CN115932005A - Electrochemical biosensor working electrode for detecting protein and preparation method thereof - Google Patents
Electrochemical biosensor working electrode for detecting protein and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biosensors, and particularly relates to a working electrode of an electrochemical biosensor for detecting protein and a preparation method thereof. The working electrode provided by the invention has the advantages of higher sensitivity, better stability, more uniform antibody reaction and better washing resistance.
Description
Technical Field
The invention belongs to the technical field of biosensors, and particularly relates to a working electrode of an electrochemical biosensor for detecting protein and a preparation method thereof.
Background
Since proteins play a very important role in many living bodies, it is very important to study proteins, interactions between proteins and other substances, and how to detect proteins.
The detection of proteins under investigation has been carried out by immunological methods, and by immunological methods using Apo (a) monoclonal antibodies. Enzyme-linked immunosorbent assay is a reliable assay method, which comprises coating a microporous plate with purified antibody to obtain solid-phase antibody, and sequentially adding antigen, antibody and HPR-labeled avidin into the micropores coated with monoclonal antibody. After thorough washing, color development was carried out with the substrate TMB. TMB is converted into blue color under the catalysis of peroxidase and is converted into final yellow color under the action of acid, and the shade of the color is in positive correlation with protein in a sample. The absorbance (OD value) was measured with a microplate reader, and the sample concentration was calculated.
The ELISA has the disadvantages of complicated operation steps, more reaction influencing factors and low sensitivity.
The electrochemical immunosensor is a biosensor developed based on antigen-antibody specific recognition function. These tend to take the form of complex molecular layers attached to the sensor, where changes in the target binding or molecules making up the sensing layer cause physical changes that can be measured by an electrochemical system. This ensures biological recognition or signal specificity and enables the physical sensor to detect biological targets that are not normally available. Therefore, it has been widely spread in recent years.
Electrochemical immunosensors have many advantages, such as high analytical sensitivity, strong specificity, and ease of use. However, the electrochemical immunosensor on the market at present has insufficient stability and is expected to be further improved.
Disclosure of Invention
Compared with the existing detection method, the working electrode of the electrochemical biosensor for detecting the protein and the preparation method thereof provided by the invention have the advantages of high sensitivity, strong specificity, convenience in operation and good stability.
In a first aspect of the present invention, a working electrode of an electrochemical biosensor for detecting a protein is provided, the working electrode is loaded with a coating layer formed by an antibody dispersion, the antibody dispersion comprises a monoclonal antibody corresponding to the protein to be detected and a dispersion, and the dispersion comprises an acetic acid solution, chitosan, nitrogen-doped graphene and water.
In the technical scheme, the single antibodies are dispersed in the dispersion liquid and coated on the surface of the electrode. The chitosan in the dispersion liquid has better viscosity and smaller steric hindrance, so that the chitosan has good biocompatibility and film-forming property, and the nitrogen-doped graphene has excellent conductivity. The combined antibody dispersion has better stability of current response.
In another embodiment of the present invention, the monoclonal antibody is labeled with a magnetic bead.
In the technical scheme, compared with the method of directly modifying the electrode by using the monoclonal antibody marked by the magnetic beads, the method has the advantages that the reaction uniformity on the surface of the electrode is better and the current is more stable. Compared with antibodies labeled by the same type of markers, such as antibodies labeled by colloidal gold, the antibodies labeled by magnetic beads have better washing resistance.
In another embodiment of the invention, the mass ratio of the chitosan to the acetic acid solution is 2-4:1.
In another embodiment of the present invention, the acetic acid solution has a mass concentration of 1 to 3%.
In another embodiment of the present invention, the amount of the nitrogen-doped graphene added is 1 to 4% by mass of the dispersion.
In another embodiment of the present invention, the ratio of the monoclonal antibody labeled with magnetic beads to the dispersion is 1:1.
In another embodiment of the present invention, in the monoclonal antibody labeled with magnetic beads, the particle size of the magnetic beads is 0.5 μm to 2.5 μm, and the antibody concentration is 0.3mg/ml to 1mg/ml.
