CN110530949B - Preparation method of immunosensor constructed based on copper nanocluster-resonance energy transfer system - Google Patents
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Abstract
The invention relates to a preparation method and application of an immunosensor constructed based on a copper nanocluster-resonance energy transfer system, belonging to the field of nano materials and resonance energy transfer (FRET); according to the invention, copper nanocluster Cu NCs wrapped by bovine serum albumin BSA are used as an energy donor, and gold nanoparticle Au NPs are grown in situ on the surface of the copper nanocluster Cu NCs are used as an energy acceptor, so that a Cu NCs-Au NPs three-dimensional resonance structure is obtained, energy is transferred between acceptor pairs through a dipole-dipole non-radiative action mode, the transfer rate is inversely proportional to the sixth power of the distance, the Cu NCs and the acceptor pairs are overlapped in high ratio of spectra and are in an ultra-close pairing distance, ECL emission of the Cu NCs is remarkably quenched, the luminous intensity is controlled within a reasonable range, a novel ECL-FRET energy transfer model is provided for trace detection of biomarkers, and a novel thought is provided for mechanism exploration of a quenching type ECL sensing system.
Description
Technical Field
The invention belongs to the field of nano science and immunoassay.
Background
The copper nanocluster is a novel nano material which is aroused by the development of nano science, and the copper nanocluster has good optical stability, large Stokes shift, high quantum yield and good biocompatibility, so that a solid foundation is laid for the application of the copper nanocluster in the field of biosensing. The copper nanoclusters can be synthesized by a plurality of micro reducing agents such as nucleic acid, ligand and protein in an alkaline environment, wherein the copper nanoclusters taking bovine serum albumin as a template show short-wavelength electro-optical signals under low potential, and meanwhile, a large number of functional group active sites are exposed from the bovine serum albumin coated on the surface of the copper nanoclusters, so that great convenience is provided for modification of biomolecules, and compared with electroluminescent bodies such as semiconductor quantum dots, the copper nanoclusters have the unique advantage in immunoassay. However, the higher electroluminescent efficiency of the sensor also makes the signal of the sensing platform difficult to control, and based on the control, a resonance energy transfer system is provided to control the luminous intensity of the sensor so that the sensor can meet the requirement of trace analysis of a target object, and a brand new thought is provided for mechanism exploration of a quenching type ECL sensing system.
Disclosure of Invention
One of the technical tasks of the invention is to provide a preparation method of a Cu NCs-Au NPs three-dimensional resonance structure in order to make up the deficiency of ECL-FRET model evidence at the present stage. The method takes three conditions to be met for realizing resonance energy transfer as a hand grip, gets rid of the fact that the traditional ECL-FRET sensing model simply emphasizes the one-sided behavior of spectrum overlapping, focuses on researching and verifying the relation between quenching efficiency and the distance between the co-acceptors, and provides a brand new thought for researching the mechanism of a quenching type ECL sensing system.
The invention has the technical task of providing the application of the ECL-FRET model in the field of biosensing, and the biosensor developed based on the model can quickly detect procalcitonin, has high sensitivity, low detection limit and good reproducibility, and has wide application prospect in clinical detection.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
1. a preparation method of an immunosensor constructed based on a copper nanocluster-resonance energy transfer system is characterized by comprising the following steps:
(1) ultrasonically cleaning glassy carbon electrode in ethanol and deionized water for 30 s respectively, and sequentially using Al of 1.0 μm, 0.3 μm and 0.05 μm2O3Polishing the glass substrate with polishing powder to make the glass substrate smooth like a mirror surface, and drying the glass substrate with nitrogen;
(2) dripping 6 mu L of Cu NCs-Au NPs solution which is uniformly dispersed and has the concentration of 1-3 mg/mL onto the surface of a glassy carbon electrode, and naturally airing at room temperature;
(3) inserting the dried glassy carbon electrode into a recombinant protein A solution of 5-10 mu g/mL, and dissolving the solution in 4 DEG CoC, incubating for 30 min, washing with ultrapure water, and naturally drying at room temperature;
(4) dripping 6 mu L of bovine serum albumin solution with the mass fraction of 0.1% to seal the nonspecific active sites, washing the surface of the electrode by phosphate buffer solution with the pH of 7.4, and naturally drying at room temperature;
(5) dripping 6 mu L of antibody standard solution with the concentration of 1-2 mu g/mL into the mixture at 4 DEGoHatching under C1 h, washing the electrode surface with phosphate buffer solution with pH 7.4, 4oC, airing;
(6) 6 μ L of an antigen solution of unknown concentration corresponding to the antibody was added dropwise thereto at 4oIncubating for 1 h under C, washing the electrode surface with phosphate buffer solution with pH of 7.4, 4oC, airing, and finishing the construction of the sensor.
