CN110441294B - Co-coated based on ferritin3O4Preparation method of biosensor with core-shell structure - Google Patents
Co-coated based on ferritin3O4Preparation method of biosensor with core-shell structure Download PDFInfo
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Abstract
The invention relates to a Fe-protein-coated Co-based coating3O4A preparation method of a biosensor with a core-shell structure, belonging to the field of novel nano materials and the technical field of biosensing; the invention is based on ECL technology, and firstly coats Co with ferritin3O4Covalently cross-linked N- (4-aminobutyl) -N-ethyl isoluminol ABEI with core-shell structure is used as a signal source, an immobilized antibody molecule with excellent biocompatibility of ferritin is utilized, and Co is utilized3O4The excellent catalysis effect of ECL reaction between the ABEI and the hydrogen peroxide can effectively amplify detection signals, the preparation method of the biosensor is simple in preparation, high in sensitivity and low in reaction energy consumption, the biosensor is applied to the detection of actual samples of parathyroid hormone, the detection limit is as low as 13 fg/mL, the linear range is as wide as 50 fg/mL-100 ng/mL, the sensitivity is high, the reproducibility is good, and the potential application value is relatively high.
Description
Technical Field
The invention belongs to the field of novel nano materials and the technical field of biosensing
Background
As a research hotspot arising from the interdigitation of various disciplines such as biology, chemistry, medicine, electronic technology and the like, the Electrochemiluminescence (ECL) immunoassay technology is the organic combination of electrochemistry, chemiluminescence and immunoassay technology, has the advantages of low cost, good selectivity, high sensitivity, high analysis speed, easy automation, miniaturization, integration and the like, and is widely applied to the fields of disease marker analysis, food safety analysis, environmental pollution analysis and the like.
Parathyroid hormone is a polypeptide of 84 amino acids secreted by the parathyroid chief cells, and changes in the level of parathyroid hormone in the serum are directly indicative of parathyroid disease. For example, hyperparathyroidism can lead to overproduction of parathyroid hormone. Closely related to the occurrence of cardiovascular and chronic kidney diseases and cancer, it is involved in the metastatic pathway through the growth and differentiation of tumor cells, and especially when breast and prostate cancers metastasize to bone, the parathyroid hormone levels in serum rise markedly. Therefore, the detection of parathyroid hormone in human blood is of great importance for monitoring the therapeutic efficacy of patients with osteoporosis, parathyroid disease, hypercalcemia associated with malignancy, and cancer. So far, only several analysis methods such as fluorescence, electrochemical analysis, and chromatography have been developed. Because the half-life of parathyroid hormone is very short, a simple, quick and accurate determination method is developed, and the method has great significance for the instant detection of parathyroid hormone (particularly parathyroid hormone in operation).
Disclosure of Invention
One of the technical tasks of the invention is to make up the defects of the prior detection technology and fully utilize ferritin to wrap Co3O4The core-shell structure has excellent biocompatibility and electrocatalytic activity, and a preparation method of the novel biosensor is provided for the first time based on the principle that the structure promotes N- (4-aminobutyl) -N-ethyl isoluminol ABEI electrochemiluminescence.
The second technical task of the invention is to provide the application of the biosensor, the sensor can rapidly detect parathyroid hormone, and has the advantages of high sensitivity, strong specificity and good reproducibility, the detection limit is 13 fg/mL, and the linear range is 50 fg/mL-50 ng/mL.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
1. co-coated based on ferritin3O4The preparation method of the biosensor with the core-shell structure comprises the following steps:
(1) polishing glassy carbon electrodes with the diameter of 4 mm by using alumina polishing powder with the diameter of 1.0 micron, 0.3 micron and 0.05 micron in sequence, and washing the polished glassy carbon electrodes with ultrapure water;
(2) dripping 6 mu L of parathyroid hormone-anti-labeled gold-hybrid graphite alkyne aerogel solution with the concentration of 2-4 mg/mL on the surface of a glassy carbon electrode to serve as a sensing substrate, and placing the sensing substrate at 37 ℃ for airing;
(3) dropwise adding 3 mu L of bovine serum albumin solution with the mass fraction of 1-3% to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode by phosphate buffer solution PBS with the pH of 7.4, and placing the electrode at 4 ℃ for airing;
(4) dripping 6 mu L of parathyroid hormone standard solution or parathyroid hormone solution with unknown concentration, incubating for 0.5-2 h at 37 ℃, washing the surface of the electrode by phosphate buffer solution PBS with pH 7.4, and placing the electrode at 4 ℃ for airing;
(5) dripping 6 mu L of parathyroid hormone secondary antibody labeled ferritin-coated Co with concentration of 2-4 mg/mL3O4And (3) placing the solution in a 37 ℃ air-drying mode, washing the surface of the electrode by phosphate buffer solution PBS (phosphate buffer solution) with the pH value of 7.4, placing the solution in a 4 ℃ air-drying mode, and finishing the construction of the sensor.