In another embodiment of the present invention, the method for preparing the monoclonal antibody labeled with the magnetic bead comprises the following steps:
1. coupling of monoclonal antibodies to magnetic beads
1) Magnetic bead cleaning
Putting 500 mu L of magnetic beads with diameters of 2 mu m modified by NHS groups into a 1.5mL EP tube, putting the EP tube into a magnetic separation frame, enriching the magnetic beads, removing supernatant, adding 1mL of precooled Wash Buffer A with the temperature of 2-8 ℃ into the 1.5mL EP tube, swirling for 15s to mix the magnetic beads uniformly, putting the EP tube into the magnetic separation frame, enriching the magnetic beads, and removing the supernatant.
2) Immobilization of biological ligands
Add 500. Mu.L of 1mg/ml mAb solution to the EP tube and vortex for 30s to mix well. The EP tube was vortexed for 15s, placed on a vertical mixer, and mixed at room temperature for 1-2 h. If vertical mixing is not uniform, 30min before reaction, remove the EP tube vortex every 5min for 15s. Thereafter, every 15min, the EP tube was removed from the vortex for 15s and the magnetic beads were enriched using a magnetic separation rack.
3) Magnetic bead sealing
Add 1mL of Blocking Buffer (100 mM Tris-HCl,150mM NaCl, pH 8.0) to the EP tube, vortex for 30s, place the EP tube in a magnetic separation rack, enrich the magnetic beads, and discard the supernatant. Adding 1mL Blocking Buffer into an EP tube, vortexing for 30s, placing the EP tube in a vertical mixer for reacting for 2h at room temperature, enriching magnetic beads, and discarding supernatant. Add 1mL of ultrapure water to the EP tube, mix well, enrich the magnetic beads with a magnetic rack, discard the supernatant.
4) Preservation of
Add 1mL of Storage Buffer (0.05% sodium azide in PBS Buffer) to the EP tube, mix well, enrich the beads with a magnetic rack, and discard the supernatant. Add 500. Mu.L of Storage Buffer to the EP tube, mix well and store at 4 ℃ until use.
In a second aspect of the present invention, there is provided a method for producing the above-mentioned antibody dispersion, comprising:
preparing water, acetic acid and chitosan into a chitosan viscous solution according to a mass ratio, adding nitrogen-doped graphene into the solution, fully and uniformly mixing, and then carrying out ultrasonic treatment on the mixed solution to obtain a uniform dispersion liquid. Mixing with the monoclonal antibody marked by the magnetic beads in proportion to obtain the antibody dispersion liquid.
In another embodiment of the present invention, the ultrasonic time is 40min to 80min, and the ultrasonic frequency is 40KHz.
In another embodiment of the present invention, the target of detection by the working electrode is a protein molecule or a cell containing a specific antibody.
Such as lipoprotein a (LP-a), serum Ferritin (SF), serum Amyloid A (SAA), C-reactive protein (CRP), and the like. The cells containing specific antibodies include platelets, leukocytes, erythrocytes, neutrophils, eosinophils, basophils, etc.
In a third aspect of the present invention, there is provided a method for preparing the working electrode, comprising the steps of:
a. grinding, polishing and drying the surface of the glassy carbon electrode to obtain a pretreated electrode;
b. and uniformly covering the surface of the glassy carbon electrode with the antibody dispersion solution, drying at room temperature, then washing with double distilled water, and drying to obtain the working electrode.
In a fourth aspect of the invention, there is provided an electrochemical biosensor for detecting a protein, comprising a working electrode as described above.
The electrochemical biosensor further comprises: auxiliary electrode, reference electrode and test base solution.
By implementing the technical scheme, the working electrode provided by the invention has the advantages of higher sensitivity, better stability, more uniform antibody reaction and better washing resistance.
Drawings
FIG. 1 is a fluorescence reaction spectrum of an electrochemical biosensor according to the present invention and a fluorescence reaction spectrum of an electrochemical biosensor according to a comparative example.
FIG. 2 is a graph showing the result of the anti-contamination performance test of the electrochemical biosensor of the present invention.
FIG. 3 is a graph showing the result of the anti-washing performance test of the electrochemical biosensor in accordance with the present invention.
FIG. 4 is a standard curve of the electrochemical biosensor of the present invention.
FIG. 5 is a graph showing the results of the reproducibility test of the electrochemical biosensor according to the present invention.
Detailed Description
The invention will be described in detail below with reference to the drawings and specific embodiments. It should be noted that the following examples are only intended to illustrate the technical solution of the present invention and are not intended to limit the present invention, and although the present invention has been described in detail by the following examples, it should be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the present invention defined by the claims.
