CN114965633B - Preparation method of ratio sensor constructed based on cysteine gold and gold platinum molybdenum disulfide - Google Patents
Preparation method of ratio sensor constructed based on cysteine gold and gold platinum molybdenum disulfide Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a ratio sensor constructed based on cysteine gold and gold platinum molybdenum disulfide, and belongs to the technical fields of novel functional nano materials, immunoassay and biosensing. Unlike conventional electrochemical sensors, which use the absolute value of a single electrical signal as an output, the ratio sensor is based on a dual signal ratio as an output to achieve accurate detection. The read-out mode of the ratio signal provides a built-in correction factor which helps to expel non-specific disturbances with significant success in improving sensor reproducibility and in addition improves the accuracy and sensitivity of the detection results.
Description
Technical Field
The invention belongs to the technical fields of novel functional nano materials, immunoassay and biosensing, and relates to a preparation method and application of a ratio sensor constructed based on cysteine gold and gold platinum molybdenum disulfide. The immunosensor is specifically constructed by adopting a novel functional nano material and is used for detecting cardiac troponin antigen.
Background
Cardiovascular and cerebrovascular diseases (CVDs) are common diseases severely threatening human health, and have the characteristics of high morbidity, high disability rate and high mortality rate, accounting for 31% of the global mortality rate. Acute Myocardial Infarction (AMI) mortality is the leading cause of cardiovascular and cerebrovascular disease. The cardiac troponin T antigen cTnT and the cardiac troponin I antigen cTnI are key markers for diagnosing the occurrence of AMI. When myocardial cells are damaged, the concentration of cTnT and cTnI can be rapidly increased to the detection level, and can be maintained for 5-7 days, so that sufficient time is provided for the re-detection of CVDs, and the diagnosis accuracy is further improved. Through detecting the contents of cTnT and cTnI in vivo, the occurrence of AMI can be effectively predicted, preventive measures can be taken as early as possible, and the death rate can be reduced. Therefore, the development of a high-sensitivity and high-accuracy method for detecting the cTnT and the cTnI has important clinical significance.
Disclosure of Invention
The invention provides a preparation method and application of a ratio sensor constructed based on cysteine gold and gold platinum molybdenum disulfide, which realize sensitive detection of myocardial troponin antigen.
The invention aims at providing a preparation method of a ratio sensor constructed based on cysteine gold and gold platinum molybdenum disulfide.
The second purpose of the invention is to construct a ratio sensor of the prepared cysteine gold and gold platinum molybdenum disulfide for detecting myocardial troponin antigen.
In order to achieve one of the above purposes, the invention discloses a preparation method of a ratio sensor constructed based on cysteine gold and gold platinum molybdenum disulfide, which is characterized by comprising the following steps:
(1) The glassy carbon electrode with the diameter of 4 mm is sequentially coated with Al with the diameters of 1.0 mu m, 0.3 mu m and 0.05 mu m 2 O 3 Polishing by polishing powder and cleaning by ultrapure water;
(2) Dripping thionine functionalized cysteine gold solution with the concentration of 3-9 mu L and the concentration of 0.5-1.5 mg/mL on the surface of an electrode, and airing in a refrigerator at the temperature of 4 ℃;
(3) Immersing the electrode into a solution of a myocardial troponin capture antibody of 5-15 mug/mL, incubating for 12 h at 4 ℃, and airing in a refrigerator at 4 ℃;
(4) Continuously dripping 2-4 mu L of 0.5-1.5 mg/mL of bovine serum albumin solution on the surface of the electrode, incubating 2-6 h in a refrigerator at 4 ℃, flushing the surface of the electrode with ultrapure water, and airing in the refrigerator at 4 ℃;
(5) Continuously immersing the electrode into a series of solutions of cardiac troponin antigens with different concentrations of 10 fg/mL-100 ng/mL, incubating at 37 ℃ for 1 h, flushing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;
(6) Immersing the electrode into 1-3 mg/mL ferrocene functionalized gold platinum molybdenum disulfide labeled myocardial troponin antigen detection antibody solution, incubating at 37 ℃ for 1 h, flushing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃ to obtain the cysteine gold and gold platinum molybdenum disulfide based construction ratio sensor.
Further, the thionine functionalized cysteine gold solution is obtained by mixing thionine and thionine in the solution to obtain thionine functionalized cysteine gold, and dispersing the thionine functionalized cysteine gold in ultrapure water to obtain the thionine functionalized cysteine gold solution.