2. Co-coated based on ferritin3O4The preparation method of the biosensor with the core-shell structure comprises the following steps of:
taking 4-6 mL of 1% HAuCl4Adding the solution and 100 mL of ultrapure water into a clean and dry 250 mL three-neck round-bottom flask, dropwise adding 10 mL of 1% sodium citrate solution slowly under continuous stirring, heating and refluxing for 15-35 min, stopping heating until the color of the solution finally becomes wine red, cooling and filtering to obtain gold nanoparticles;
preparing 20 mL of a graphite alkyne solution with the concentration of 1.5-3.5 mg/mL, adding 3-5 mL of gold nanoparticles under uniform stirring, oscillating for 6 h, dialyzing for 8 h with ultrapure water, and then freeze-drying to obtain the gold hybrid graphite alkyne aerogel with the 3D structure;
preparing a 2 mg/mL gold-hybrid graphite alkyne aerogel solution, adding 100-300 mu L of parathyroid hormone primary antibody solution with the concentration of 10 mg/mL, oscillating and incubating for 6-18 h at 4 ℃, centrifuging, dispersing into 1 mL of phosphate buffer solution with the pH of 7.4 to obtain parathyroid hormone primary antibody labeled gold-hybrid graphite alkyne aerogel solution, and storing for later use at 4 ℃.
3. Co-coated based on ferritin3O4Preparation method of biosensor with core-shell structure, wherein Co is wrapped by ferritin marked by parathyroid hormone secondary antibody3O4The solution is prepared by the following steps:
carrying out N-pass on 25 mL of apoferritin solution with the concentration of 50-150 mu g/mL by using a capillary tube2Treating for 1 h, carrying out constant-temperature water bath on the solution, keeping the temperature at 65 ℃, adjusting the pH to 9.5 by using 0.1 mol/L sodium hydroxide solution, then adding 25 mL of cobalt nitrate hexahydrate with the concentration of 0.05 mol/L, stirring for 1 h, adding 0.4-1.2 mL of hydrogen peroxide solution with the concentration of 10 mmol/L, reacting for 5 min, adding 10 mL of sodium citrate solution with the concentration of 10 mmol to chelate free cobalt ions which do not enter the interior of apoferritin, and obtaining the ferritin-coated Co after centrifugation, dialysis and purification3O4A solution;
2 mL of ferritin coated Co3O4Adding 0.5-2.5 mL of N- (4-aminobutyl) -N-ethyl isoluminol solution with the concentration of 10 mmol/L and 50-150 uL of glutaraldehyde solution with the mass fraction of 50%, centrifugally separating, mixing, adding 100-300 mu L of parathyroid hormone secondary antibody solution with the concentration of 10 mg/mL, oscillating and incubating for 6-18 h at 4 ℃, centrifuging, dispersing into 1 mL of phosphate buffer solution with the pH of 7.4 to obtain parathyroid hormone secondary antibody labeled ferritin-coated Co with the parathyroid hormone secondary antibody3O4The solution was stored at 4 ℃ until use.