The monoclonal antibody labeled with the magnetic beads described in each example was obtained according to the following preparation method.
1. Coupling of monoclonal antibodies to magnetic beads
1) Magnetic bead cleaning
Taking 500 mu L of magnetic beads with diameters of 2 mu m modified by NHS groups to be placed in a 1.5mL EP tube, placing the EP tube in a magnetic separation frame, enriching the magnetic beads, removing supernatant, adding 1mL of precooled Wash Buffer A with the temperature of 2-8 ℃ to the 1.5mL EP tube, swirling for 15s to mix the magnetic beads uniformly, placing the EP tube in the magnetic separation frame, enriching the magnetic beads, and removing the supernatant.
2) Immobilization of biological ligands
Add 500. Mu.L of 1mg/ml mAb solution to the EP tube and vortex for 30s to mix well. The EP tube was vortexed for 15s, placed on a vertical mixer, and mixed at room temperature for 1-2 h. If the vertical mixing is not uniform, 30min before the reaction, the EP tube vortex is removed every 5min for 15s. Thereafter, every 15min, the EP tube was removed from the vortex for 15s and the magnetic beads were enriched using a magnetic separation rack.
3) Magnetic bead sealing
Add 1mL of Blocking Buffer (100 mM Tris-HCl,150mM NaCl, pH 8.0) to the EP tube, vortex for 30s, place the EP tube in a magnetic separation rack, enrich the magnetic beads, and discard the supernatant. Adding 1mL Blocking Buffer into an EP tube, vortexing for 30s, placing the EP tube in a vertical mixer for reacting for 2h at room temperature, enriching magnetic beads, and discarding supernatant. Add 1mL of ultrapure water to the EP tube, mix well, enrich the magnetic beads with a magnetic rack, discard the supernatant.
4) Preservation of
Add 1mL of Storage Buffer (0.05% sodium azide in PBS Buffer) to the EP tube, mix well, enrich the beads with a magnetic rack, and discard the supernatant. Add 500. Mu.L of Storage Buffer to the EP tube, mix well and store at 4 ℃ until needed.
Example 1
This example provides a working electrode of an electrochemical biosensor for detecting lipoprotein a, the working electrode being loaded with a coating layer formed of an antibody dispersion prepared as follows: preparing water, 3% acetic acid and chitosan into a chitosan viscous solution according to the mass ratio of 95. After mixing, the mixture was mixed with magnetic bead-labeled LPA at a ratio of 1:1 (magnetic bead particle size 2 μm, antibody concentration 0.5 mg/ml). And uniformly covering the mixed material with a working electrode, naturally drying at room temperature, cleaning with double distilled water, and drying to obtain the working electrode.
Example 2
This example provides a working electrode of an electrochemical biosensor for detecting lipoprotein a, the working electrode being loaded with a coating layer formed of an antibody dispersion prepared as follows: preparing water, 2% acetic acid and chitosan into a chitosan viscous solution according to the mass ratio of 96. After mixing, the mixture was mixed with magnetic bead-labeled LPA at a ratio of 1:1 (magnetic bead particle size 2 μm, antibody concentration 0.5 mg/ml). And uniformly covering the mixed material with a working electrode, naturally drying at room temperature, washing with double distilled water, and drying to obtain the working electrode.
Example 3
This example provides a working electrode of an electrochemical biosensor for detecting lipoprotein a, the working electrode being loaded with a coating layer formed of an antibody dispersion prepared as follows: preparing a chitosan viscous solution from water, 2% acetic acid and chitosan according to the mass ratio of 97. After mixing, the mixture was mixed with magnetic bead-labeled LPA at a ratio of 1:1 (magnetic bead particle size 2 μm, antibody concentration 0.5 mg/ml). And uniformly covering the mixed material with a working electrode, naturally drying at room temperature, washing with double distilled water, and drying to obtain the working electrode.