Further, the preparation method of the thionine functionalized cysteine gold solution comprises the following specific steps:
(1) Preparation of gold cysteine solution
Adding 5-15 mL of HAuCl with mass fraction of 1% into cysteine solution with concentration of 2 mmol/L under stirring of 15-25 and 25 mL 4 Continuously stirring and rapidly adding 1-2 mL NaBH with concentration of 0.01 mmol/L 4 Stirring the solution at room temperature for reaction 2 h, centrifuging at 8000 r/min for 10 min, washing with ultrapure water for 3 times, and drying in a blast drying oven to obtain cysteine gold;
(2) Preparation of thionine functionalized cysteine gold solution
Dispersing 2-6 mg thionine in 2-4 mL of the cysteine gold solution prepared by 2 mg/mL, continuously stirring, adding 2-4 mL of 10 mmol/L EDC and 2-4 mL mmol/L NHS, stirring 24 h, centrifugally washing, and drying in a vacuum drying oven at 60 ℃ to prepare thionine functionalized cysteine gold;
and dispersing 0.5-1.5 mg of the prepared thionine functionalized cysteine gold in 1 mL ultrapure water to prepare a thionine functionalized cysteine gold solution.
Further, the ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution is prepared by mixing a gold platinum solution and a molybdenum disulfide solution, separating and drying the mixture to obtain gold platinum molybdenum disulfide, combining the gold platinum molybdenum disulfide with ferrocene, centrifugally washing the combination of the gold platinum molybdenum disulfide and the ferrocene to obtain ferrocene functionalized gold platinum molybdenum disulfide, dispersing the ferrocene functionalized gold platinum molybdenum disulfide in ultrapure water, and mixing the dispersion of the ferrocene functionalized gold platinum molybdenum disulfide with the cardiac troponin antigen detection antibody solution to obtain the ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution.
Further, the specific preparation steps of the ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution are as follows:
(1) Preparation of gold platinum solution
1) Mixing 1-3 mL of HAuCl with the mass fraction of 1% 4 Adding into 100 mL ultrapure water, rapidly adding 2-4 mL sodium citrate solution with mass fraction of 1% under stirring, and slowly adding NaBH 4 Until the solution turns from light yellow to reddish brown, stirring overnight to obtain solution A;
2) 2 to 4 of mL with the mass fraction of 1 percent of H 2 PtCl 6 Adding the solution A into the boiled solution A, continuously adding 1-3 mL of ascorbic acid with the concentration of 0.1 mol/L, and heating for 30 min to obtain a gold-platinum solution;
(2) Preparation of gold platinum molybdenum disulfide solution
Adding 10-30 mL mass percent of polydiene dimethyl ammonium chloride solution into 10-30 mL molybdenum disulfide dispersion liquid under stirring, stirring for 30 min, centrifugally separating, washing 3 times with ultrapure water, adding 5-15 mL gold platinum solution prepared in the step (1), stirring for 1 h, carrying out ultrasonic treatment for 15 min, centrifugally washing for 10 min under 4000-8000 r/min, and drying in a vacuum drying oven at 40 ℃ to obtain gold platinum molybdenum disulfide;
dispersing 6 mg gold platinum molybdenum disulfide in 1 mL ultrapure water to prepare 6 mg/mL gold platinum molybdenum disulfide solution for later use;
(3) Preparation of ferrocene functionalized gold platinum molybdenum disulfide labeled myocardial troponin antigen detection antibody solution
Mixing 1-3 mL of gold platinum molybdenum disulfide solution prepared in the step (2) with 1-3 mL of 2.5% glutaraldehyde solution, adding 10-30 mg ferrocene, stirring 6 h, centrifugally washing, dispersing in 1-3 mL of 10 mug/mL of myocardial troponin antigen detection antibody solution, vibrating overnight at 4 ℃, centrifugally washing to prepare ferrocene-functionalized gold platinum molybdenum disulfide labeled myocardial troponin antigen detection antibody, dispersing in PBS buffer solution with pH of 7.4 to prepare 1-3 mg/mL of ferrocene-functionalized gold platinum molybdenum disulfide labeled myocardial troponin antigen detection antibody solution, and preserving at 4 ℃ for later use.
Further, the cardiac troponin antigen is selected from one of cardiac troponin T antigen cTnT, cardiac troponin I antigen cTnI.
To achieve the second object, the invention discloses an application of a ratio sensor constructed based on cysteine gold and gold platinum molybdenum disulfide, which is used for detecting myocardial troponin antigen.
The specific steps are as follows:
(1) Using an electrochemical workstation to test in a three-electrode system, wherein a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, and the prepared sensor is used as a working electrode and is tested in a phosphate buffer solution with the pH of 10 mL and 0.1 mol/L and 7.4;
(2) Detecting the analyte by using a differential pulse voltammetry, wherein the scanning potential voltage is-0.6V to 0.6V, sharp dual-signal peaks are respectively obtained at-0.3V and 0.2V, the differential pulse voltammetry dual-signal current intensities corresponding to the cardiac troponin antigen solutions with different concentrations are recorded, and the dual-signal current ratio is calculated to be used as an output signal;
(3) And obtaining the concentration of the cardiac troponin antigen in the sample to be detected by using a working curve method.