4. The prepared biosensor is used for detecting the concentration of parathyroid hormone.
5. The parathyroid hormone concentration detection steps are as follows:
(1) setting parameters: the high voltage of a photomultiplier of the ultra-weak electrochemiluminescence instrument is set to be 600V, the cyclic volt-ampere scanning potential range of the electrochemical workstation is set to be 0-0.6V, and the scanning rate is set to be 0.1V/s;
(2) and (3) testing: taking a silver/silver chloride electrode as a reference electrode, a platinum wire electrode as a counter electrode, taking the sensor prepared by the method as a working electrode, carrying out electrochemiluminescence test in 10 mL of phosphate buffer solution containing 45-75 mmol/L of hydrogen peroxide to obtain corresponding electrochemiluminescence signal intensity when the parathyroid hormone with different concentrations is incubated, and drawing a working curve, wherein the detection limit is 13 fg/mL, and the linear range is 50 fg/mL-50 ng/mL;
(3) and testing the electrochemiluminescence sensor for hatching the parathyroid hormone actual sample with unknown concentration to obtain corresponding signal intensity, and calculating according to the working curve to obtain the parathyroid hormone concentration in the actual sample.
Advantageous results of the invention
(1) Co is coated based on ferritin for the first time3O4The principle that the isoluminol ABEI electrochemiluminescence is promoted by a core-shell structure provides a novel and reliable immunosensing technology. Because the ferritin wraps the Co3O4The core-shell structure has excellent electrocatalysis performance, can carry out high-efficiency catalytic decomposition on hydrogen peroxide to generate superoxide anions and hydroxyl free radicals, provides sufficient co-reaction free radicals for ECL excitation of ABEI, and realizes effective amplification of ECL signals;
(2) the method solves the problems of complex operation, low sensitivity and poor reproducibility of the existing electrochemical detection technology, is applied to sample detection of the parathyroid hormone, has the detection limit of 36 fg/mL and the linear range of 100 fg/mL-50 ng/mL, and has the advantages of high response speed, high sensitivity, good reproducibility, simple preparation, low cost and environmental protection.
Detailed Description
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.
Example 1A ferritin-based coated Co3O4The preparation method of the biosensor with the core-shell structure comprises the following steps:
(1) polishing glassy carbon electrodes with the diameter of 4 mm by using alumina polishing powder with the diameter of 1.0 micron, 0.3 micron and 0.05 micron in sequence, and washing the polished glassy carbon electrodes with ultrapure water;
(2) dripping 6 mu L of parathyroid hormone-anti-labeled gold-hybrid graphite alkyne aerogel solution with the concentration of 2 mg/mL on the surface of a glassy carbon electrode to serve as a sensing substrate, and placing the sensing substrate at 37 ℃ for airing;
(3) dropwise adding 3 mu L of bovine serum albumin solution with the mass fraction of 1% to seal the nonspecific active sites on the surface of the electrode, washing the surface of the electrode with phosphate buffer solution PBS with the pH of 7.4, and placing the electrode at 4 ℃ for airing;
(4) dripping 6 mu L of parathyroid hormone standard solution or parathyroid hormone solution with unknown concentration, incubating at 37 ℃ for 0.5 h, washing the surface of the electrode with phosphate buffer solution PBS with pH 7.4, placing at 4 ℃ and drying in the air;
(5) dripping 6 mu L of parathyroid hormone secondary antibody labeled ferritin coated Co with concentration of 2 mg/mL3O4And (3) placing the solution in a 37 ℃ air-drying mode, washing the surface of the electrode by phosphate buffer solution PBS (phosphate buffer solution) with the pH value of 7.4, placing the solution in a 4 ℃ air-drying mode, and finishing the construction of the sensor.