Example 4
This example provides a working electrode of an electrochemical biosensor for detecting lipoprotein a, the working electrode being loaded with a coating layer formed of an antibody dispersion prepared as follows: preparing water, 1% acetic acid and chitosan into a chitosan viscous solution according to the mass ratio of 97 to 2, adding 2 per thousand of nitrogen-doped graphene into the solution, fully and uniformly mixing, and then carrying out ultrasonic treatment on the mixed solution for about 1 hour to obtain a uniform dispersion liquid. After mixing, the mixture was mixed with magnetic bead-labeled LPA at a ratio of 1:1 (magnetic bead particle size 2 μm, antibody concentration 0.5 mg/ml). And uniformly covering the mixed material with a working electrode, naturally drying at room temperature, washing with double distilled water, and drying to obtain the working electrode.
Comparative example 1
This comparative example provides a working electrode of an electrochemical biosensor for detecting lipoprotein a, the working electrode being loaded with a coating layer formed of an antibody dispersion prepared as follows: preparing water, 1% acetic acid and chitosan into a chitosan viscous solution according to the mass ratio of 97 to 2, adding 3 per thousand of nitrogen-doped graphene into the solution, fully and uniformly mixing, and then carrying out ultrasonic treatment on the mixed solution for about 50min to obtain a uniform dispersion liquid. After mixing, the mixture was mixed with LPA monoclonal antibody at a ratio of 1:1 (magnetic bead size 2 μm, antibody concentration 0.5 mg/ml). And uniformly covering the mixed material with a working electrode, naturally drying at room temperature, cleaning with double distilled water, and drying to obtain the working electrode. Compared with example 4, this comparative example directly uses LPA mab without magnetic bead coupling.
Comparative example 2
This example provides a working electrode of an electrochemical biosensor for detecting lipoprotein a, the working electrode being loaded with a coating layer formed of an antibody dispersion prepared as follows: preparing water, 2% acetic acid and chitosan into a chitosan viscous solution according to the mass ratio of 95. After mixing, the mixture was mixed with protein A at a ratio of 1:1 (magnetic bead diameter: 2 μm, antibody concentration: 0.5 mg/ml). And uniformly covering the mixed material with a working electrode, naturally drying at room temperature, washing with double distilled water, and drying to obtain the working electrode.
Comparative example 3
This example provides a working electrode of an electrochemical biosensor for detecting lipoprotein a, the working electrode being loaded with a coating layer formed of an antibody dispersion prepared as follows: preparing water, 1% acetic acid and chitosan into a chitosan viscous solution according to the mass ratio of 97 to 2, adding 2 per thousand of nitrogen-doped graphene into the solution, fully and uniformly mixing, and then carrying out ultrasonic treatment on the mixed solution for about 1 hour to obtain a uniform dispersion liquid. After mixing, the mixture was mixed with LPA Jin Oulian gel at a ratio of 1:1 (magnetic bead size 2 μm, antibody concentration 0.5 mg/ml). And uniformly covering the mixed material with a working electrode, naturally drying at room temperature, cleaning with double distilled water, and drying to obtain the working electrode. In contrast to example 4, this comparative example employed colloidal gold conjugated LPA mab.
Example 5
This example provides four electrochemical biosensors for detecting lipoprotein a, the sensor composition comprising a working electrode, an auxiliary electrode, a reference electrode, and a test base solution. Working electrodes obtained in example 4 and comparative examples 1 to 3 were used as the working electrodes, respectively; the auxiliary electrode adopts a carbon electrode, and the reference electrode adopts Ag/AgCl; the test base solution was a solution containing 0.5mol/L potassium ferricyanide and having a pH of 7.4. The resulting electrochemical biosensor, corresponding to example 4 and comparative examples 1-3, was labeled A, B, C, D.
The electrochemical biosensor A, B, C, D is subjected to performance detection.
1. Fluorescence reaction Using verification antibodies
The verification method comprises the following steps: 10ul Dyl488-secondary antibody (100 ug/ml) was added dropwise to the working electrode of the prepared sensor A, B, C, D. After incubation at 37 ℃ for 30 minutes, the working electrode surface incubated Dyl-secondary antibody was washed with 1x PBS. The Dyl-secondary antibody was observed to react and link with the LPA monoclonal antibody, the antibody of Protein a, on the surface of the working electrode. By means of the fluorescence distribution expression of the surface of the working electrode, the distribution condition of the antibody on the surface of the electrode can be obtained after the reactive antibody dispersion liquid is dripped on the surface of the working electrode and dried.