Advantageous results of the invention
(1) The invention designs a ratio sensor based on cysteine gold and gold platinum molybdenum disulfide, which is different from the traditional electrochemical sensor in that the absolute value of a single electric signal is used as output, and the ratio sensor is used for realizing accurate detection based on the ratio of double signals as output. The read-out mode of the ratio signal provides a built-in correction factor, is favorable for discharging nonspecific interference, has remarkable effect in improving the reproducibility of the sensor, and improves the accuracy and the sensitivity of a detection result;
(2) According to the invention, thionine functionalized cysteine gold is used as a substrate material for immobilizing the cardiac troponin antigen capture antibody, the cysteine gold has good conductivity and abundant active sites, can be tightly combined with the high-density cardiac troponin antigen capture antibody, and can generate a sharp signal peak as a signal probe. The ferrocene functionalized gold platinum molybdenum disulfide is used as a marking material for immobilizing the cardiac troponin antigen detection antibody, has good biocompatibility, large specific surface area and good dispersibility, and a large number of binding sites can firmly bind with the high-density cardiac troponin antigen detection antibody, so that a good basis is provided for the subsequent specific binding with the cardiac troponin antigen, and in addition, the higher conductivity is also favorable for obtaining a stronger differential pulse voltammetry signal;
(3) The thionine functionalized cysteine gold and the ferrocene functionalized gold platinum molybdenum disulfide respectively show sharp signal peaks at-0.3V and 0.2V in electrochemical detection. As the concentration of cardiac troponin antigen increases, the amount of bound cardiac troponin antigen detection antibody also increases, resulting in an increase in the peak of oxidative signal produced by ferrocene and a decrease in the peak of oxidative signal produced by thionine. Based on the ratio of the double signals and the currentΔI=ΔI Thionine (S) /ΔI Ferrocene ) The linear relation between the myocardial troponin antigen and the myocardial troponin antigen concentration realizes high-accuracy detection of the myocardial troponin antigen;
(4) A ratio sensor based on cysteine gold and gold platinum molybdenum disulfide is used for detecting cardiac troponin antigens, wherein the linear range of the ratio sensor is 10 fg/mL-100 ng/mL, the detection limit of the ratio sensor is 3.33 fg/mL, the linear range of the ratio sensor is 10 fg/mL-100 ng/mL, and the detection limit of the ratio sensor based on cysteine gold and gold platinum molybdenum disulfide is 3.34fg/mL, so that the aim of accurately detecting cardiac troponin antigens can be achieved.
Drawings
FIG. 1 shows a graph of differential pulse voltammetric double signal obtained from detection of a range of concentration gradient cardiac troponin antigen solutions.
Fig. 2 is a standard curve with a dual signal ratio (Δi=Δi thionine/Δi ferrocene) on the ordinate and antigen concentration vs. value on the abscissa.
Detailed Description
The invention will now be further illustrated by means of specific embodiments, without being limited thereto.
The raw materials used in the invention can be purchased from chemical reagent companies or biopharmaceutical companies.
1. Preparation of thionine functionalized cysteine gold solution
Example 1
The preparation of the thionine functionalized cysteine gold solution comprises the following steps:
(1) Preparation of gold cysteine solution
5 mL% HAuCl by mass was added to 15 mL,2 mmol/L cysteine solution with stirring 4 Stirring was continued and 1 mL was added rapidly, naBH at a concentration of 0.01 mmol/L 4 Stirring the solution at room temperature for reaction 2 h, centrifuging at 8000 r/min for 10 min, washing with ultrapure water for 3 times, and drying in a blast drying oven to obtain cysteine gold;
(2) Preparation of thionine functionalized cysteine gold solution
Dispersing 2 mg thionine in 2 mL,2 mg/mL of the prepared cysteine gold solution by ultrasonic, continuously stirring, adding 2 mL,10 mmol/L EDC and 2 mL,2 mmol/L NHS, stirring 24 h, centrifugally washing, and drying in a vacuum drying oven at 60 ℃ to prepare thionine functionalized cysteine gold;
the thionine functionalized cysteine gold prepared by 0.5. 0.5 mg is dispersed in 1 mL ultrapure water to prepare a thionine functionalized cysteine gold solution.
Example 2
The preparation of the thionine functionalized cysteine gold solution comprises the following steps:
(1) Preparation of gold cysteine solution
To 20 mL of cysteine solution having a concentration of 2 mmol/L, 10 mL of HAuCl having a mass fraction of 1% was added with stirring 4 Stirring was continued and 1.5. 1.5 mL NaBH was added rapidly at a concentration of 0.01 mmol/L 4 Stirring the solution at room temperature for reaction 2 h, centrifuging at 8000 r/min for 10 min, washing with ultrapure water for 3 times, and drying in a blast drying oven to obtain cysteine gold;
(2) Preparation of thionine functionalized cysteine gold solution
Dispersing 4 mg thionine in 3 mL,2 mg/mL of the prepared cysteine gold solution by ultrasonic, continuously stirring, adding 3 mL,10 mmol/L EDC and 3 mL,2 mmol/L NHS, stirring 24 h, centrifugally washing, and drying in a vacuum drying oven at 60 ℃ to prepare thionine functionalized cysteine gold;
taking 1 mg to obtain the thionine functionalized cysteine gold, dispersing the thionine functionalized cysteine gold into 1 mL ultrapure water to obtain the thionine functionalized cysteine gold solution.
