CN114965633A - Preparation method and application of ratio sensor constructed based on gold cysteine and gold platinum molybdenum disulfide - Google Patents
Preparation method and application of ratio sensor constructed based on gold cysteine 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 gold cysteine and gold platinum molybdenum disulfide, and belongs to the technical field of novel functional nano materials, immunoassay and biosensing. Unlike traditional electrochemical sensors that use the absolute value of a single electrical signal as output, ratiometric sensors are based on the ratio of two signals as output to achieve accurate detection. The read-out pattern of the ratio signal provides a built-in correction factor that helps to reject non-specific interference, 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 field of novel functional nano materials, immunoassay and biosensing, and relates to a preparation method and application of a ratio sensor constructed on the basis of gold cysteine and gold platinum molybdenum disulfide. Specifically, the immunosensor is constructed by adopting a novel functional nano material and is used for detecting the cardiac troponin antigen.
Background
Cardiovascular and cerebrovascular diseases (CVDs) are common diseases seriously threatening human health, have the characteristics of high morbidity, high disability rate and high mortality rate, and account for 31 percent of the global mortality rate. The mortality rate of Acute Myocardial Infarction (AMI) is the first of cardiovascular and cerebrovascular diseases. The cardiac troponin T antigen cTnT and the cardiac troponin I antigen cTnI are key markers for diagnosing the occurrence of AMI. When the myocardial cells are damaged, the concentrations of cTnT and cTnI can be quickly increased to the detection level and can be maintained for 5-7 days, so that sufficient time is provided for the retest of CVDs, and the diagnosis accuracy is further improved. By detecting the content of cTnT and cTnI in vivo, the occurrence of AMI can be effectively predicted, preventive measures can be taken as soon as possible, and the death rate is reduced. Therefore, the development of a method with high sensitivity and high accuracy for detecting cTnT and cTnI has important clinical significance.
Disclosure of Invention
The invention provides a preparation method and application of a ratio sensor constructed on the basis of gold cysteine and gold platinum molybdenum disulfide, and realizes sensitive detection of a cardiac troponin antigen.
One of the purposes of the invention is to provide a preparation method for constructing a ratio sensor based on cysteine gold and gold platinum molybdenum disulfide.
The second purpose of the invention is to construct a ratio sensor by using the prepared cysteine gold and the gold-platinum molybdenum disulfide for detecting the cardiac troponin antigen.
In order to realize one of the purposes, the invention discloses a preparation method for constructing a ratio sensor based on cysteine gold and gold platinum molybdenum disulfide, which is characterized by comprising the following steps:
(1) a glassy carbon electrode with a diameter of 4 mm was used in the order of 1.0 μm, 0.3 μm, 0.05 μm Al 2 O 3 Polishing the polishing powder, and cleaning with ultrapure water;
(2) dropwise adding 3-9 muL and 0.5-1.5 mg/mL thionine functionalized cysteine gold solution to the surface of the electrode, and airing in a refrigerator at 4 ℃;
(3) immersing the electrode into a 5-15 mug/mL solution of a cardiac troponin capture antibody, incubating for 12 h at 4 ℃, and airing in a refrigerator at 4 ℃;
(4) continuously dropwise adding 2-4 muL of bovine serum albumin solution of 0.5-1.5 mg/mL to the surface of the electrode, incubating in a refrigerator at 4 ℃ for 2-6 h, washing 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, wherein the solutions are 10 fg/mL-100 ng/mL, incubating for 1 h at 37 ℃, washing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;
(6) immersing the electrode into a 1-3 mg/mL cardiac troponin antigen detection antibody solution marked by ferrocene functionalized gold platinum molybdenum disulfide, incubating at 37 ℃ for 1 h, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃ to obtain the ratio sensor constructed on the basis of gold cysteine and gold platinum molybdenum disulfide.