Example 2A ferritin-based coated Co3O4The preparation method of the biosensor with the core-shell structure comprises the following steps:
(1) polishing glassy carbon electrodes with the diameter of 4 mm by using alumina polishing powder with the diameter of 1.0 micron, 0.3 micron and 0.05 micron in sequence, and washing the polished glassy carbon electrodes with ultrapure water;
(2) dripping 6 mu L of parathyroid hormone-anti-labeled gold-hybrid graphite alkyne aerogel solution with the concentration of 3 mg/mL on the surface of a glassy carbon electrode to serve as a sensing substrate, and placing the sensing substrate at 37 ℃ for airing;
(3) dropwise adding 3 mu L of 2% bovine serum albumin solution to seal the nonspecific active sites on the surface of the electrode, washing the surface of the electrode with phosphate buffer solution PBS (pH 7.4), and airing at 4 ℃;
(4) dripping 6 mu L of parathyroid hormone standard solution or parathyroid hormone solution with unknown concentration, incubating at 37 ℃ for 1.5 h, washing the surface of the electrode with phosphate buffer solution PBS with pH 7.4, placing at 4 ℃ and drying in the air;
(5) dripping 6 mu L of parathyroid hormone secondary antibody marked ferritin coated Co with concentration of 3 mg/mL3O4And (3) placing the solution in a 37 ℃ air-drying mode, washing the surface of the electrode by phosphate buffer solution PBS (phosphate buffer solution) with the pH value of 7.4, placing the solution in a 4 ℃ air-drying mode, and finishing the construction of the sensor.
Example 3A ferritin-based coated Co3O4The preparation method of the biosensor with the core-shell structure comprises the following steps:
(1) polishing glassy carbon electrodes with the diameter of 4 mm by using alumina polishing powder with the diameter of 1.0 micron, 0.3 micron and 0.05 micron in sequence, and washing the polished glassy carbon electrodes with ultrapure water;
(2) dripping 6 mu L of parathyroid hormone-anti-labeled gold-hybrid graphite alkyne aerogel solution with the concentration of 4 mg/mL on the surface of a glassy carbon electrode to serve as a sensing substrate, and placing the sensing substrate at 37 ℃ for airing;
(3) dropwise adding 3 mu L of bovine serum albumin solution with the mass fraction of 3% to seal the nonspecific active sites on the surface of the electrode, washing the surface of the electrode with phosphate buffer solution PBS with the pH of 7.4, and placing the electrode at 4 ℃ for airing;
(4) dripping 6 mu L of parathyroid hormone standard solution or parathyroid hormone solution with unknown concentration, incubating for 0.5-2 h at 37 ℃, washing the surface of the electrode by phosphate buffer solution PBS with pH 7.4, and placing the electrode at 4 ℃ for airing;
(5) dripping 6 mu L of parathyroid hormone secondary antibody labeled ferritin coated Co with concentration of 4 mg/mL3O4And (3) placing the solution in a 37 ℃ air-drying mode, washing the surface of the electrode by phosphate buffer solution PBS (phosphate buffer solution) with the pH value of 7.4, placing the solution in a 4 ℃ air-drying mode, and finishing the construction of the sensor.
Example 4. the parathyroid hormone-anti-labeled gold-hybrid graphdine aerogel solution was prepared by the following steps:
4 mL of 1% HAuCl was taken4Adding the solution and 100 mL of ultrapure water into a clean and dry 250 mL three-neck round-bottom flask; slowly adding 10 mL of 1% sodium citrate solution dropwise under stirring, heating and refluxing for 15 min, and stopping heating until the solution color finally turns into wine red; obtaining gold nanoparticles through cooling and filtering;
preparing 20 mL of a graphite alkyne solution with the concentration of 1.5 mg/mL, adding 3 mL of gold nanoparticles under uniform stirring, oscillating for 6 h, dialyzing for 8 h with ultrapure water, and then freeze-drying to obtain the gold hybrid graphite alkyne aerogel with the 3D structure;
preparing 2 mg/mL gold-hybrid graphite alkyne aerogel solution, adding 100 mu L parathyroid hormone primary antibody solution with the concentration of 10 mg/mL, oscillating and incubating for 6 h at 4 ℃, centrifuging, dispersing into 1 mL phosphate buffer solution with the pH of 7.4 to obtain parathyroid hormone primary antibody labeled gold-hybrid graphite alkyne aerogel solution, and storing at 4 ℃ for later use.