Example 3
The preparation of the thionine functionalized cysteine gold solution comprises the following steps:
(1) Preparation of gold cysteine solution
To 25 mL of cysteine solution at a concentration of 2 mmol/L, 15 mL of HAuCl at a mass fraction of 1% was added with stirring 4 Stirring was continued and 2 mL was added rapidly, naBH at a concentration of 0.01 mmol/L 4 Stirring the solution at room temperature for reaction 2 h, centrifuging at 8000 r/min for 10 min, washing with ultrapure water for 3 times, and drying in a blast drying oven to obtain cysteine gold;
(2) Preparation of thionine functionalized cysteine gold solution
Dispersing 6 mg thionine in 4mL, 2 mg/mL of the prepared cysteine gold solution by ultrasonic, continuously stirring, adding 4mL, 10 mmol/L EDC and 4mL, 2 mmol/L NHS, stirring 24 h, centrifugally washing, and drying in a vacuum drying oven at 60 ℃ to prepare thionine functionalized cysteine gold;
taking 1.5mg of the thionine functionalized cysteine gold prepared above, dispersing the thionine functionalized cysteine gold in 1 mL ultrapure water to prepare a thionine functionalized cysteine gold solution.
2. Preparation of ferrocene functionalized gold platinum molybdenum disulfide labeled myocardial troponin antigen detection antibody solution
Example 4
The preparation of the ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution comprises the following steps:
(1) Preparation of gold platinum solution
1) Mixing 1 mL with 1% HAuCl 4 Adding into 100 mL ultrapure water, rapidly adding 2 mL sodium citrate solution with mass fraction of 1% under stirring, and slowly adding NaBH 4 Until the solution turns from light yellow to reddish brown, and stirring overnight to obtainSolution A;
2) Mixing 2. 2 mL with 1% H by mass 2 PtCl 6 Adding into boiled solution A, adding 1. 1 mL,0.1 mol/L ascorbic acid, and heating for 30 min to obtain gold platinum solution;
(2) Preparation of gold platinum molybdenum disulfide solution
Adding 10 mL mass percent of polydiene dimethyl ammonium chloride solution with stirring into 10 mL molybdenum disulfide dispersion liquid, stirring for 30 min, centrifugally separating, washing with ultrapure water for 3 times, adding 5 mL gold platinum solution prepared in the step (1), stirring for 1 h, carrying out ultrasonic treatment for 15 min, centrifugally washing for 10 min at 4000 r/min, and drying in a vacuum drying oven at 40 ℃ to obtain gold platinum molybdenum disulfide;
dispersing 6 mg gold platinum molybdenum disulfide in 1 mL ultrapure water to prepare 6 mg/mL gold platinum molybdenum disulfide solution for later use;
(3) Preparation of ferrocene functionalized gold platinum molybdenum disulfide labeled myocardial troponin antigen detection antibody solution
Mixing 1 mL of the 6 mg/mL gold platinum molybdenum disulfide solution prepared in the step (2) with 1 mL of 2.5% glutaraldehyde solution, adding 10 mg ferrocene, stirring 6 h, centrifugally washing, redispersing in 1 mL of 10 mug/mL of myocardial troponin antigen detection antibody solution, vibrating overnight at 4 ℃, centrifugally washing to prepare ferrocene-functionalized gold platinum molybdenum disulfide labeled myocardial troponin antigen detection antibody, dispersing in PBS buffer solution with pH of 7.4 to prepare 1 mg/mL of ferrocene-functionalized gold platinum molybdenum disulfide labeled myocardial troponin antigen detection antibody solution, and preserving at 4 ℃ for later use.