2. The preparation method of the immunosensor constructed based on the copper nanocluster-resonance energy transfer system comprises the following steps of:
at room temperature, fully mixing 1 mL of copper sulfate solution with the concentration of 5-10 mmol/L and 1 mL of bovine serum albumin solution with the concentration of 1-2 mg/mL, after vigorously stirring for 2-5 min, adding 0.1-1 mL of sodium hydroxide solution with the concentration of 1-2 mol/L, and then placing the mixed solution in a 65-mol/L containeroAnd C, performing water bath for 12 hours until the color of the solution is changed from light blue to brown, wherein copper ions are successfully reduced by BSA at the moment, and obtaining the BSA coated copper nanocluster Cu NCs. And mixing the prepared Cu NCs with an L-ascorbic acid solution with the concentration of 10 mmol/L, adding 5 mL of deionized water for dilution, adding 50-150 uL of chloroauric acid solution with the mass fraction of 2% into the solution, and continuously stirring for 2 h to obtain the purple-black Cu NCs-Au NPs three-dimensional resonance nanostructure.
3. The preparation method of the immunosensor constructed based on the copper nanocluster-resonance energy transfer system is characterized in that the antigen is procalcitonin which is a marker of acute bacterial infection and sepsis of a human body.
4. The sensor prepared by the preparation method of the immunosensor constructed based on the copper nanocluster-resonance energy transfer system is used for detecting the concentration of procalcitonin.
5. The method for detecting the concentration of procalcitonin is characterized by comprising the following steps:
(1) the method comprises the following steps of using a three-electrode system of an electrochemical workstation as an excitation source, using an Ag/AgCl electrode as a reference electrode, using a platinum electrode as a counter electrode, using a prepared electroluminescent sensor as a working electrode, combining the electrochemical workstation with an ultra-weak light detector, setting the high voltage of a photomultiplier to be 800V, setting the cyclic voltammetry scanning potential to be 0-1.5V, and setting the scanning speed to be 200 mV/s;
(2) detecting the electroluminescence signal intensity of the sensor under a series of antigen modification states of different concentrations of an object to be detected by an electroluminescence system in 10 mL of triethylamine phosphate buffer solution with the concentration of 50-80 mmol/L and the pH of 7.4, and drawing a working curve;
(3) and (3) replacing the antigen to be detected with the actual serum sample for detection.
Advantageous results of the invention
(1) The three-dimensional resonance ECL sensing model Cu NCs-Au NPs are prepared for the first time based on the copper nanoclusters, and the ECL strength of the copper nanoclusters can be effectively controlled by the structure through energy transfer in a dipole-dipole non-radiation action mode between receptor pairs;
(2) the ECL sensing platform is constructed for detecting the actual sample of procalcitonin based on the Cu NCs-Au NPs with the three-dimensional resonance structure for the first time, the sensor is high in sensitivity and wide in detection range, and the detection limit is as low as 2.9 fg/mL.
The specific implementation mode is as follows:
the invention will now be further illustrated by reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention.
Embodiment 1. a method for preparing an immunosensor based on a copper nanocluster-resonance energy transfer system, comprising the following steps:
(1) ultrasonically cleaning glassy carbon electrode in ethanol and deionized water for 30 s respectively, and sequentially using Al of 1.0 μm, 0.3 μm and 0.05 μm2O3Polishing the glass substrate with polishing powder to make the glass substrate smooth like a mirror surface, and drying the glass substrate with nitrogen;
(2) dripping 6 mu L of Cu NCs-Au NPs solution which is uniformly dispersed and has the concentration of 1 mg/mL onto the surface of a glassy carbon electrode, and naturally airing at room temperature;
(3) the dried glassy carbon electrode was inserted into a 5. mu.g/mL recombinant protein A solution at 4oC, incubating for 30 min, washing with ultrapure water, and naturally drying at room temperature;
(4) dripping 6 mu L of bovine serum albumin solution with the mass fraction of 0.1% to seal the nonspecific active sites, washing the surface of the electrode by phosphate buffer solution with the pH of 7.4, and naturally drying at room temperature;
(5) 6. mu.L of a standard antibody solution having a concentration of 1. mu.g/mL was added dropwise thereto at 4oIncubating for 1 h under C, washing the electrode surface with phosphate buffer solution with pH of 7.4, 4oC, airing;
(6) 6 μ L of an antigen solution of unknown concentration corresponding to the antibody was added dropwise thereto at 4oIncubating for 1 h under C, washing the electrode surface with phosphate buffer solution with pH of 7.4, 4oC, airing, and finishing the construction of the sensor.