And (4) verification result: see figure 1. The higher fluorescence intensity at the periphery of sensor panels a and B compared to the interior was observed, indicating that there was only one antibody component in the material and that the sensor surface reaction was not uniform. The sensors C and D effectively solve the problem of uniform distribution of effective antibody components in the coupling material by introducing colloidal gold and magnetic beads.
2. Anti-pollution Performance test
Electrochemical biosensor a was used in this test.
The verification method comprises the following steps: in the electrode test of the magnetic bead antibody coupling component, 10ul FITC-labeled secondary fluorescent antibody (100 ug/ml), 10ul FITC-labeled CD62p primary antibody (100 ug/ml) and 10ul FITC-labeled AFP primary antibody (100 ug/ml) were respectively added dropwise to the working electrode. After incubation at 37 ℃ for 30 minutes, the working electrode was washed with 1 × PBS, and the fluorescence state of the surface of the working electrode was observed using a fluorescence microscope.
And (4) verification result: only the FITC-labeled secondary antibody reacted with LPA showed visible fluorescence in the electrode of the magnetic bead component, observed using a fluorescence microscope. The electrode specificity of the magnetic bead component is better, and the magnetic bead component has the anti-pollution performance. See figure 2.
3. Resistance to cleaning test
The verification method comprises the following steps: the prepared electrode material mixed by the colloidal gold-LPA and the electrode material mixed by the magnetic bead-LPA are dripped to the surface of the electrode, and then 10ul of the electrode material and FITC label secondary antibody (100 ug/ml) are respectively dripped to incubate for 30 minutes at 37 ℃. After incubation, the working electrode was washed 1, 5 and 10 times with PBS to observe the fluorescence intensity on the electrode surface.
And (4) verification result: the colloidal gold was observed by overuse to decrease the fluorescence intensity with increasing number of washes. The magnetic bead-LPA has better washing resistance. See figure 3.
4. Drawing of standard curve
The electrochemical biosensor corresponding to example 4 was used.
1mg/ml of LP (a) antigen was diluted separately with PBS to concentrations: 700ug/ml, 500ug/ml, 300ug/ml, 200ug/ml, 100ug/ml, 0ug/ml. The diluted 6 parts of the standard solution were respectively sucked 10ul and dropped on the surface of the prepared electrode sensor, and the current was measured using an electrochemical workstation, and the results are as follows in table 1:
TABLE 1 sensor response Current for different concentrations of antigen
The standard curve of the electrochemical sensor is plotted as shown in fig. 4.
5. Reproducibility test
The electrochemical biosensor corresponding to example 4 was used. Under the same conditions, the results of the measurement using 5 electrodes of different concentrations of LPA antigen prepared in the same batch of the present invention are detailed in FIG. 5, and the reproducibility Relative Standard Deviation (RSD) of 700ug/ml, 500ug/ml, 300ug/ml, 200ug/ml, 100ug/ml, 0ug/ml is 3.37%, 2.75%, 2.23%, 2.1%, 2.37 and 1.41%, respectively, which indicates that the difference in sensor batch is small and the reproducibility is good.
6. Electrochemical sensor related application
1) Cell detection
To demonstrate that electrochemical sensors can be used for cell detection, the sensors are applied by introducing cytometric detection. 6 indexes of platelets, white blood cells, red blood cells, neutrophils, eosinophils and basophils are selected for application. Relevant cellular information is in table 2:
TABLE 2 cell information
The specific antibody corresponding to each cell and the magnetic bead are coupled by the antibody magnetic bead coupling method described in the above example 1, the prepared coupling material is added on the working electrode by the method described in the example 2, the sensor is used for detection, the sensor response current is taken as the X axis, the number of the test cells is taken as the Y axis, and a curve equation is fitted.
a) Platelet count
Repeatedly detecting standard substance solution with platelet count concentration of 0, 75, 150, 295, 585 and 1156 (unit is 10^ 9/L) to obtain average value of sensor response current, establishing a standard curve by taking the sensor response current as abscissa and the cell number as ordinate, and fitting a curve equation to meet the requirements: r 2 Not less than 0.99 (Table 3.)