Further, the thionine functionalized cysteine gold solution is obtained by mixing cysteine gold and thionine in a 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 specific preparation steps of the thionine functionalized gold cysteine solution are as follows:
(1) preparation of gold cysteine solution
Adding 5-15 mL of 1% HAuCl into 15-25 mL of cysteine solution with the concentration of 2 mmol/L under stirring 4 Continuously stirring and rapidly adding 1-2 mL of NaBH with the concentration of 0.01 mmol/L 4 Stirring the solution at room temperature for reaction for 2 h, centrifuging at 8000 r/min for 10 min, washing with ultrapure water for 3 times, and drying in an air-blast drying oven to obtain cysteine gold;
(2) preparation of thionine functionalized gold cysteine solution
Ultrasonically dispersing 2-6 mg of thionine into 2-4 mL of the cysteine gold solution prepared at the concentration of 2 mg/mL, continuously stirring, adding 2-4 mL of EDC at the concentration of 10 mmol/L, 2-4 mL of NHS at the concentration of 2 mmol/L, stirring for 24 hours, centrifugally washing, and drying in a vacuum drying oven at the temperature of 60 ℃ to prepare thionine functionalized cysteine gold;
and (3) dispersing 0.5-1.5 mg of the prepared thionine functionalized gold cysteine into 1 mL of ultrapure water to prepare a thionine functionalized gold cysteine solution.
Further, the ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution is obtained by mixing a gold platinum solution and a molybdenum disulfide solution, separating and drying to obtain gold platinum molybdenum disulfide, combining the gold platinum molybdenum disulfide and ferrocene, centrifuging and washing to obtain ferrocene functionalized gold platinum molybdenum disulfide, dispersing the ferrocene functionalized gold platinum molybdenum disulfide in ultrapure water, and mixing the dispersed 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 concrete 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) 1-3 mL of HAuCl with the mass fraction of 1% 4 Adding 100 mL of ultrapure water, rapidly adding 2-4 mL of 1% sodium citrate solution with stirring, and slowly adding NaBH 4 Stirring overnight until the solution turns reddish brown from light yellow to obtain solution A;
2) 2-4 mL of 1% H 2 PtCl 6 Adding the solution into the boiled solution A, continuously adding 1-3 mL of 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 of polydiene dimethyl ammonium chloride solution with the mass fraction of 20% into 10-30 mL of molybdenum disulfide dispersion liquid under stirring, stirring for 30 min, carrying out centrifugal separation, washing with ultrapure water for 3 times, adding 5-15 mL of gold platinum solution prepared in the step (1), stirring for 1 h, carrying out ultrasonic treatment for 15 min, carrying out centrifugal washing on the obtained solution 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 of gold platinum molybdenum disulfide in 1 mL of ultrapure water to prepare a 6 mg/mL gold platinum molybdenum disulfide solution for later use;
(3) preparation of ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution
Mixing 1-3 mL of the 6 mg/mL gold platinum molybdenum disulfide solution prepared in the step (2) with 1-3 mL of 2.5% glutaraldehyde solution, adding 10-30 mg of ferrocene, stirring for 6 h, centrifugally washing, dispersing in 1-3 mL of 10 mu g/mL cardiac troponin antigen detection antibody solution, shaking overnight at 4 ℃, centrifugally washing to prepare the ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody, dispersing the ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody in a PBS (phosphate buffered saline) buffer solution with the pH of 7.4 to prepare 1-3 mg/mL of ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution, and storing at 4 ℃ for later use.
Further, the cardiac troponin antigen is selected from one of cardiac troponin T antigen cTnT and cardiac troponin I antigen cTnI.
In order to achieve the second purpose, the invention discloses the application of constructing a ratio sensor based on gold cysteine and gold platinum molybdenum disulfide, and the ratio sensor is used for detecting a cardiac troponin antigen.