Embodiment 2. a method for preparing an immunosensor based on a copper nanocluster-resonance energy transfer system, comprising the following steps:
(1) ultrasonically cleaning glassy carbon electrode in ethanol and deionized water for 30 s respectively, and sequentially using Al of 1.0 μm, 0.3 μm and 0.05 μm2O3Polishing the glass substrate with polishing powder to make the glass substrate smooth like a mirror surface, and drying the glass substrate with nitrogen;
(2) dripping 6 mu L of Cu NCs-Au NPs solution which is uniformly dispersed and has the concentration of 2 mg/mL onto the surface of a glassy carbon electrode, and naturally airing at room temperature;
(3) the above-mentioned dried glassy carbon electrode was inserted into 7.5. mu.g/mL of recombinant protein A solution at 4oC, incubating for 30 min, washing with ultrapure water, and naturally drying at room temperature;
(4) dripping 6 mu L of bovine serum albumin solution with the mass fraction of 0.1% to seal the nonspecific active sites, washing the surface of the electrode by phosphate buffer solution with the pH of 7.4, and naturally drying at room temperature;
(5) 6. mu.L of a standard antibody solution having a concentration of 1.5. mu.g/mL was added dropwise thereto at 4oIncubating for 1 h under C, washing the electrode surface with phosphate buffer solution with pH of 7.4, 4oC, airing;
(6) 6 μ L of an antigen solution of unknown concentration corresponding to the antibody was added dropwise thereto at 4oIncubating for 1 h under C, washing the electrode surface with phosphate buffer solution with pH of 7.4, 4oC, airing, and finishing the construction of the sensor.
Embodiment 3. a method for preparing an immunosensor based on a copper nanocluster-resonance energy transfer system, comprising the following steps:
(1) ultrasonically cleaning glassy carbon electrode in ethanol and deionized water for 30 s respectively, and sequentially using Al of 1.0 μm, 0.3 μm and 0.05 μm2O3Polishing the glass substrate with polishing powder to make the glass substrate smooth like a mirror surface, and drying the glass substrate with nitrogen;
(2) dripping 6 mu L of Cu NCs-Au NPs solution which is uniformly dispersed and has the concentration of 3 mg/mL onto the surface of a glassy carbon electrode, and naturally airing at room temperature;
(3) the dried glassy carbon electrode was inserted into 10. mu.g/mL recombinant protein A solution at 4oC, incubating for 30 min, washing with ultrapure water, and naturally drying at room temperature;
(4) dripping 6 mu L of bovine serum albumin solution with the mass fraction of 0.1% to seal the nonspecific active sites, washing the surface of the electrode by phosphate buffer solution with the pH of 7.4, and naturally drying at room temperature;
(5) 6. mu.L of an antibody standard solution having a concentration of 2. mu.g/mL was added dropwise thereto at 4oIncubating for 1 h under C, washing the electrode surface with phosphate buffer solution with pH of 7.4, 4oC, airing;
(6) 6 μ L of an antigen solution of unknown concentration corresponding to the antibody was added dropwise thereto at 4oIncubating for 1 h under C, washing the electrode surface with phosphate buffer solution with pH of 7.4, 4oC, airing, and finishing the construction of the sensor.
Example 4. Cu NCs-Au NPs solution, prepared by the following steps:
mixing 1 mL of 5 mmol/L copper sulfate solution and 1 mL of 1 mg/mL bovine serum albumin solution at room temperature, stirring vigorously for 2 min, adding 0.1 mL of 1 mol/L sodium hydroxide solution, and placing the mixture in 65oAnd C, performing water bath for 12 hours until the color of the solution is changed from light blue to brown, wherein copper ions are successfully reduced by BSA at the moment, and obtaining the BSA coated copper nanocluster Cu NCs. The prepared Cu NCs were mixed with a 10 mmol/L-ascorbic acid solution, followed by addition of 5 mL of deionized water for dilution, and then, the solution was addedAnd adding 50 uL of chloroauric acid solution with the mass fraction of 2% into the solution, and continuously stirring for 2 hours to obtain the purple-black Cu NCs-Au NPs three-dimensional resonance nano structure.
