ab modified substrate: dissolving 0.5 g of polyvinyl alcohol (PVA) solid in 10 mL of ultrapure water, adding 2mL of glycerol, and heating for 20 minutes under continuous stirring to finally obtain uniform hydrosol; then, treating the hydrosol with 1 mL of NaOH solution with the concentration of 0.5 mol/L for 1 h at 40 ℃, adding 1 mL of cyanuric chloride with the mass fraction of 1 percent dissolved in 1, 4-dioxane, and reacting for 1 h; then, acetic acid solution with the mass fraction of 3% is dripped to neutralize the redundant alkali; then transferring the hydrosol to a 96-pore plate with each pore being 100 mu L, and cooling to room temperature to obtain PVA hydrogel used as a flexible temperature-sensitive conductive device SED; in order to further enlarge the adsorption area, 30 microliter of HCl with the mass fraction of 10% is added on the surface of the SED of the flexible temperature-sensitive conductive equipment for etching for 10 minutes and is washed for several times by a phosphoric acid buffer solution with the pH value of 7.4; then 5. mu.L of human anti-asialoglycoprotein receptor human anti-ASGPR antibody at a concentration of 1.6U/mL was added and incubated at 4 ℃ for 40 min to wash away unsuccessfully adsorbed antibody Ab with phosphate buffered solution pH 7.4; blocking the nonspecific sites by bovine serum albumin BSA solution with the mass fraction of 1.0 wt.% to obtain an SED/Ab modified substrate;
（2）SED/Ab/Ag-Ti 3 C 2 preparation of @ CuNCs modified substrate: adding 250 mu L of Ti 3 C 2 The @ CuNCs complex was dispersed in 250. mu.L of a 10mg/mL solution of 1-aminoethyl-3-methylimidazolium bromide and sonicated 30min to Ti 3 C 2 Carrying out surface amination treatment on the @ CuNCs compound; subsequently, 50. mu.L of human asialoglycoprotein receptor human anti-ASGPR provided by Jianglai Biotech Ltd of Shanghai at a concentration of 1.6U/mL and 10. mu.L of glutaraldehyde having a mass fraction of 25 wt% were added dropwise to the aminated Ti 3 C 2 The @ CuNCs solution is mixed and stirred for 12 hours at room temperature; centrifuging the mixed solution at 5000r/min for 10min, removing supernatant, separating weak binding molecules, and re-dispersing with 250 μ L of phosphoric acid buffer solution with pH of 7.4 to obtain Ag-Ti 3 C 2 @ CuNCs probe; the human anti-asialoglycoprotein receptor human anti-ASGPR standard solution and the labeled probe Ag-Ti which are provided by Shanghai Jianglai Biotechnology Limited company and have different concentrations 3 C 2 Dropping the mixed solution of @ CuNCs into the SED/Ab modified substrate, incubating for 40 min at 4 ℃, and combining the antibody fixed on the SED surface of the flexible temperature-sensitive conductive equipment by using a competitive reaction; washing the surface of the substrate with a phosphate buffer solution with pH 7.4 and naturally drying at room temperature to obtain SED/Ab/Ag-Ti 3 C 2 @ CuNCs modified substrates;
(3) detection of human anti-asialoglycoprotein receptor human anti-ASGPR: dripping 30 μ L developer Leuco-MB on SED/Ab/Ag-Ti 3 C 2 The reaction is carried out for 90s on the substrate modified by @ CuNCs, so that the Leuco-MB developer is oxidized to generate methylene blue MB to obtain SED/Ab/Ag-Ti 3 C 2 Modification of substrate with @ CuNCs/MB, and observation of 1X 10 -6 U/mL–1×10 -2 Color change between U/mL; SED/Ab/Ag-Ti collected by smart phone under different concentrations 3 C 2 The picture of the substrate modified by @ CuNCs/MB is converted into red, green and blue RGB images by professional drawing software PowerPoint, and 1 × 10 is calculated -6 U/mL–1×10 -2 Color values between U/mL; the color change is defined by: color value = red value R + green value G × 256 + blue value B × 65536; using 808nm laser at optical power density of 5W cm -2 The following pairs SED/Ab/Ag-Ti 3 C 2 Irradiating the modified substrate of @ CuNCs/Leuco-MB for 1min, and measuring by 1 × 10 with electronic thermometer -8 U/mL–1×10 -3 Temperature change between U/mLMelting; subjecting SED/Ab/Ag-Ti 3 C 2 A @ CuNCs/MB modified substrate is connected with a digital multimeter, and 808nm laser is used for measuring the optical power density of 5W cm -2 The following pairs SED/Ab/Ag-Ti 3 C 2 Irradiating the substrate modified with @ CuNCs/MB for 1min, recording 1X 10 -8 U/mL–1×10 -2 Resistance change between U/mL; drawing a working curve by recording different temperature changes, resistance changes and color value changes generated before and after 808nm laser radiation; and replacing the human asialoglycoprotein receptor (asialoglycoprotein receptor) anti-ASGPR standard solution with the sample solution to be detected for detection, and checking the detection result through a working curve.