The method comprises the following specific steps:
(1) an electrochemical workstation is used for testing in a three-electrode system, a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, the prepared sensor is used as a working electrode, and the test is carried out in 10 mL of 0.1 mol/L phosphate buffer solution with the pH value of 7.4;
(2) detecting an analyte by using a differential pulse voltammetry, wherein the scanning potential voltage is between-0.6V and 0.6V, sharp double signal peaks are obtained at-0.3V and 0.2V respectively, the current intensity of the differential pulse voltammetry double signals corresponding to the cardiac troponin antigen solutions with different concentrations is recorded, and the ratio of the double signal current is calculated to serve as an output signal;
(3) and obtaining the concentration of the myocardial 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 constructed based on gold cysteine 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 a double-signal ratio value as output. The read-out mode of the ratio signal provides a built-in correction factor, which is beneficial to discharging nonspecific interference, has obvious effect on improving the reproducibility of the sensor, and also 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 the thionine is used as a signal probe and can generate a sharp signal peak. The ferrocene functionalized gold platinum molybdenum disulfide is used as a labeling material for immobilizing a cardiac troponin antigen detection antibody, has good biocompatibility, large specific surface area and good dispersibility, a large number of binding sites can be firmly combined with the high-density cardiac troponin antigen detection antibody, a good foundation is provided for subsequent specific binding with the cardiac troponin antigen, and in addition, the higher conductivity of the ferrocene functionalized gold platinum molybdenum disulfide is also beneficial to obtaining a stronger differential pulse voltammetric signal;
(3) the thionine functionalized gold cysteine and the ferrocene functionalized gold platinum molybdenum disulfide respectively have 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 ferrocene oxidation signal peak and a decrease in the thionine oxidation signal peak. Based on the ratio of the currents of the two signals (ΔI=ΔI Viola root /ΔI Ferrocene ) The linear relation between the concentration of the antigen and the concentration of the cardiac troponin realizes the high-accuracy detection of the cardiac troponin antigen;
(4) a ratio sensor constructed based on gold cysteine and molybdenum disulfide gold platinum is used for detecting a cardiac troponin antigen, wherein the linear range of the detection on the cardiac troponin T antigen cTnT is 10 fg/mL-100 ng/mL, the detection limit is at least 3.33 fg/mL, the linear range of the detection on the cardiac troponin I antigen cTnI is 10 fg/mL-100 ng/mL, and the detection limit is at least 3.34 fg/mL, so that the aim of accurately determining the cardiac troponin antigen can be achieved by constructing the ratio sensor based on the gold cysteine and the molybdenum disulfide gold platinum.
Drawings
FIG. 1 is a graph of differential pulse voltammetric dual signals obtained by testing a series of concentration gradient cardiac troponin antigen solutions.
Figure 2 standard curve with double signal ratio (Δ I = Δ I thionine/Δ I ferrocene) plotted on the ordinate versus the antigen concentration logarithm plotted on the abscissa.
Detailed Description
The present invention will now be further illustrated by, but not limited to, specific embodiments thereof.
The raw materials used in the present invention are all available from chemical agents companies or biopharmaceutical companies.
Preparation of thionine functionalized cysteine gold solution
Example 1
The preparation method of the thionine functionalized gold cysteine solution comprises the following steps:
(1) preparation of gold cysteine solution
5 mL of 1% HAuCl was added to 15 mL of 2 mmol/L cysteine solution with stirring 4 While stirring was continued, 1 mL of NaBH at 0.01 mmol/L was added rapidly 4 Stirring the solution at room temperature for reaction for 2 h, centrifuging at 8000 r/min for 10 min, washing with ultrapure water for 3 times, and drying in an air-blast drying oven to obtain cysteine gold;
(2) preparation of thionine functionalized gold cysteine solution
Ultrasonically dispersing 2 mg of thionine into 2 mL of the cysteine gold solution prepared at the concentration of 2 mg/mL, continuously stirring, adding 2 mL of EDC at the concentration of 10 mmol/L, 2 mL of NHS at the concentration of 2 mmol/L, stirring for 24 hours, centrifugally washing, and drying in a vacuum drying oven at the temperature of 60 ℃ to prepare thionine functionalized cysteine gold;
0.5 mg of the prepared thionine functionalized gold cysteine is dispersed in 1 mL of ultrapure water to prepare a thionine functionalized gold cysteine solution.
Example 2
The preparation method of the thionine functionalized cysteine gold solution comprises the following steps:
(1) preparation of gold cysteine solution
Adding 10 mL of HAuCl with the mass fraction of 1% into 20 mL of cysteine solution with the concentration of 2 mmol/L under stirring 4 Stirring was continued and 1.5 mL of NaBH at a concentration of 0.01 mmol/L was added rapidly 4 Stirring the solution at room temperature for reaction for 2 h, centrifuging at 8000 r/min for 10 min, washing with ultrapure water for 3 times, and drying in an air-blast drying oven to obtain cysteine gold;
(2) preparation of thionine functionalized cysteine gold solution
Ultrasonically dispersing 4 mg of thionine into 3 mL and 2 mg/mL of the prepared cysteine gold solution, continuously stirring, adding 3 mL, 10 mmol/L of EDC, 3 mL and 2 mmol/L of NHS, stirring for 24 hours, centrifugally washing, and drying in a vacuum drying oven at 60 ℃ to prepare thionine functionalized cysteine gold;
1 mg of the prepared thionine functionalized cysteine gold is dispersed in 1 mL of ultrapure water to prepare a thionine functionalized cysteine gold solution.