Example 5. the parathyroid hormone-anti-labeled gold-hybrid graphdine aerogel solution was prepared by the following steps:
5 mL of 1% HAuCl was taken4Adding the solution and 100 mL of ultrapure water into a clean and dry 250 mL three-neck round-bottom flask; slowly adding 10 mL of 1% sodium citrate solution dropwise under stirring, heating and refluxing for 25 min, and stopping heating until the solution color finally turns into wine red; obtaining gold nanoparticles through cooling and filtering;
preparing 20 mL of a graphite alkyne solution with the concentration of 2.5 mg/mL, adding 4 mL of gold nanoparticles under uniform stirring, oscillating for 6 h, dialyzing for 8 h with ultrapure water, and then freeze-drying to obtain the gold hybrid graphite alkyne aerogel with the 3D structure;
preparing 2 mg/mL gold-hybrid graphite alkyne aerogel solution, adding 200 mu L parathyroid hormone primary antibody solution with the concentration of 10 mg/mL, oscillating and incubating for 12 h at 4 ℃, dispersing into 1 mL phosphate buffer solution with the pH of 7.4 after centrifugation to obtain parathyroid hormone primary antibody labeled gold-hybrid graphite alkyne aerogel solution, and storing at 4 ℃ for later use.
Example 6. the parathyroid hormone-anti-labeled gold-hybrid graphdine aerogel solution was prepared by the following steps:
6 mL of 1% HAuCl was taken4Adding the solution and 100 mL of ultrapure water into a clean and dry 250 mL three-neck round-bottom flask; slowly adding 10 mL of 1% sodium citrate solution dropwise under stirring, heating and refluxing for 35 min, and stopping heating until the solution color finally turns into wine red; obtaining gold nanoparticles through cooling and filtering;
preparing 20 mL of 3.5 mg/mL graphite alkyne solution, adding 5 mL of gold nanoparticles under uniform stirring, oscillating for 6 h, dialyzing for 8 h with ultrapure water, and then freeze-drying to obtain the 3D-structure gold hybrid graphite alkyne aerogel;
preparing 2 mg/mL gold-hybrid graphite alkyne aerogel solution, adding 300 mu L parathyroid hormone primary antibody solution with the concentration of 10 mg/mL, oscillating and incubating for 18 h at 4 ℃, centrifuging, dispersing into 1 mL phosphate buffer solution with the pH of 7.4 to obtain parathyroid hormone primary antibody labeled gold-hybrid graphite alkyne aerogel solution, and storing at 4 ℃ for later use.
Example 7 the parathyroid hormone Secondary antibody-labeled ferritin-coated Co3O4The solution is prepared by the following steps:
carrying out N-pass on 25 mL of apoferritin solution with the concentration of 50 mu g/mL by using a capillary tube2Treating for 1 h, performing constant-temperature water bath on the solution, keeping the temperature at 65 ℃, adjusting the pH to 9.5 by using 0.1 mol/L sodium hydroxide solution, then adding 25 mL of cobalt nitrate hexahydrate with the concentration of 0.05 mol/L, stirring for 1 h, adding 0.4 mL of hydrogen peroxide solution with the concentration of 10 mmol/L, reacting for 5 min, adding 10 mL of sodium citrate solution with the concentration of 10 mmol to chelate free cobalt ions which do not enter the interior of apoferritin, and obtaining the ferritin-coated Co after centrifugation, dialysis and purification3O4A solution;
2 mL of ferritin coated Co3O4Adding 0.5 mL of concentrated solutionCentrifuging 10 mmol/L N- (4-aminobutyl) -N-ethyl isoluminol solution and 50 uL of 50% glutaraldehyde solution, mixing, adding 100 uL parathyroid hormone secondary antibody solution with concentration of 10 mg/mL, oscillating and incubating at 4 ℃ for 6 h, centrifuging, dispersing into 1 mL phosphate buffer solution with pH 7.4 to obtain parathyroid hormone secondary antibody labeled ferritin-coated Co3O4The solution was stored at 4 ℃ until use.