TABLE 3 corresponding Current values of different platelet count sensors
Randomly selecting 5 samples to perform sensor response current detection, substituting the corresponding current value obtained by detection into the curve equation, calculating the corresponding cell number, repeating each sample for 5 times, and obtaining the data shown in the following table:
TABLE 4 detection of platelet Standard cell number
The standard sample with the confirmed cell number is measured and repeated for 5 times, and the obtained response current value is substituted into the fitted equation to calculate the corresponding cell number. The variation coefficient of 5 times of test results is less than or equal to 0.942%.
b) White blood cell count
Repeatedly detecting standard substance solutions with the leucocyte counting concentrations of 21.97, 10.44, 4.82, 2.52, 1.34 and 0.00 (the unit is 10^ 9/L) to obtain an average value of the response current of the sensor, establishing a standard curve by taking the response current of the sensor as a horizontal coordinate and the cell number as a vertical coordinate, and fitting the equation of the curve to meet the requirements: r is 2 Not less than 0.99 (Table 5.)
TABLE 5 corresponding Current values of different leukocyte count sensors
Randomly selecting 5 samples to perform sensor response current detection, substituting the corresponding current value obtained by detection into the curve equation, calculating the corresponding cell number, repeating each sample for 5 times, and obtaining the data shown in the following table:
TABLE 6 measurement of the number of leukocytes in standard samples
The standard sample with the confirmed cell number is measured and repeated for 5 times, and the obtained response current value is substituted into the fitted equation to calculate the corresponding cell number. The variation coefficient of 5 times of test results is less than or equal to 0.929%.
c) Erythrocyte count
Repeatedly detecting standard substance solutions with the red blood cell count concentration of 6.52, 3.36, 1.61, 0.83, 0.43 and 0.00 (the unit is 10^ 12/L) to obtain an average value of sensor response current, establishing a standard curve by taking the sensor response current as a horizontal coordinate and the cell number as a vertical coordinate, and fitting the curve equation requirement: r 2 Not less than 0.99 (Table 7.)
TABLE 7 corresponding Current values for different red blood cell count sensors
Randomly selecting 5 samples to perform sensor response current detection, substituting the corresponding current value obtained by detection into the curve equation, calculating the corresponding cell number, repeating each sample for 5 times, and obtaining the data shown in the following table:
TABLE 8 erythrocyte Standard cell number assay
The standard sample with the confirmed cell number is measured and repeated for 5 times, and the obtained response current value is substituted into the fitted equation to calculate the corresponding cell number. The variation coefficient of 5 times of test results is less than or equal to 0.881 percent.
d) Neutrophil count
Repeatedly detecting standard substance solutions with the neutrophil count concentration of 5.95, 2.84, 1.15, 0.66, 0.30 and 0.00 (the unit is 10^ 9/L) to obtain an average value of sensor response current, establishing a standard curve by taking the sensor response current as a horizontal coordinate and the cell number as a vertical coordinate, and fitting the equation of the curve to meet the requirements: r 2 Not less than 0.99 (Table 9.)
TABLE 9 corresponding Current values for different neutrophil count sensors
Randomly selecting 5 samples to perform sensor response current detection, substituting the corresponding current value obtained by detection into the curve equation, calculating the corresponding cell number, repeating each sample for 5 times, and obtaining the data shown in the following table:
TABLE 10 neutrophil standards cell number assay
The standard sample with the confirmed cell number is measured and repeated for 5 times, and the obtained response current value is substituted into the fitted equation to calculate the corresponding cell number. The variation coefficient of the 5 times of test results is less than or equal to 1.298%.
e) Eosinophil count
Repeatedly detecting standard substance solution with eosinophil count concentration of 1.87, 0.89, 0.52, 0.11, 0.05, 0.02 and 0.00 (unit is 10^ 9/L) to obtain average value of sensor response current, establishing a standard curve by taking the sensor response current as abscissa and the cell number as ordinate, and fitting the curve equation requirement:R 2 not less than 0.99 (Table 11.)
TABLE 11 corresponding Current values for different eosinophil count sensors
Randomly selecting 5 samples to perform sensor response current detection, substituting the corresponding current value obtained by detection into the curve equation, calculating the corresponding cell number, repeating each sample for 5 times, and obtaining the data shown in the following table:
TABLE 12 eosinophil Standard cell number assay
The standard sample with the confirmed cell number is measured and repeated for 5 times, and the obtained response current value is substituted into the fitted equation to calculate the corresponding cell number. The coefficient of variation of the 5 test results is not more than 5.357%.
f) Basophil enumeration
Repeatedly detecting standard substance solution with basophil counting concentration of 0.90, 0.15, 0.08, 0.04, 0.02 and 0.00 (unit is 10^ 9/L) to obtain the average value of sensor response current, establishing a standard curve by taking the sensor response current as a horizontal coordinate and the cell number as a vertical coordinate, establishing the standard curve, and fitting the curve equation requirement: r 2 Not less than 0.99 (Table 13.)