Example 3
The preparation method of the thionine functionalized cysteine gold solution comprises the following steps:
(1) preparation of gold cysteine solution
Adding 15 mL of HAuCl with the mass fraction of 1% into 25 mL of cysteine solution with the concentration of 2 mmol/L under stirring 4 Stirring was continued and 2 mL of NaBH at a concentration of 0.01 mmol/L was added rapidly 4 Stirring the solution at room temperature for reaction for 2 h, centrifuging at 8000 r/min for 10 min, washing with ultrapure water for 3 times, and drying in a forced air drying oven to obtain cysteine gold;
(2) preparation of thionine functionalized cysteine gold solution
Ultrasonically dispersing 6 mg of thionine into 4 mL and 2 mg/mL of the prepared cysteine gold solution, continuously stirring, adding 4 mL, 10 mmol/L of EDC, 4 mL and 2 mmol/L of NHS, stirring for 24 hours, centrifugally washing, and drying in a vacuum drying oven at 60 ℃ to prepare thionine functionalized cysteine gold;
1.5mg of the prepared thionine functionalized cysteine gold is dispersed in 1 mL of ultrapure water to prepare a thionine functionalized cysteine gold solution.
Preparation of ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution
Example 4
The preparation method 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) 1 mL of HAuCl with the mass fraction of 1 percent 4 Adding 100 mL of ultrapure water, rapidly adding 2 mL of 1% sodium citrate solution with stirring, and slowly adding NaBH 4 Stirring overnight until the solution turns reddish brown from light yellow to obtain solution A;
2) 2 mL of H with the mass fraction of 1% 2 PtCl 6 Adding into the boiled solution A, adding 1 mL of ascorbic acid 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 mL of polydiene dimethyl ammonium chloride solution with the mass fraction of 20% into 10 mL of molybdenum disulfide dispersion liquid under stirring, stirring for 30 min, carrying out centrifugal separation, washing for 3 times by using ultrapure water, adding 5 mL of gold and platinum solution prepared in the step (1), stirring for 1 h, carrying out ultrasonic treatment for 15 min, carrying out centrifugal washing for 10 min under 4000 r/min on the obtained solution, and drying in a vacuum drying oven at 40 ℃ to obtain gold and platinum molybdenum disulfide;
dispersing 6 mg of gold platinum molybdenum disulfide in 1 mL of ultrapure water to prepare a 6 mg/mL gold platinum molybdenum disulfide solution for later use;
(3) preparation of ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution
Mixing 1 mL of the gold platinum molybdenum disulfide solution prepared in the step (2) with 1 mL of 2.5% glutaraldehyde solution, adding 10 mg of ferrocene, stirring for 6 h, centrifugally washing, dispersing in 1 mL of 10 mu g/mL cardiac troponin antigen detection antibody solution, shaking overnight at 4 ℃, centrifugally washing to prepare the ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody, dispersing in a PBS buffer solution with the pH of 7.4 to prepare 1 mg/mL of ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution, and storing at 4 ℃ for later use.
Example 5
The preparation method 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 of HAuCl with the mass fraction of 1 percent 4 Adding 100 mL of ultrapure water, rapidly adding 3 mL of sodium citrate solution with the mass fraction of 1% while stirring, and slowly adding NaBH 4 Stirring overnight until the solution turns reddish brown from light yellow to obtain solution A;
2) 3 mL of 1 percent of H 2 PtCl 6 Adding into the boiled solution A, adding 2 mL of ascorbic acid 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 20 mL of polydiene dimethyl ammonium chloride solution with the mass fraction of 20% into 20 mL of molybdenum disulfide dispersion liquid under stirring, stirring for 30 min, carrying out centrifugal separation, washing for 3 times by using ultrapure water, adding 10 mL of gold and platinum solution prepared in the step (1), stirring for 1 h, carrying out ultrasonic treatment for 15 min, carrying out centrifugal washing for 10 min at 6000 r/min on the obtained solution, and drying in a vacuum drying oven at 40 ℃ to obtain gold and platinum molybdenum disulfide;
dispersing 6 mg of gold platinum molybdenum disulfide in 1 mL of ultrapure water to prepare a 6 mg/mL gold platinum molybdenum disulfide solution for later use;
(3) preparation of ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution
Mixing 2 mL of the gold platinum molybdenum disulfide solution prepared in the step (2) with 2 mL of 2.5% glutaraldehyde solution, adding 20 mg of ferrocene, stirring for 6 h, centrifugally washing, dispersing in 2 mL of 10 mu g/mL cardiac troponin antigen detection antibody solution, shaking overnight at 4 ℃, centrifugally washing to prepare the ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody, dispersing in a PBS buffer solution with the pH of 7.4 to prepare the 2 mg/mL ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution, and storing at 4 ℃ for later use.