Example 8 the parathyroid hormone Secondary antibody-labeled ferritin-coated Co3O4The solution is prepared by the following steps:
carrying out N-pass on 25 mL apoferritin solution with the concentration of 100 mu g/mL by using a capillary tube2Treating for 1 h, performing constant-temperature water bath on the solution, keeping the temperature at 65 ℃, adjusting the pH to 9.5 by using 0.1 mol/L sodium hydroxide solution, then adding 25 mL of cobalt nitrate hexahydrate with the concentration of 0.05 mol/L, stirring for 1 h, adding 0.8 mL of hydrogen peroxide solution with the concentration of 10 mmol/L, reacting for 5 min, adding 10 mL of sodium citrate solution with the concentration of 10 mmol to chelate free cobalt ions which do not enter the interior of apoferritin, and obtaining the ferritin-coated Co after centrifugation, dialysis and purification3O4A solution;
2 mL of ferritin coated Co3O4Adding 1.5 mL of N- (4-aminobutyl) -N-ethyl isoluminol solution with the concentration of 10 mmol/L and 100 uL of glutaraldehyde solution with the mass fraction of 50%, centrifugally separating, mixing, adding 200 mu L of parathyroid hormone secondary antibody solution with the concentration of 10 mg/mL, oscillating and incubating at 4 ℃ for 12 h, centrifuging, dispersing into 1 mL of phosphate buffer solution with the pH of 7.4 to obtain parathyroid hormone secondary antibody labeled ferritin-coated Co3O4The solution was stored at 4 ℃ until use.
Example 9 the parathyroid hormone Secondary antibody-labeled ferritin-coated Co3O4The solution is prepared by the following steps:
carrying out N-pass on 25 mL apoferritin solution with the concentration of 150 mu g/mL by using a capillary tube2Treatment, after 1 h the solution was subjected to a thermostatic water bath, the temperature being maintained at 65 ℃ and the mixture was usedAdjusting the pH value to 9.5 by 0.1 mol/L sodium hydroxide solution, then adding 25 mL of cobalt nitrate hexahydrate with the concentration of 0.05 mol/L, stirring for 1 h, adding 1.2 mL of hydrogen peroxide solution with the concentration of 10 mmol/L, reacting for 5 min, adding 10 mL of sodium citrate solution with the concentration of 10 mmol to chelate free cobalt ions which do not enter the interior of the apoferritin, and obtaining the ferritin-coated Co after centrifugation, dialysis and purification3O4A solution;
2 mL of ferritin coated Co3O4Adding 2.5 mL of N- (4-aminobutyl) -N-ethyl isoluminol solution with the concentration of 10 mmol/L and 150 uL of glutaraldehyde solution with the mass fraction of 50%, centrifugally separating, mixing, adding 300 mu L of parathyroid hormone secondary antibody solution with the concentration of 10 mg/mL, oscillating and incubating at 4 ℃ for 18 h, centrifuging, dispersing into 1 mL of phosphate buffer solution with the pH of 7.4 to obtain parathyroid hormone secondary antibody labeled ferritin-coated Co with secondary antibody3O4The solution was stored at 4 ℃ until use.
Example 10 the sensor is used for detecting the concentration of parathyroid hormone, and the operation steps are as follows:
(1) setting parameters: the high voltage of a photomultiplier of the ultra-weak electrochemiluminescence instrument is set to be 600V, the cyclic volt-ampere scanning potential range of the electrochemical workstation is set to be 0-0.6V, and the scanning rate is set to be 0.1V/s;
(2) and (3) testing: taking a silver/silver chloride electrode as a reference electrode, a platinum wire electrode as a counter electrode, taking the sensor prepared by the method as a working electrode, carrying out electrochemiluminescence test in 10 mL of phosphate buffer solution containing 45 mmol/L hydrogen peroxide to obtain corresponding electrochemiluminescence signal intensity when the parathyroid hormone with different concentrations is incubated, and drawing a working curve, wherein the detection limit is 13 fg/mL, and the linear range is 50 fg/mL-50 ng/mL;
(3) and testing the electrochemiluminescence sensor for hatching the parathyroid hormone actual sample with unknown concentration to obtain corresponding signal intensity, and calculating according to the working curve to obtain the parathyroid hormone concentration in the actual sample.