TABLE 13 corresponding Current values for different basophil number sensors
Randomly selecting 5 samples to perform sensor response current detection, substituting the corresponding current value obtained by detection into the curve equation, calculating the corresponding cell number, repeating each sample for 5 times, wherein the data is shown in the following table:
TABLE 14 basophil standard cell number assay
The standard sample with the confirmed cell number is measured and repeated for 5 times, and the obtained response current value is substituted into the fitted equation to calculate the corresponding cell number. The coefficient of variation of the 5 test results is not more than 6.501.
And (4) conclusion:
the cell number of 6 groups of different cells is detected, and the coefficient of variation of all detection values meets the requirement, so that the electrochemical sensor can be used for detecting the cells with the diameters of 2-20 micrometers (microns).
2) Protein detection
The sensor is introduced for the detection of protein content. Selecting lipoprotein a (LP-a), serum Ferritin (SF), serum Amyloid A (SAA) and C Reactive Protein (CRP) for application. The relevant protein information is shown in table 15:
TABLE 15 protein information
The specific antibody of each protein was coupled to the magnetic beads by the antibody magnetic bead coupling method described in example 1, the prepared coupling material was added to the working electrode by the method described in example 2, detection was performed by the sensor, and a curve equation was fitted with the sensor response current as the X-axis and the test protein concentration as the Y-axis.
a) Lipoprotein a (LP-a)
Repeatedly detecting the standard protein solutions with the standard protein concentrations of 700, 500, 300, 200, 100 and 0 (the unit is mg/L) to obtain the average value of the response current of the sensor, establishing a standard curve by taking the response current of the sensor as a horizontal coordinate and the protein concentration as a vertical coordinate, and fitting the requirements of a curve equation: r2 is more than or equal to 0.99 (Table 16.)
TABLE 16 corresponding Current values for different concentrations of protein sensor
Selecting 5 diluted standard samples to perform sensor response current detection, substituting the corresponding current value obtained by detection into the curve equation, calculating the corresponding protein concentration, repeating each sample for 5 times, and obtaining the data shown in the following table:
TABLE 17 lipoprotein a Standard detection
The concentration of the standard substance was determined, repeated 5 times, and the obtained response current value was substituted into the above fitted equation to calculate the corresponding concentration. The coefficient of variation of the 5 test results is not more than 3.609.
b) Serum Ferritin (SF)
Repeatedly detecting standard substance solutions with the protein standard concentrations of 0.000, 0.013, 0.025, 0.050, 0.100 and 0.200 (the unit is mg/L) to obtain an average value of the response current of the sensor, establishing a standard curve by taking the response current of the sensor as an abscissa and the protein concentration as an ordinate, and fitting the equation of the curve to require that: r2 is more than or equal to 0.99 (Table 18.)
TABLE 18 corresponding Current values for different concentrations of protein sensor
Selecting 5 diluted standard samples to perform sensor response current detection, substituting corresponding current values obtained by detection into the curve equation, calculating corresponding protein concentration, repeating each sample for 5 times, wherein the data is shown in the following table:
TABLE 19 serum ferritin Standard assay
And (3) measuring the standard substance with confirmed protein concentration, repeating the test for 5 times, substituting the current value into the fitted equation, and calculating the corresponding concentration value. The deviation between the protein concentration and the standard substance concentration corresponding to the 5 times of test results is less than or equal to 6.2363.
c) Serum Amyloid A (SAA)
Repeatedly detecting the standard solutions with the protein concentrations of 0, 12.5, 25, 50, 100 and 200 (the unit is mg/L) to obtain the average value of the response current of the sensor, establishing a standard curve by taking the response current of the sensor as a horizontal coordinate and the protein concentration as a vertical coordinate, and fitting the requirements of a curve equation: r2 is more than or equal to 0.99 (in the table 20.)