Example 6
The preparation method 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 of HAuCl with the mass fraction of 1 percent 4 Adding 100 mL of ultrapure water, rapidly adding 4 mL of sodium citrate solution with the mass fraction of 1% while stirring, and slowly adding NaBH 4 Stirring overnight until the solution turns reddish brown from light yellow to obtain solution A;
2) 4 mL of 1 percent of H 2 PtCl 6 Adding into the boiled solution A, adding 3 mL of ascorbic acid of 0.1 mol/L, and heating for 30 min to obtain a gold platinum solution;
(2) preparation of gold platinum molybdenum disulfide
Adding 30 mL of polydiene dimethyl ammonium chloride solution with the mass fraction of 20% into 30 mL of molybdenum disulfide dispersion liquid under stirring, stirring for 30 min, carrying out centrifugal separation, washing for 3 times by using ultrapure water, adding 15 mL of gold platinum solution prepared in the step (1), stirring for 1 h, carrying out ultrasonic treatment for 15 min, carrying out centrifugal washing for 10 min at 8000 r/min on the obtained solution, and drying in a vacuum drying oven at 40 ℃ to obtain gold platinum molybdenum disulfide;
dispersing 6 mg of platinum disulfide molybdenum in 1 mL of ultrapure water to prepare a 6 mg/mL gold platinum disulfide molybdenum solution for later use;
(3) preparation of ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution
Mixing 3 mL of the gold platinum molybdenum disulfide solution prepared in the step (2) with 3 mL of a glutaraldehyde solution with the concentration of 2.5%, adding 30 mg of ferrocene, stirring for 6 h, centrifugally washing, dispersing in a cardiac troponin antigen detection antibody solution with the concentration of 3 mL and the concentration of 10 mu g/mL, shaking overnight at 4 ℃, centrifugally washing to prepare a ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody, dispersing the ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody in a PBS (phosphate buffered saline) buffer solution with the pH value of 7.4 to prepare a 3 mg/mL of ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution, and storing at 4 ℃ for later use.
Preparation of ratio sensor constructed based on cysteine gold and gold platinum molybdenum disulfide
Example 7
A preparation method for constructing a ratio sensor based on cysteine gold and gold-platinum molybdenum disulfide comprises the following steps:
(1) a glassy carbon electrode with a diameter of 4 mm was used in the order of 1.0 μm, 0.3 μm, 0.05 μm Al 2 O 3 Polishing the polishing powder, and cleaning with ultrapure water;
(2) dropwise adding 0.5 mg/mL thionine functionalized cysteine gold solution which is 3 mu L and prepared in the embodiment 1 to the surface of the electrode, and airing in a refrigerator at 4 ℃;
(3) immersing the electrode into a 5 microgram/mL solution of the cardiac troponin capture antibody, incubating at 4 ℃ for 12 h, and airing in a refrigerator at 4 ℃;
(4) continuously dropwise adding 2 muL of bovine serum albumin solution of 0.5 mg/mL to the surface of the electrode, incubating for 2 h in a refrigerator at 4 ℃, washing the surface of the electrode with ultrapure water, and airing in the refrigerator at 4 ℃;
(5) continuously immersing the electrode into a solution of a cardiac troponin antigen, incubating for 1 h at 37 ℃, washing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;
(6) immersing the electrode into a 1 mg/mL cardiac troponin antigen detection antibody solution marked by ferrocene functionalized gold platinum molybdenum disulfide prepared in embodiment 4, incubating at 37 ℃ for 1 h, rinsing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃ to prepare the ratio sensor constructed on the basis of gold cysteine and gold platinum molybdenum disulfide.