Example 5
The preparation of the ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution comprises the following steps:
(1) Preparation of gold platinum solution
1) 2 mL HAuCl with mass fraction of 1% 4 Adding into 100 mL ultrapure water, rapidly adding 3 mL sodium citrate solution with mass fraction of 1% under stirring, and slowly adding NaBH 4 Until the solution turns from yellowish to reddish brown, and stirring overnightObtaining a solution A;
2) 3 mL, 1% H 2 PtCl 6 Adding into the boiled solution A, continuously adding 2 mL and 0.1 mol/L ascorbic acid, and heating for 30 min to obtain gold-platinum solution;
(2) Preparation of gold platinum molybdenum disulfide solution
Adding 20 mL mass percent of polydiene dimethyl ammonium chloride solution into 20 mL molybdenum disulfide dispersion liquid under stirring, stirring for 30 min, centrifugally separating, washing with ultrapure water for 3 times, adding 10 mL gold platinum solution prepared in the step (1), stirring for 1 h, carrying out ultrasonic treatment for 15 min, centrifugally washing for 10 min under 6000 r/min, and drying in a vacuum drying oven at 40 ℃ to obtain gold platinum molybdenum disulfide;
dispersing 6 mg gold platinum molybdenum disulfide in 1 mL ultrapure water to prepare 6 mg/mL gold platinum molybdenum disulfide solution for later use;
(3) Preparation of ferrocene functionalized gold platinum molybdenum disulfide labeled myocardial troponin antigen detection antibody solution
Mixing 2 mL of the 6 mg/mL gold platinum molybdenum disulfide solution prepared in the step (2) with 2 mL of 2.5% glutaraldehyde solution, adding 20 mg ferrocene, stirring 6 h, centrifugally washing, redispersing in 2 mL of 10 mug/mL of myocardial troponin antigen detection antibody solution, vibrating overnight at 4 ℃, centrifugally washing to prepare ferrocene-functionalized gold platinum molybdenum disulfide labeled myocardial troponin antigen detection antibody, dispersing in PBS buffer solution with pH of 7.4 to prepare 2 mg/mL of ferrocene-functionalized gold platinum molybdenum disulfide labeled myocardial troponin antigen detection antibody solution, and preserving at 4 ℃ for later use.
Example 6
The preparation of the ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution comprises the following steps:
(1) Preparation of gold platinum solution
1) 3 mL HAuCl with mass fraction of 1% 4 Adding into 100 mL ultrapure water, rapidly adding 4mL sodium citrate solution with mass fraction of 1% under stirring, and slowly adding NaBH 4 Until the solution turns from light yellow to reddish brown, stirringOvernight to obtain solution A;
2) Mixing 4. 4mL with 1% H 2 PtCl 6 Adding into the boiled solution A, continuously adding 3 mL and 0.1 mol/L ascorbic acid, and heating for 30 min to obtain gold-platinum solution;
(2) Preparation of gold platinum molybdenum disulfide
Adding 30 mL mass percent of polydiene dimethyl ammonium chloride solution into 30 mL molybdenum disulfide dispersion liquid under stirring, stirring for 30 min, centrifugally separating, washing with ultrapure water for 3 times, adding 15 mL gold platinum solution prepared in the step (1), stirring for 1 h, carrying out ultrasonic treatment for 15 min, centrifugally washing for 10 min at 8000 r/min, and drying in a vacuum drying oven at 40 ℃ to obtain gold platinum molybdenum disulfide;
dispersing 6 mg platinum molybdenum disulfide in 1 mL ultrapure water to prepare 6 mg/mL gold platinum molybdenum disulfide solution for later use;
(3) Preparation of ferrocene functionalized gold platinum molybdenum disulfide labeled myocardial troponin antigen detection antibody solution
Mixing 3 mL of the 6 mg/mL gold platinum molybdenum disulfide solution prepared in the step (2) with 3 mL of 2.5% glutaraldehyde solution, adding 30 mg ferrocene, stirring 6 h, centrifugally washing, redispersing in 3 mL of 10 mug/mL of myocardial troponin antigen detection antibody solution, vibrating overnight at 4 ℃, centrifugally washing to prepare ferrocene-functionalized gold platinum molybdenum disulfide labeled myocardial troponin antigen detection antibody, dispersing in PBS buffer solution with pH of 7.4 to prepare 3 mg/mL of ferrocene-functionalized gold platinum molybdenum disulfide labeled myocardial troponin antigen detection antibody solution, and preserving at 4 ℃ for later use.
3. Preparation of ratio sensor based on cysteine gold and gold platinum molybdenum disulfide construction
Example 7
A preparation method for constructing a ratio sensor based on cysteine gold and gold platinum molybdenum disulfide comprises the following steps:
(1) The glassy carbon electrode with the diameter of 4 mm is sequentially coated with Al with the diameters of 1.0 mu m, 0.3 mu m and 0.05 mu m 2 O 3 Polishing by polishing powder and cleaning by ultrapure water;
(2) Dripping 0.5 mg/mL of thionine functionalized cysteine gold solution prepared in the embodiment 1 with the volume of 3 mu L onto the surface of an electrode, and airing in a refrigerator with the temperature of 4 ℃;
(3) Immersing the electrode into a solution of a myocardial troponin capture antibody with the concentration of 5 mug/mL, incubating for 12 hours at 4 ℃, and airing in a refrigerator at 4 ℃;
(4) Continuously dripping 2 mu L of 0.5 mg/mL of bovine serum albumin solution on the surface of the electrode, incubating for 2 h in a refrigerator at 4 ℃, flushing the surface of the electrode with ultrapure water, and airing in the refrigerator at 4 ℃;
(5) Immersing the electrode into a solution of cardiac troponin antigen, incubating at 37 ℃ for 1 h, flushing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;
(6) Immersing the electrode into 1 mg/mL ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution prepared in embodiment 4, incubating at 37 ℃ for 1 h, flushing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃ to prepare the cysteine gold and gold platinum molybdenum disulfide construction ratio sensor.