2. The method of claim 1, wherein said Ti is selected from the group consisting of Ti, and Ti 3 C 2 The @ CuNCs complex is prepared by the following method: 250 μ L of Ti with a concentration of 5mg/mL 3 C 2 MXene nanosheets were dissolved in N, N-dimethylformamide and 100uL of CuCl with a concentration of 100mM was injected 2 And 20uL of ascorbic acid AA with a concentration of 50mM, and stirring well; subsequently, 20. mu.L of copper nanocluster CuNCs having a concentration of 0.25mg/mL are added to the mixture and refluxed at 100 ℃ for 12h until the mixture changes color from gray to brown; then, centrifuging the obtained mixture for 10min at 5000r/min, collecting the precipitate, and washing away free ions by using deionized water; dissolving the precipitate with 250 μ L deionized water to obtain Ti 3 C 2 @ CuNCs plasma nano hybrid;
the Ti 3 C 2 The MXene nanosheet material is prepared by the following method:
50mg of Ti 3 AlC 2 MXene powder was dissolved in 3mL of 50 wt% hydrofluoric acid HF and stirred at room temperature for 48 hours; centrifuging the resulting solution at 10000 r/min for 10 minutes to remove supernatant and harvest the product, and washing with 0.5M NaOH after sonication to neutralize excess acid; then, washing the collected precipitate with deionized water for multiple times until the solution becomes neutral; drying the product at 70 ℃ overnight to obtain Ti 3 C 2 MXene nanosheets;
the copper nanocluster Cu NCs are prepared by the following method:
100 mg of yeast extract was dissolved in 8mL of ultrapure water and mixed with 2mL of 100mM CuCl 2 Mixing the solutions at room temperature, and stirring for 2-3 min; refluxing the mixture at 100 deg.C for 12h until the solution changes color from light blue to dark green; finally, centrifuging the obtained sample for 10min under 10000rmp, and taking supernatant to obtain a purified copper nanocluster Cu NCs solution;
the Leuco-MB color developing agent is prepared by the following method:
mixing 5 mM of methylene blue MB and 50mM of ascorbic acid AA according to the volume ratio of 1:1, and shaking for 20min until the color of the solution is changed from dark blue to light blue, thus obtaining the Leuco-MB color developing agent.