Example 8
A preparation method for constructing a ratio sensor based on cysteine gold and gold-platinum molybdenum disulfide comprises the following steps:
(1) a glassy carbon electrode with a diameter of 4 mm was used in the order of 1.0 μm, 0.3 μm, 0.05 μm Al 2 O 3 Polishing the polishing powder, and cleaning with ultrapure water;
(2) dropwise adding 6 muL of thionine functionalized cysteine gold solution of 1 mg/mL prepared in the embodiment 2 to the surface of the electrode, and airing in a refrigerator at 4 ℃;
(3) immersing the electrode into a solution of a cardiac troponin capture antibody of 10 mug/mL, incubating at 4 ℃ for 12 h, and airing in a refrigerator at 4 ℃;
(4) continuously dropwise adding a bovine serum albumin solution of 3 muL and 1 mg/mL to the surface of the electrode, incubating for 4 hours in a refrigerator at 4 ℃, washing the surface of the electrode with ultrapure water, and airing in the refrigerator at 4 ℃;
(5) continuously immersing the electrode into a solution of a cardiac troponin antigen, incubating for 1 h at 37 ℃, washing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;
(6) immersing the electrode into a cardiac troponin antigen detection antibody solution which is prepared in the embodiment 5 and marked by 2 mg/mL of ferrocene functionalized gold platinum molybdenum disulfide, incubating at 37 ℃ for 1 h, rinsing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃ to prepare the ratio sensor constructed on the basis of gold cysteine and gold platinum molybdenum disulfide.
Example 9
A preparation method for constructing a ratio sensor based on cysteine gold and gold-platinum molybdenum disulfide comprises the following steps:
(1) a glassy carbon electrode with a diameter of 4 mm was used with 1.0 μm, 0.3 μm, and 0.05 μm Al in this order 2 O 3 Polishing the polishing powder, and cleaning with ultrapure water;
(2) dropwise adding 9 muL of thionine functionalized cysteine gold solution of 1.5 mg/mL prepared in the embodiment 3 to the surface of the electrode, and airing in a refrigerator at 4 ℃;
(3) immersing the electrode into a 15 mug/mL solution of a cardiac troponin capture antibody, incubating at 4 ℃ for 12 h, and airing at 4 ℃ in a refrigerator;
(4) continuously dropwise adding a bovine serum albumin solution of 4 muL and 1.5 mg/mL to the surface of the electrode, incubating for 6 h in a refrigerator at 4 ℃, washing the surface of the electrode with ultrapure water, and airing in the refrigerator at 4 ℃;
(5) continuously incubating the electrode cardiac troponin antigen solution at 37 ℃ for 1 h, washing the electrode surface with ultrapure water, and drying in a refrigerator at 4 ℃;
(6) immersing the electrode into 3 mg/mL of ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution prepared in embodiment 6, incubating at 37 ℃ for 1 h, rinsing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃ to prepare the ratio sensor constructed on the basis of gold cysteine and gold platinum molybdenum disulfide.
Fourthly, drawing a detection working curve of cardiac troponin T antigen cTnT
Example 10
(1) Preparing a standard solution, namely preparing a series of solutions of cardiac troponin antigens with different concentrations, wherein the solutions are 10 fg/mL-100 ng/mL;
(2) a ratio sensor based on gold cysteine and gold platinum molybdenum disulfide containing different concentrations of the cardiac troponin antigen cTnT was obtained using standard solutions following the procedure of example 7;
(3) testing by using an electrochemical workstation in a three-electrode system, taking a saturated calomel electrode as a reference electrode and a platinum wire electrode as an auxiliary electrode, taking the ratio sensor which contains different cardiac troponin antigen concentrations and is constructed based on cysteine gold and gold platinum molybdenum disulfide as a working electrode, and testing in 10 mL of 0.1 mol/L pH 7.4 phosphate buffer solution;
(4) detecting an analyte by using a differential pulse voltammetry, wherein the scanning potential voltage is between-0.6V and 0.6V, sharp double signal peaks are obtained at-0.3V and 0.2V respectively, the current intensity of the differential pulse voltammetry double signals corresponding to the cardiac troponin antigen solutions with different concentrations is recorded, and the ratio of the double signal current is calculated to serve as an output signal;
(5) acquiring corresponding relations between different concentrations of cardiac troponin antigens and output signals;
(6) and drawing a working curve according to the linear relation between the ratio of the obtained differential pulse volt-ampere double signals and the concentration of cTnT, wherein the measured linear range is 10 fg/mL-100 ng/mL, and the detection limit is 3.33 fg/mL.
Detection of cardiac troponin T antigen cTnT
Example 11
The sensor prepared by the procedure of example 8 was used to detect the cardiac troponin I antigen cTnI in the sample, and the dual signal ratio was obtained according to the procedures (3) and (4) as in example 10, and finally the concentration of the cardiac troponin I antigen cTnI was calculated from the working curve.