Example 8
A preparation method for constructing a ratio sensor based on cysteine gold and gold platinum molybdenum disulfide comprises the following steps:
(1) The glassy carbon electrode with the diameter of 4 mm is sequentially coated with Al with the diameters of 1.0 mu m, 0.3 mu m and 0.05 mu m 2 O 3 Polishing by polishing powder and cleaning by ultrapure water;
(2) Dripping 6 mu L of the thionine functionalized cysteine gold solution with the concentration of 1 mg/mL prepared in the embodiment 2 onto the surface of an electrode, and airing in a refrigerator at the temperature of 4 ℃;
(3) Immersing the electrode into a solution of a myocardial troponin capture antibody of 10 mug/mL, incubating for 12 hours at 4 ℃, and airing in a refrigerator at 4 ℃;
(4) Continuously dripping 3 mu L of 1 mg/mL of bovine serum albumin solution on the surface of the electrode, incubating for 4 h in a refrigerator at 4 ℃, flushing the surface of the electrode with ultrapure water, and airing in the refrigerator at 4 ℃;
(5) Immersing the electrode into a solution of cardiac troponin antigen, incubating at 37 ℃ for 1 h, flushing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;
(6) Immersing the electrode into 2 mg/mL ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution prepared in embodiment 5, incubating at 37 ℃ for 1 h, flushing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃ to prepare the cysteine gold and gold platinum molybdenum disulfide construction ratio sensor.
Example 9
A preparation method for constructing a ratio sensor based on cysteine gold and gold platinum molybdenum disulfide comprises the following steps:
(1) The glassy carbon electrode with the diameter of 4 mm is sequentially coated with Al with the diameters of 1.0 mu m, 0.3 mu m and 0.05 mu m 2 O 3 Polishing by polishing powder and cleaning by ultrapure water;
(2) Taking 9 mu L of the thionine functionalized cysteine gold solution of 1.5 mg/mL prepared in the embodiment 3, dripping the thionine functionalized cysteine gold solution onto the surface of an electrode, and airing the thionine functionalized cysteine gold solution in a refrigerator at 4 ℃;
(3) Immersing the electrode into a solution of a myocardial troponin capture antibody of 15 mug/mL, incubating for 12 hours at 4 ℃, and airing in a refrigerator at 4 ℃;
(4) Continuously dripping 4 mu L of 1.5 mg/mL of bovine serum albumin solution on the surface of the electrode, incubating for 6 h in a refrigerator at 4 ℃, flushing the surface of the electrode with ultrapure water, and airing in the refrigerator at 4 ℃;
(5) Continuously incubating 1 h in the solution of the electrode cardiac troponin antigen at 37 ℃, flushing the electrode surface with ultrapure water, and drying in a refrigerator at 4 ℃;
(6) Immersing the electrode into 3 mg/mL ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution prepared in embodiment 6, incubating at 37 ℃ for 1 h, flushing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃ to prepare the cysteine gold and gold platinum molybdenum disulfide construction ratio sensor.
4. Drawing a detection working curve of cardiac troponin T antigen cTnT
Example 10
(1) Preparing standard solution, namely preparing a series of solutions of cardiac troponin antigens with different concentrations of 10 fg/mL-100 ng/mL;
(2) A ratio sensor based on gold cysteine and gold platinum molybdenum disulfide build-up with different concentrations of cardiac troponin antigen cTnT was obtained following the procedure of example 7 using standard solutions;
(3) Using an electrochemical workstation to test by using a three-electrode system, using a saturated calomel electrode as a reference electrode and a platinum wire electrode as an auxiliary electrode, using the cysteine gold-based and gold platinum molybdenum disulfide construction ratio sensor containing different myocardial troponin antigen concentrations as a working electrode, and testing in a pH 7.4 phosphate buffer solution of 10 mL and 0.1 mol/L;
(4) Detecting the analyte by using a differential pulse voltammetry, wherein the scanning potential voltage is-0.6V to 0.6V, sharp dual-signal peaks are respectively obtained at-0.3V and 0.2V, the differential pulse voltammetry dual-signal current intensities corresponding to the cardiac troponin antigen solutions with different concentrations are recorded, and the dual-signal current ratio is calculated to be used as an output signal;
(5) Obtaining the corresponding relation between different myocardial troponin antigen concentrations and output signals;
(6) And drawing a working curve according to the linear relation between the obtained differential pulse voltammetric double-signal ratio and the concentration of cTnT, and measuring the linear range to be 10 fg/mL-100 ng/mL and the detection limit to be 3.33 fg/mL.