Example 11. the sensor is used for the detection of parathyroid hormone concentration, and the operation steps are as follows:
(1) setting parameters: the high voltage of a photomultiplier of the ultra-weak electrochemiluminescence instrument is set to be 600V, the cyclic volt-ampere scanning potential range of the electrochemical workstation is set to be 0-0.6V, and the scanning rate is set to be 0.1V/s;
(2) and (3) testing: taking a silver/silver chloride electrode as a reference electrode, a platinum wire electrode as a counter electrode, taking the sensor prepared by the method as a working electrode, carrying out electrochemiluminescence test in 10 mL of phosphate buffer solution containing 65 mmol/L hydrogen peroxide to obtain corresponding electrochemiluminescence signal intensity when the parathyroid hormone with different concentrations is incubated, and drawing a working curve, wherein the detection limit is 13 fg/mL, and the linear range is 50 fg/mL-50 ng/mL;
(3) and testing the electrochemiluminescence sensor for hatching the parathyroid hormone actual sample with unknown concentration to obtain corresponding signal intensity, and calculating according to the working curve to obtain the parathyroid hormone concentration in the actual sample.
Example 12 the sensor is used for detecting the concentration of parathyroid hormone, and the operation steps are as follows:
(1) setting parameters: the high voltage of a photomultiplier of the ultra-weak electrochemiluminescence instrument is set to be 600V, the cyclic volt-ampere scanning potential range of the electrochemical workstation is set to be 0-0.6V, and the scanning rate is set to be 0.1V/s;
(2) and (3) testing: taking a silver/silver chloride electrode as a reference electrode, a platinum wire electrode as a counter electrode, taking the sensor prepared by the method as a working electrode, carrying out electrochemiluminescence test in 10 mL of phosphate buffer solution containing 75 mmol/L hydrogen peroxide to obtain corresponding electrochemiluminescence signal intensity when the parathyroid hormone with different concentrations is incubated, and drawing a working curve, wherein the detection limit is 13 fg/mL, and the linear range is 50 fg/mL-50 ng/mL;
(3) and testing the electrochemiluminescence sensor for hatching the parathyroid hormone actual sample with unknown concentration to obtain corresponding signal intensity, and calculating according to the working curve to obtain the parathyroid hormone concentration in the actual sample.
Claims (5)
1. Co-coated based on ferritin3O4The preparation method of the biosensor with the core-shell structure is characterized by comprising the following steps:
(1) polishing glassy carbon electrodes with the diameter of 4 mm by using alumina polishing powder with the diameter of 1.0 micron, 0.3 micron and 0.05 micron in sequence, and washing the polished glassy carbon electrodes with ultrapure water;
(2) dripping 6 mu L of parathyroid hormone-anti-labeled gold-hybrid graphite alkyne aerogel solution with the concentration of 2-4 mg/mL on the surface of a glassy carbon electrode to serve as a sensing substrate, and placing the sensing substrate at 37 ℃ for airing;
(3) dropwise adding 3 mu L of bovine serum albumin solution with the mass fraction of 1-3% to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode by phosphate buffer solution PBS with the pH of 7.4, and placing the electrode at 4 ℃ for airing;
(4) dripping 6 mu L of parathyroid hormone standard solution or parathyroid hormone solution with unknown concentration, incubating for 0.5-2 h at 37 ℃, washing the surface of the electrode by phosphate buffer solution PBS with pH 7.4, and placing the electrode at 4 ℃ for airing;
(5) dripping 6 mu L of parathyroid hormone secondary antibody labeled ferritin-coated Co with concentration of 2-4 mg/mL3O4And (3) placing the solution in a 37 ℃ air-drying mode, washing the surface of the electrode by phosphate buffer solution PBS (phosphate buffer solution) with the pH value of 7.4, placing the solution in a 4 ℃ air-drying mode, and finishing the construction of the sensor.