Sixthly, drawing a detection working curve of cardiac troponin T antigen cTnI
Example 12
(1) Preparing a series of solutions of the cardiac troponin antigen with different concentrations, namely (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, and (i) 100 pg/mL, of 10 fg/mL;
(2) a ratio sensor based on gold cysteine and gold platinum molybdenum disulfide containing different concentrations of cardiac troponin antigen cTnI was obtained using standard solutions following the procedure of example 7;
(3) testing in a three-electrode system by using an electrochemical workstation, taking a saturated calomel electrode as a reference electrode and a platinum wire electrode as an auxiliary electrode, taking the ratio sensor which contains different cardiac troponin antigen concentrations and is constructed on the basis of cysteine gold and gold platinum molybdenum disulfide as a working electrode, and testing in 10 mL of 0.1 mol/L pH 7.4 phosphate buffer solution;
(4) the analyte was detected by differential pulse voltammetry at a scanning potential voltage of-0.6V to 0.6V, and sharp dual signal peaks were obtained at-0.3V and 0.2V, respectively, where the signal peak at-0.3V was generated by Thi redox and the signal peak at 0.2V was Fc redox. Recording the current intensity of the differential pulse voltammetry dual signals corresponding to the cardiac troponin antigen solutions with different concentrations and calculating the current ratio of the dual signals as output signals;
(5) obtaining the corresponding relation between different cardiac troponin antigen concentrations and output signals, as shown in figure 1;
(6) according to the linear relation between the obtained differential pulse voltammetry dual signal ratio and the cTnI concentration, a working curve is drawn, as shown in FIG. 2, a standard curve with the dual signal ratio (Δ I = Δ I thionine/Δ I ferrocene) as the ordinate and the antigen concentration logarithm as the abscissa, the linear equation Δ I = 0.57-0.12 lgC (ng/mL), and the linear correlation coefficient is 0.992. The linear range is 10 fg/mL-100 ng/mL, and the detection limit is 3.34 fg/mL.
Detection of seven, cardiac troponin I antigen cTnI
Example 13
The sensor prepared by the procedure of example 8 was used to detect the cardiac troponin I antigen cTnI in the sample, and the dual signal ratio was obtained according to the procedures (3) and (4) as in example 12, and finally the concentration of the cardiac troponin I antigen cTnI was calculated from the working curve.
Claims (8)
1. A preparation method for constructing a 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 with ultrapure water;
(2) dropwise adding 3-9 muL and 0.5-1.5 mg/mL thionine functionalized cysteine gold solution to the surface of the electrode, and airing in a refrigerator at 4 ℃;
(3) immersing the electrode into a 5-15 mug/mL solution of a cardiac troponin capture antibody, incubating for 12 h at 4 ℃, and airing in a refrigerator at 4 ℃;
(4) continuously dropwise adding 2-4 muL of bovine serum albumin solution of 0.5-1.5 mg/mL to the surface of the electrode, incubating in a refrigerator at 4 ℃ for 2-6 h, washing the surface of the electrode with ultrapure water, and airing in the refrigerator at 4 ℃;
(5) continuously immersing the electrode into a solution of a cardiac troponin antigen, incubating for 1 h at 37 ℃, washing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;
(6) immersing the electrode into a 1-3 mg/mL solution of a ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody, incubating at 37 ℃ for 1 h, washing the surface of the electrode with ultrapure water, and placing in a refrigerator at 4 ℃ for airing to obtain the ratio sensor constructed on the basis of gold cysteine and gold platinum molybdenum disulfide.
2. The method of claim 1, wherein the thionine functionalized gold cysteine solution is obtained by: mixing the gold cysteine and the thionine in the solution to obtain the thionine functionalized gold cysteine, and dispersing the thionine functionalized gold cysteine in ultrapure water to prepare the thionine functionalized gold cysteine solution.
3. The method of claim 2, wherein the ratio sensor is constructed from gold cysteine and molybdenum disulfide platinum gold, and wherein the ratio sensor comprises: the specific preparation steps of the thionine functionalized cysteine gold solution are as follows:
(1) preparation of gold cysteine solution
Adding 5-15 mL of 1 mass percent HAuCl into 15-25 mL of cysteine solution with the concentration of 2 mmol/L under stirring 4 Continuously stirring and rapidly adding 1-2 mL of NaBH with the concentration of 0.01 mmol/L 4 Stirring the solution at room temperature for reaction for 2 h, centrifuging at 8000 r/min for 10 min, washing with ultrapure water for 3 times, and drying in an air-blast drying oven to obtain cysteine gold;
(2) preparation of thionine functionalized gold cysteine solution
Ultrasonically dispersing 2-6 mg of thionine into 2-4 mL of the cysteine gold solution prepared at the concentration of 2 mg/mL, continuously stirring, adding 2-4 mL of EDC at the concentration of 10 mmol/L, 2-4 mL of NHS at the concentration of 2 mmol/L, stirring for 24 hours, centrifugally washing, and drying in a vacuum drying oven at the temperature of 60 ℃ to prepare thionine functionalized cysteine gold;
(3) and (3) dispersing 0.5-1.5 mg of the prepared thionine functionalized cysteine gold in 1 mL of ultrapure water to prepare a thionine functionalized cysteine gold solution.