Example 5. Cu NCs-Au NPs solution, prepared by the following steps:
mixing 1 mL of copper sulfate solution with concentration of 7.5 mmol/L and 1 mL of bovine serum albumin solution with concentration of 1.5 mg/mL at room temperature, stirring vigorously for 3.5 min, adding 0.5 mL of sodium hydroxide solution with concentration of 1.5 mol/L, and placing the mixture in 65oAnd C, performing water bath for 12 hours until the color of the solution is changed from light blue to brown, wherein copper ions are successfully reduced by BSA at the moment, and obtaining the BSA coated copper nanocluster Cu NCs. And mixing the prepared Cu NCs with an L-ascorbic acid solution with the concentration of 10 mmol/L, then adding 5 mL of deionized water for dilution, then adding 100 uL of chloroauric acid solution with the mass fraction of 2% into the solution, and continuously stirring for 2 h to obtain the purple-black Cu NCs-Au NPs three-dimensional resonance nanostructure.
Example 6 Cu NCs-Au NPs solution, prepared by the following steps:
mixing 1 mL of 10 mmol/L copper sulfate solution and 1 mL of 2 mg/mL bovine serum albumin solution at room temperature, stirring vigorously for 5 min, adding 1 mL of 2 mol/L sodium hydroxide solution, and placing the mixture in 65oAnd C, performing water bath for 12 hours until the color of the solution is changed from light blue to brown, wherein copper ions are successfully reduced by BSA at the moment, and obtaining the BSA coated copper nanocluster Cu NCs. And mixing the prepared Cu NCs with an L-ascorbic acid solution with the concentration of 10 mmol/L, then adding 5 mL of deionized water for dilution, then adding 150 uL of chloroauric acid solution with the mass fraction of 2% into the solution, and continuously stirring for 2 h to obtain the purple-black Cu NCs-Au NPs three-dimensional resonance nanostructure.
Example 7 detection of Procalcitonin concentration
(1) The method comprises the following steps of using a three-electrode system of an electrochemical workstation as an excitation source, using an Ag/AgCl electrode as a reference electrode, using a platinum electrode as a counter electrode, using a prepared electroluminescent sensor as a working electrode, combining the electrochemical workstation with an ultra-weak light detector, setting the high voltage of a photomultiplier to be 800V, setting the cyclic voltammetry scanning potential to be 0-1.5V, and setting the scanning speed to be 200 mV/s;
(2) detecting the electroluminescence signal intensity of the sensor under a series of antigen modification states of objects to be detected with different concentrations through an electroluminescence system in 10 mL of triethylamine phosphate buffer solution with the concentration of 50 mmol/L and the pH value of 7.4, and drawing a working curve;
(3) and (3) replacing the antigen to be detected with the actual serum sample for detection.
Example 8 detection of Procalcitonin concentration
(1) The method comprises the following steps of using a three-electrode system of an electrochemical workstation as an excitation source, using an Ag/AgCl electrode as a reference electrode, using a platinum electrode as a counter electrode, using a prepared electroluminescent sensor as a working electrode, combining the electrochemical workstation with an ultra-weak light detector, setting the high voltage of a photomultiplier to be 800V, setting the cyclic voltammetry scanning potential to be 0-1.5V, and setting the scanning speed to be 200 mV/s;
(2) detecting the electroluminescent signal intensity of the sensor under a series of antigen modification states of objects to be detected with different concentrations through an electroluminescent system in 10 mL of triethylamine phosphate buffer solution with the concentration of 65 mmol/L and the pH value of 7.4, and drawing a working curve;
(3) and (3) replacing the antigen to be detected with the actual serum sample for detection.