2. The ferritin-coated Co-based material of claim 13O4The preparation method of the biosensor with the core-shell structure is characterized in that the parathyroid hormone-anti-labeled gold-hybrid grapyne aerogel solution is prepared by the following steps:
taking 4-6 mL of 1% HAuCl4Adding the solution and 100 mL of ultrapure water into a clean and dry 250 mL three-neck round-bottom flask, dropwise adding 10 mL of 1% sodium citrate solution slowly under continuous stirring, heating and refluxing for 15-35 min, stopping heating until the solution color finally becomes wine red, cooling and filtering to obtain gold nanoparticlesA seed;
preparing 20 mL of a graphite alkyne solution with the concentration of 1.5-3.5 mg/mL, adding 3-5 mL of gold nanoparticles under uniform stirring, oscillating for 6 h, dialyzing for 8 h with ultrapure water, and then freeze-drying to obtain the gold hybrid graphite alkyne aerogel with the 3D structure;
preparing a 2 mg/mL gold-hybrid graphite alkyne aerogel solution, adding 100-300 mu L of parathyroid hormone primary antibody solution with the concentration of 10 mg/mL, oscillating and incubating for 6-18 h at 4 ℃, centrifuging, dispersing into 1 mL of phosphate buffer solution with the pH of 7.4 to obtain parathyroid hormone primary antibody labeled gold-hybrid graphite alkyne aerogel solution, and storing for later use at 4 ℃.
3. The ferritin-coated Co-based material of claim 13O4The preparation method of the biosensor with the core-shell structure is characterized in that the parathyroid hormone secondary antibody marked ferritin wraps Co3O4The solution is prepared by the following steps:
carrying out N-pass on 25 mL of apoferritin solution with the concentration of 50-150 mu g/mL by using a capillary tube2Treating for 1 h, carrying out constant-temperature water bath on the solution, keeping the temperature at 65 ℃, adjusting the pH to 9.5 by using 0.1 mol/L sodium hydroxide solution, then adding 25 mL of cobalt nitrate hexahydrate with the concentration of 0.05 mol/L, stirring for 1 h, adding 0.4-1.2 mL of hydrogen peroxide solution with the concentration of 10 mmol/L, reacting for 5 min, adding 10 mL of sodium citrate solution with the concentration of 10 mmol to chelate free cobalt ions which do not enter the interior of apoferritin, and obtaining the ferritin-coated Co after centrifugation, dialysis and purification3O4A solution;
2 mL of ferritin coated Co3O4Adding 0.5-2.5 mL of N- (4-aminobutyl) -N-ethyl isoluminol solution with the concentration of 10 mmol/L and 50-150 uL of glutaraldehyde solution with the mass fraction of 50%, centrifugally separating, mixing, adding 100-300 mu L of parathyroid hormone secondary antibody solution with the concentration of 10 mg/mL, oscillating and incubating for 6-18 h at 4 ℃, centrifuging, and dispersing into 1 mL of phosphate buffer solution with the pH of 7.4 to obtain the parathyroid hormone secondary antibody labeled ferritin bagWrapped with Co3O4The solution was stored at 4 ℃ until use.
4. Use of the biosensor prepared by the preparation method according to claim 1 for detecting parathyroid hormone concentration.
5. The use of claim 4, wherein the steps of operating are as follows:
(1) setting parameters: the high voltage of a photomultiplier of the ultra-weak electrochemiluminescence instrument is set to be 600V, the cyclic volt-ampere scanning potential range of the electrochemical workstation is set to be 0-0.6V, and the scanning rate is set to be 0.1V/s;
(2) and (3) testing: taking a silver/silver chloride electrode as a reference electrode, a platinum wire electrode as a counter electrode, taking the sensor prepared by the method as a working electrode, carrying out electrochemiluminescence test in 10 mL of phosphate buffer solution containing 45-75 mmol/L of hydrogen peroxide to obtain corresponding electrochemiluminescence signal intensity when the parathyroid hormone with different concentrations is incubated, and drawing a working curve, wherein the detection limit is 13 fg/mL, and the linear range is 50 fg/mL-50 ng/mL;
(3) and testing the electrochemiluminescence sensor for hatching the parathyroid hormone actual sample with unknown concentration to obtain corresponding signal intensity, and calculating according to the working curve to obtain the parathyroid hormone concentration in the actual sample.
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