4. The method of claim 1, wherein the ratio sensor is constructed from gold cysteine and molybdenum disulfide platinum gold, and wherein the ratio sensor comprises: 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 to obtain gold platinum molybdenum disulfide, combining the gold platinum molybdenum disulfide and ferrocene, centrifuging and washing to obtain ferrocene functionalized gold platinum molybdenum disulfide, dispersing the ferrocene functionalized gold platinum molybdenum disulfide in ultrapure water, and mixing 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 according to claim 4, wherein the ratio sensor is constructed based on gold cysteine and molybdenum disulfide platinum,
the method is characterized in that the concrete 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) 1-3 mL of HAuCl with the mass fraction of 1% 4 Adding 100 mL of ultrapure water, rapidly adding 2-4 mL of 1% sodium citrate solution with stirring, and slowly adding NaBH 4 Stirring overnight until the solution turns from light yellow to reddish brown to obtain solution A;
2) 2-4 mL of 1% H 2 PtCl 6 Adding the solution A into the boiled solution A, continuously adding 1-3 mL of 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 of polydiene dimethyl ammonium chloride solution with the mass fraction of 20% into 10-30 mL of molybdenum disulfide dispersion liquid under stirring, stirring for 30 min, carrying out centrifugal separation, washing with ultrapure water for 3 times, adding 5-15 mL of gold platinum solution prepared in the step (1), stirring for 1 h, carrying out ultrasonic treatment for 15 min, carrying out centrifugal washing on the obtained solution 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 of gold platinum molybdenum disulfide in 1 mL of ultrapure water to prepare a 6 mg/mL gold platinum molybdenum disulfide solution for later use;
(3) preparation of ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution
Mixing 1-3 mL of the gold platinum molybdenum disulfide solution prepared in the step (2) with 1-3 mL of 2.5% glutaraldehyde solution, adding 10-30 mg of ferrocene, stirring for 6 h, centrifugally washing, dispersing in 1-3 mL of 10 microgram/mL cardiac troponin antigen detection antibody solution, shaking overnight at 4 ℃, centrifugally washing to obtain a ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody, dispersing in a PBS buffer solution with the pH of 7.4 to obtain the ferrocene functionalized gold platinum molybdenum disulfide labeled cardiac troponin antigen detection antibody solution with 1-3 mg/mL of ferrocene, and storing at 4 ℃ for later use.
6. The method for preparing a ratiometric sensor of any one of claims 1 to 5, wherein the cardiac troponin antigen is at least one selected from the group consisting of cardiac troponin T antigen cTnT and cardiac troponin I antigen cTnI.
7. Use of gold cysteine and molybdenum disulfide gold platinum gold disulfide for the construction of a ratiometric sensor based on the preparation process as claimed in any one of claims 1 to 6, for the detection of cardiac troponin antigens.
8. The use of gold cysteine and molybdenum disulfide gold platinum gold cysteine to construct a ratiometric sensor according to claim 7, comprising the steps of:
(1) testing in a three-electrode system by using an electrochemical workstation, taking a saturated calomel electrode as a reference electrode, a platinum wire electrode as an auxiliary electrode, and the prepared sensor as a working electrode in 10 mL of 0.1 mol/L phosphate buffer solution with the pH value of 7.4;
(2) detecting an analyte by using a differential pulse voltammetry, wherein the scanning potential voltage is between-0.6V and 0.6V, sharp double signal peaks are obtained at-0.3V and 0.2V respectively, the current intensity of the differential pulse voltammetry double signals corresponding to the cardiac troponin antigen solutions with different concentrations is recorded, and the ratio of the double signal current is calculated to serve as an output signal;
(3) and obtaining the concentration of the myocardial troponin antigen in the sample to be detected by using a working curve method.
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