Example 9 detection of Procalcitonin concentration
(1) The method comprises the following steps of using a three-electrode system of an electrochemical workstation as an excitation source, using an Ag/AgCl electrode as a reference electrode, using a platinum electrode as a counter electrode, using a prepared electroluminescent sensor as a working electrode, combining the electrochemical workstation with an ultra-weak light detector, setting the high voltage of a photomultiplier to be 800V, setting the cyclic voltammetry scanning potential to be 0-1.5V, and setting the scanning speed to be 200 mV/s;
(2) detecting the electroluminescence signal intensity of the sensor under a series of antigen modification states of objects to be detected with different concentrations through an electroluminescence system in 10 mL of triethylamine phosphate buffer solution with the concentration of 80 mmol/L and the pH value of 7.4, and drawing a working curve;
(3) and (3) replacing the antigen to be detected with the actual serum sample for detection.
Claims (3)
1. A preparation method of an immunosensor constructed based on a copper nanocluster-resonance energy transfer system is characterized by comprising the following steps:
(1) ultrasonically cleaning glassy carbon electrode in ethanol and deionized water for 30 s respectively, and sequentially using Al of 1.0 μm, 0.3 μm and 0.05 μm2O3Polishing the glass surface to be smooth like a mirror surface by polishing powder, and drying the glass surface by using nitrogen;
(2) dripping 6 mu L of Cu NCs-Au NPs solution which is uniformly dispersed and has the concentration of 1-3 mg/mL onto the surface of a glassy carbon electrode, and naturally airing at room temperature;
(3) inserting the dried glassy carbon electrode into a recombinant protein A solution of 5-10 mu g/mL, and dissolving the solution in a solvent of 4 DEG CoC, incubating for 30 min, washing with ultrapure water, and naturally drying at room temperature;
(4) dripping 6 mu L of bovine serum albumin solution with the mass fraction of 0.1% to seal the nonspecific active sites, washing the surface of the electrode by phosphate buffer solution with the pH of 7.4, and naturally drying at room temperature;
(5) dripping 6 mu L of antibody standard solution with the concentration of 1-2 mu g/mL into the mixture at 4 DEGoIncubating for 1 h under C, washing the electrode surface with phosphate buffer solution with pH of 7.4, 4oC, airing;
(6) 6 μ L of an antigen solution of unknown concentration corresponding to the antibody was added dropwise thereto at 4oIncubating for 1 h under C, washing the electrode surface with phosphate buffer solution with pH of 7.4, 4oC, drying in the air, and finishing the construction of the sensor;
the Cu NCs-Au NPs solution is prepared by the following steps:
at room temperature, fully mixing 1 mL of copper sulfate solution with the concentration of 5-10 mmol/L and 1 mL of bovine serum albumin solution with the concentration of 1-2 mg/mL, after vigorously stirring for 2-5 min, adding 0.1-1 mL of sodium hydroxide solution with the concentration of 1-2 mol/L, and then placing the mixed solution in a 65-mol/L containeroC, in a water bath for 12 hours until the color of the solution is changed from light blue to brown, at the moment, the copper ions are successfully reduced by bovine serum albumin, and copper nanocluster Cu NCs coated by the bovine serum albumin are obtained;
and mixing the prepared Cu NCs with an L-ascorbic acid solution with the concentration of 10 mmol/L, adding 5 mL of deionized water for dilution, adding 50-150 uL of chloroauric acid solution with the mass fraction of 2% into the solution, and continuously stirring for 2 h to obtain the purple-black Cu NCs-Au NPs three-dimensional resonance nanostructure.
2. The method for preparing the immunosensor based on the copper nanocluster-resonance energy transfer system construction of claim 1, wherein the antigen is procalcitonin which is a marker of acute bacterial infection and sepsis in humans.
3. The method for preparing the immunosensor based on the copper nanocluster-resonance energy transfer system construction of claim 1, wherein the immunosensor is used for detection of procalcitonin, and the detection steps are as follows:
(1) the method comprises the following steps of using a three-electrode system of an electrochemical workstation as an excitation source, using an Ag/AgCl electrode as a reference electrode, using a platinum electrode as a counter electrode, using a prepared immunosensor as a working electrode, combining the electrochemical workstation with an ultra-weak light detector, setting the high voltage of a photomultiplier to be 800V, setting the cyclic voltammetry scanning potential to be 0-1.5V, and setting the scanning speed to be 200 mV/s;
(2) detecting the electroluminescent signal intensity of the immunosensor in 10 mL of triethylamine phosphate buffer solution with the pH value of 7.4 and the concentration of 50-80 mmol/L through an electroluminescent system under the antigen modification state of a series of objects to be detected with different concentrations, and drawing a working curve;
(3) and (3) replacing the antigen to be detected with the actual serum sample for detection.
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