5. Detection of cardiac troponin T antigen cTnT
Example 11
The sensor prepared using the procedure of example 8 was used to detect cardiac troponin I antigen cTnI in a sample, and a two-signal ratio was obtained as in steps (3) and (4) of example 10, and finally the concentration of cardiac troponin I antigen cTnI was calculated from the working curve.
6. Drawing a detection working curve of cardiac troponin T antigen cTnI
Example 12
(1) Preparing standard solutions, namely preparing a series of solutions of cardiac troponin antigens with different concentrations of 10 fg-100 ng/mL, wherein the solutions comprise (a) 10 fg/mL, (b) 100 fg/mL, (c) 1 pg/mL, (d) 10 pg/mL, (e) 50 pg/mL, (f) 100 pg/mL, (g) 1 ng/mL, (h) 10 pg/mL, (i) 100 pg/mL;
(2) A ratio sensor based on gold cysteine and gold platinum molybdenum disulfide build up containing different concentrations of cardiac troponin antigen cTnI was obtained following the procedure of example 7 using standard solutions;
(3) Using an electrochemical workstation to test by using a three-electrode system, using a saturated calomel electrode as a reference electrode and a platinum wire electrode as an auxiliary electrode, using the cysteine gold-based and gold platinum molybdenum disulfide construction ratio sensor containing different myocardial troponin antigen concentrations as a working electrode, and testing in a pH 7.4 phosphate buffer solution of 10 mL and 0.1 mol/L;
(4) Analytes were detected by differential pulse voltammetry with scanning potential voltages from-0.6V to 0.6V, sharp double signal peaks at-0.3V and 0.2V, respectively, with signal peak at-0.3V being the signal peak generated by Thi redox and Fc redox signal peak at 0.2V. Recording differential pulse volt-ampere dual-signal current intensities corresponding to myocardial troponin antigen solutions with different concentrations, and calculating a dual-signal current ratio to be used as an output signal;
(5) Obtaining the corresponding relation between different myocardial troponin antigen concentrations and output signals, as shown in figure 1;
(6) According to the linear relation between the obtained differential pulse voltammetric double signal ratio and the cTnI concentration, a working curve is drawn, as shown in fig. 2, a double signal ratio (delta I=delta I thionine/delta I ferrocene) is taken as a standard curve with a ordinate and an antigen concentration logarithmic value as an abscissa, and a linear equation delta I=0.57-0.12 lgC (ng/mL) is taken as a linear correlation coefficient 0.992. The linear range is measured to be 10 fg/mL-100 ng/mL, and the detection limit is 3.34fg/mL.
7. Detection of cardiac troponin I antigen cTnI
Example 13
The sensor prepared using the procedure of example 8 was used to detect cardiac troponin I antigen cTnI in a sample, and a two-signal ratio was obtained as in steps (3) and (4) of example 12, and finally the concentration of cardiac troponin I antigen cTnI was obtained from the calculation of the working curve.
Claims (6)
1. The preparation method of the ratio sensor based on cysteine gold and gold platinum molybdenum disulfide is characterized by comprising the following steps:
(1) Polishing the glassy carbon electrode, and cleaning by using ultrapure water;
(2) Dripping thionine functionalized cysteine gold solution with the concentration of 3-9 mu L and the concentration of 0.5-1.5 mg/mL on the surface of an electrode, and airing in a refrigerator at the temperature of 4 ℃;
(3) Immersing the electrode into a solution of a myocardial troponin capture antibody of 5-15 mug/mL, incubating for 12 h at 4 ℃, and airing in a refrigerator at 4 ℃;
(4) Continuously dripping 2-4 mu L of 0.5-1.5 mg/mL of bovine serum albumin solution on the surface of the electrode, incubating 2-6 h in a refrigerator at 4 ℃, flushing the surface of the electrode with ultrapure water, and airing in the refrigerator at 4 ℃;
(5) Immersing the electrode into a solution of cardiac troponin antigen, incubating at 37 ℃ for 1 h, flushing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;
(6) Immersing the electrode into 1-3 mg/mL ferrocene functionalized gold platinum molybdenum disulfide labeled myocardial troponin antigen detection antibody solution, incubating at 37 ℃ for 1 h, flushing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃ to obtain the cysteine gold and gold platinum molybdenum disulfide based construction ratio sensor.
2. The method for preparing the ratio sensor based on cysteine gold and gold platinum molybdenum disulfide according to claim 1, wherein the thionine functionalized cysteine gold solution is obtained by the following steps: and mixing the cysteine gold and the thionine in the solution to obtain thionine functionalized cysteine gold, and dispersing the thionine functionalized cysteine gold in ultrapure water to obtain the thionine functionalized cysteine gold solution.