3. A multimode immune instant analysis sensor excited by nano titanium carbide hybrid is characterized by comprising SED/Ab/Ag-Ti 3 C 2 @ CuNCs modified substrate, SED/Ab/Ag-Ti 3 C 2 The @ CuNCs modified substrate is prepared by the following method: 1) preparation of SED/Ab modified substrate: dissolving 0.5 g of polyvinyl alcohol (PVA) solid in 10 mL of ultrapure water, adding 2mL of glycerol, and heating for 20 minutes under continuous stirring to finally obtain uniform hydrosol; then, treating the hydrosol with 1 mL of NaOH solution with the concentration of 0.5 mol/L for 1 h at 40 ℃, and adding 1 mL of cyanuric chloride with the mass fraction of 1% for reaction for 1 h; then, acetic acid solution with the mass fraction of 3% is dripped to neutralize redundant alkali; then transferring the hydrosol to a 96-pore plate with each pore being 100 mu L, and cooling to room temperature to obtain PVA hydrogel used as a flexible temperature-sensitive conductive device SED; to further expand the adsorption area, 30 μ L of 10% by mass HCl was added to the SED surface and etched for 10 minutes and washed several times with pH 7.4 phosphate buffer; then 5. mu.L of an antibody against the asialoglycoprotein receptor human anti-ASGPR provided by Jianglai Biotech Co., Ltd., Shanghai was added thereto at a concentration of 1.6U/mL, and the mixture was incubated at 4 ℃ for 40 min, and the unsuccessfully adsorbed antibody was washed away with a phosphate buffer solution of pH 7.4; blocking the nonspecific sites with 1.0 wt.% bovine serum albumin BSA solution to obtain SED/Ab modified substrate, 2) adding 250. mu.L Ti 3 C 2 The @ CuNCs complex was dispersed in 250. mu.L of 10mM 1-aminoethyl-3-methylimidazolammonium bromideIn solution, and ultrasonic treatment is carried out for 30min to treat Ti 3 C 2 Carrying out surface amination treatment on the @ CuNCs compound to obtain Ti with aminated surface 3 C 2 @ CuNCs solution; then, 50. mu.L of human asialoglycoprotein receptor human anti-ASGPR provided by Jianglai Biotech Ltd of Shanghai at a concentration of 1.6U/mL and 10. mu.L of glutaraldehyde having a mass fraction of 25 wt% were dropped into the surface-aminated Ti 3 C 2 @ CuNCs solution to obtain a mixed solution, and stirring at room temperature for 12 hours; centrifuging the mixed solution at 5000r/min for 10min, removing supernatant, separating weak binding molecules, and re-dispersing with 250 μ L of phosphoric acid buffer solution with pH of 7.4 to obtain Ag-Ti 3 C 2 @ CuNCs probe; the human anti-asialoglycoprotein receptor human anti-ASGPR standard solution and the labeled probe Ag-Ti which are provided by Shanghai Jianglai Biotechnology Limited company and have different concentrations 3 C 2 The mixed solution of @ CuNCs is dripped into the SED/Ab modified substrate to be incubated for 40 min at 4 ℃, and the antibody fixed on the surface of the SED/Ab modified substrate is combined by using a competitive reaction; washing the SED/Ab modified substrate surface with phosphate buffer solution with pH 7.4, and naturally drying at room temperature to obtain SED/Ab/Ag-Ti 3 C 2 @ CuNCs modifies the substrate.
4. A non-disease diagnosis multimode immune instant analysis method excited by nano titanium carbide hybrid is characterized by comprising the following steps: 1) dropping 30. mu.L of Leuco-MB developer on the SED/Ab/Ag-Ti of claim 3 3 C 2 The reaction is carried out for 90s on the substrate modified by @ CuNCs, so that the developer Leuco-MB is oxidized to generate methylene blue MB to obtain SED/Ab/Ag-Ti 3 C 2 Modifying a substrate with @ CuNCs/MB, and laser irradiating SED/Ab/Ag-Ti with 808nm 3 C 2 Irradiating a @ CuNCs/MB modified substrate; 2) measurement of human anti-asialoglycoprotein receptor human anti-ASGPR: collecting SED/Ab/Ag-Ti at different concentrations through smart phone 3 C 2 @ CuNCs/MB modifies the photograph of the substrate and converts it into red, green and blue RGB images using the professional drawing software PowerPoint, calculates 1X 10 -6 U/mL–1×10 -2 Color values between U/mL; the color change may be defined by: colour(s)The value Color value = red value R + green value G × 256 + blue value B × 65536; using 808nm laser to SED/Ab/Ag-Ti 3 C 2 @ CuNCs/MB modified substrate with optical power density of 5W cm -2 Irradiating for 1min, and measuring by electronic thermometer to obtain 1 × 10 -8 U/mL–1×10 -3 Temperature change between U/mL; subjecting SED/Ab/Ag-Ti 3 C 2 The @ CuNCs/MB modified substrate is connected with a digital multimeter, and SED/Ab/Ag-Ti is subjected to laser alignment at 808nm 3 C 2 @ CuNCs/MB modified substrate with optical power density of 5W cm -2 Irradiating for 1min, and recording with digital multimeter to obtain 1 × 10 -8 U/mL–1×10 -2 Resistance change between U/mL; drawing a working curve by recording different temperature changes, resistance changes and color value changes generated before and after 808nm laser radiation; and replacing the human asialoglycoprotein receptor (asialoglycoprotein receptor) anti-ASGPR standard solution with the sample solution to be detected for detection, and checking the detection result through a working curve.
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