TABLE 20 corresponding Current values of serum amyloid A Sensors at different concentrations
Selecting 5 diluted standard samples to perform sensor response current detection, substituting the corresponding current value obtained by detection into the curve equation, calculating the corresponding protein concentration, repeating each sample for 5 times, and obtaining the data shown in the following table:
TABLE 21 serum ferritin Standard test
And (3) measuring the standard substance with confirmed protein concentration, repeating the test for 5 times, substituting the current value into the fitted equation, and calculating the corresponding concentration value. The deviation between the protein concentration and the standard substance concentration corresponding to the 5 times of test results is less than or equal to 2.299.
d) C Reactive Protein (CRP)
Repeatedly detecting the standard substance solution with the protein concentration of 5.95, 2.84, 1.15, 0.66, 0.30 and 0.00 (unit is mg/L) to obtain the average value of the response current of the sensor, establishing a standard curve by taking the response current of the sensor as the abscissa and the protein concentration as the ordinate, and fitting the equation of the curve to require that: r2 is more than or equal to 0.99 (Table 22.)
TABLE 22 corresponding Current values for different concentrations of C-reactive protein sensor
Selecting 5 diluted standard samples to perform sensor response current detection, substituting the corresponding current value obtained by detection into the curve equation, calculating the corresponding protein concentration, repeating each sample for 5 times, and obtaining the data shown in the following table:
TABLE 23C reactive protein Standard assay
The measurement of the standard substance with confirmed protein concentration was repeated 5 times, and the current value was substituted into the above fitted equation to calculate the corresponding concentration value. The deviation between the protein concentration and the standard substance concentration corresponding to the 5 times of test results is less than or equal to 2.123.
And (4) conclusion:
through the detection of 4 histones, the electrochemical sensor can be used for detecting the protein content in milligram.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006133137A (en) * | 2004-11-08 | 2006-05-25 | Eiichi Tamiya | Method for detecting material to be inspected |
| CN102749373A (en) * | 2012-07-06 | 2012-10-24 | 济南大学 | Preparation method and application of environmental estrogen electrochemical immunosensor |
| CN104297309A (en) * | 2014-10-29 | 2015-01-21 | 江苏出入境检验检疫局动植物与食品检测中心 | Electrochemical transducer, and preparation method and application for detecting pathogenic bacteria |
| CN106996971A (en) * | 2017-04-28 | 2017-08-01 | 江南大学 | Detect the kit and method of human peripheral EVs TRPC5 contents |
| CN109060918A (en) * | 2018-08-30 | 2018-12-21 | 华南理工大学 | Hydroquinone biosensor and its preparation and application based on the compound horseradish peroxidase of nitrogen-doped graphene |
| CN109270140A (en) * | 2018-11-01 | 2019-01-25 | 安阳师范学院 | The preparation method and application of high dispersive graphene/Zn Base Metal organic framework composite material electrochemical sensor |
| GB202201006D0 (en) * | 2022-01-26 | 2022-03-09 | Paragraf Ltd | A biosensor device and a method of manufacturing a biosensor device |
-
2022
- 2022-12-08 CN CN202211568626.3A patent/CN115932005A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006133137A (en) * | 2004-11-08 | 2006-05-25 | Eiichi Tamiya | Method for detecting material to be inspected |
| CN102749373A (en) * | 2012-07-06 | 2012-10-24 | 济南大学 | Preparation method and application of environmental estrogen electrochemical immunosensor |
| CN104297309A (en) * | 2014-10-29 | 2015-01-21 | 江苏出入境检验检疫局动植物与食品检测中心 | Electrochemical transducer, and preparation method and application for detecting pathogenic bacteria |
| CN106996971A (en) * | 2017-04-28 | 2017-08-01 | 江南大学 | Detect the kit and method of human peripheral EVs TRPC5 contents |
| CN109060918A (en) * | 2018-08-30 | 2018-12-21 | 华南理工大学 | Hydroquinone biosensor and its preparation and application based on the compound horseradish peroxidase of nitrogen-doped graphene |
| CN109270140A (en) * | 2018-11-01 | 2019-01-25 | 安阳师范学院 | The preparation method and application of high dispersive graphene/Zn Base Metal organic framework composite material electrochemical sensor |
| GB202201006D0 (en) * | 2022-01-26 | 2022-03-09 | Paragraf Ltd | A biosensor device and a method of manufacturing a biosensor device |
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