3. The method for preparing the ratio sensor based on cysteine gold and gold platinum molybdenum disulfide is characterized in that: the preparation method of the thionine functionalized cysteine gold solution comprises the following specific steps:
(1) Preparation of gold cysteine solution
Adding 5-15 mL% HAuCl with mass fraction of 1% into cysteine solution with concentration of 2 mmol/L at 15-25-mL under stirring 4 Continuously stirring and rapidly adding 1-2 mL NaBH with concentration of 0.01 mmol/L 4 Stirring the solution at room temperature for reaction 2 h, centrifuging at 8000 r/min for 10 min, washing with ultrapure water for 3 times, and drying in a blast drying oven to obtain cysteine gold;
(2) Preparation of thionine functionalized cysteine gold solution
Dispersing 2-6 mg thionine in 2-4 mL of 2 mg/mL of the prepared cysteine gold solution by ultrasonic, adding 2-4 mL,10 mmol/L EDC and 2-4 mL and 2 mmol/L NHS while stirring, stirring for 24-h, centrifugally washing, and drying in a vacuum drying oven at 60 ℃ to prepare thionine functionalized cysteine gold;
(3) And dispersing 0.5-1.5 mg of the prepared thionine functionalized cysteine gold in 1 mL ultrapure water to prepare a thionine functionalized cysteine gold solution.
4. The method for preparing the ratio sensor based on cysteine gold and gold platinum molybdenum disulfide is characterized in that: the ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution is prepared by mixing a gold platinum solution and a molybdenum disulfide solution, separating and drying the mixture to obtain gold platinum molybdenum disulfide, combining the gold platinum molybdenum disulfide with ferrocene, centrifugally washing the combination of the gold platinum molybdenum disulfide and the ferrocene to obtain ferrocene functionalized gold platinum molybdenum disulfide, dispersing the ferrocene functionalized gold platinum molybdenum disulfide in ultrapure water, and mixing the dispersion with the cardiac troponin antigen detection antibody solution to obtain the ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution.
5. The method for manufacturing a ratio sensor based on cysteine gold and gold platinum molybdenum disulfide according to claim 4,
the preparation method is characterized by comprising the following specific preparation steps of a ferrocene functionalized gold platinum molybdenum disulfide labeled myocardial troponin antigen detection antibody solution:
(1) Preparation of gold platinum solution
1) Mixing 1-3 mL of HAuCl with the mass fraction of 1% 4 Adding into 100 mL ultrapure water, rapidly adding 2-4 mL sodium citrate solution with mass fraction of 1% under stirring, and slowly adding NaBH 4 Until the solution turns from light yellow to reddish brown, stirring overnight to obtain solution A;
2) 2 to 4 of mL with the mass fraction of 1 percent of H 2 PtCl 6 Adding into the boiled solution A, continuously adding 1-3 mL and 0.1 mol/L ascorbic acid, and heating for 30 min to obtain a gold-platinum solution;
(2) Preparation of gold platinum molybdenum disulfide solution
Adding 10-30 mL mass percent of polydiene dimethyl ammonium chloride solution into 10-30 mL molybdenum disulfide dispersion liquid under stirring, stirring for 30 min, centrifugally separating, washing 3 times with ultrapure water, adding 5-15 mL gold platinum solution prepared in the step (1), stirring for 1 h, carrying out ultrasonic treatment for 15 min, centrifugally washing for 10 min under 4000-8000 r/min, and drying in a vacuum drying oven at 40 ℃ to obtain gold platinum molybdenum disulfide;
dispersing 6 mg gold platinum molybdenum disulfide in 1 mL ultrapure water to prepare 6 mg/mL gold platinum molybdenum disulfide solution for later use;
(3) Preparation of ferrocene functionalized gold platinum molybdenum disulfide labeled myocardial troponin antigen detection antibody solution
Mixing 1-3 mL of gold platinum molybdenum disulfide solution prepared in the step (2) with 1-3 mL of 2.5% glutaraldehyde solution, adding 10-30 mg ferrocene, stirring 6 h, centrifugally washing, dispersing in 1-3 mL of 10 mug/mL of myocardial troponin antigen detection antibody solution, vibrating overnight at 4 ℃, centrifugally washing to prepare ferrocene-functionalized gold platinum molybdenum disulfide labeled myocardial troponin antigen detection antibody, dispersing in PBS buffer solution with pH of 7.4 to prepare 1-3 mg/mL of ferrocene-functionalized gold platinum molybdenum disulfide labeled myocardial troponin antigen detection antibody solution, and preserving at 4 ℃ for later use.
6. The method for preparing a ratio sensor based on cysteine gold and gold platinum molybdenum disulfide according to any one of claims 1 to 5, wherein the cardiac troponin antigen is at least one selected from cardiac troponin T antigen cTnT and cardiac troponin I antigen cTnI.
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