CN112213482B - Method for electrochemically detecting antigen content in sample based on immunohistochemical space - Google Patents
Method for electrochemically detecting antigen content in sample based on immunohistochemical space Download PDFInfo
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Abstract
The invention relates to a method for electrochemically detecting the content of an antigen in a sample based on an immunohistochemical space, which comprises the following steps of: modifying an electrochemical signal (ferrocene/quantum dot and the like) on the antibody, carrying out antigen-antibody combination, removing the unbound antibody, detecting the electrochemical signal in the solution by using an electrochemical instrument, and reflecting the content of the antigen in the cancer tissue/cell by the strength of the signal. Compared with the prior art, the invention can realize the simultaneous detection of different antigens by using different electrochemical signals to modify the antibody; the electrochemical signal value directly corresponds to the quantity of the antigen by the measurement of a standard curve, and the full quantitative detection is realized; compared with immunohistochemistry, the electrochemical detection step is simple to operate and has low technical requirements on operators; the modified electrode can greatly improve the sensitivity and realize the trace detection of the antigen.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for electrochemically detecting the content of an antigen in a sample based on an immunohistochemical space.
Background
Immunohistochemical detection, abbreviated as immunohistochemical detection, is a method of determining histocyte antigens (polypeptides, proteins, pathogens, etc.) by applying the immunological basic principle, namely the antigen-antibody reaction, i.e., the principle of specific binding of antigen and antibody, and developing color development agents (fluorescein, enzyme, metal ions, isotopes, etc.) of labeled antibodies through chemical reaction, and performing research on the histocyte antigens (polypeptides, proteins, pathogens, etc.) in positioning, qualitative and relative quantification, wherein the research is divided into immunohistochemical technology (immunohistochemistry) and immunocytochemical technology (immunocytochemistry). Immunohistochemistry can be broadly classified into immunofluorescence cytochemistry, immunoenzyme cytochemistry, and immunocolloidal gold techniques, depending on the kind of the marker.
In conventional pathological diagnosis of tumors, accurate diagnosis of the morphology of some cases is difficult by HE staining alone. The immunohistochemistry can make definite diagnosis on a plurality of difficult tumors by means of the combination of specific antigens and antibodies, and the differential diagnosis accuracy rate of the immunohistochemical diagnosis on the poorly differentiated/undifferentiated tumors reaches 50-75%. Immunohistochemistry can be mainly used for differential diagnosis of malignant tumors, determination of primary parts of metastatic malignant tumors, pathological typing of tumors, treatment of soft tissue tumors, discovery of micrometastases and the like.
Immunohistochemistry has become the "gold standard" in medical tumor diagnosis in recent years due to its strong specificity, high sensitivity, accurate localization, and other advantages. But because of its complex operation: the technical requirements for the pathology technicians are high; the sensitivity is relatively low: due to optical limitations, trace amounts of antigen sometimes cannot be accurately observed; the method cannot quantify: immunohistochemical tablets can only be relatively quantitative, and a plurality of experts are needed to give a unified conclusion, so that more human factors exist; time consuming: due to the fact that the immunohistochemical operation process is multiple, 2-3 days are needed to give a prepared pathological report to clinical patients, and subsequent treatment progress is easily influenced.
Therefore, the immunohistochemical detection in the prior art also has the defects of incomplete quantification, complex operation and the like.
Disclosure of Invention
The invention aims to provide a method for electrochemically detecting the content of an antigen in a sample based on immunohistochemistry and a method for electrochemically detecting the content of the antigen in the sample based on an immunohistochemical space.
The invention provides possibility for quantitative detection of immunohistochemistry by the content of electrochemical signal strong and weak reaction antigen and realizing space matching.
The purpose of the invention can be realized by the following technical scheme:
the invention provides a method for detecting the content of an antigen in a sample based on immunohistochemical electrochemistry, which comprises the following steps: modifying an electrochemical signal on an antibody, mixing the antibody modified by the electrochemical signal with a sample with the antigen content to be detected, combining the antigen and the antibody, detecting the electrochemical signal of the antibody combined with the antigen or detecting the electrochemical signal of the antibody not combined with the antigen by using an electrochemical instrument, comparing the detected electrochemical signal with a standard curve of the electrochemical signal, and calculating to obtain the antigen content in the sample to be detected.
Further, when the sample with the antigen content to be detected is the tissue with the antigen content to be detected, mixing the tissue with the antigen content to be detected with the antibody modified by the electrochemical signal, incubating, combining the antigen and the antibody, removing the tissue with the antigen content to be detected, detecting the electrochemical signal of the antibody which is not combined by using a differential pulse voltammetry, comparing the detected electrochemical signal with a standard curve of the electrochemical signal, and calculating to obtain the antigen content in the tissue with the antigen content to be detected.
Further, when the sample with the antigen content to be detected is the cell with the antigen content to be detected, mixing the cell with the antigen content to be detected and the antibody modified by the electrochemical signal, incubating, combining the antigen and the antibody, centrifuging to remove the unbound antibody, detecting the electrochemical signal on the surface of the cell by using a differential pulse voltammetry method, comparing the detected electrochemical signal with a standard curve of the electrochemical signal, and calculating to obtain the antigen content in the tissue with the antigen content to be detected.
When an electrochemical instrument is used for detecting an electrochemical signal of the antibody combined with the antigen, the detected electrochemical signal is compared with a standard curve of the electrochemical signal, the content of the antibody combined with the antigen is obtained through calculation, and the content of the antigen in the detection sample is obtained through calculation according to the content of the antibody combined with the antigen;
when an electrochemical instrument is used for detecting an electrochemical signal of the antibody which is not combined with the antigen, the detected electrochemical signal is compared with a standard curve of the electrochemical signal, the content of the antibody which is not combined with the antigen is calculated, the content of the antibody which is combined with the antigen is calculated according to the total amount of the antibody modified by the electrochemical signal before the antibody is mixed with the antigen sample to be detected, and the content of the antigen in the detected sample is calculated according to the content of the antibody which is combined with the antigen.
Further, a standard curve of electrochemical signals was prepared by the following method: and detecting electrochemical signals with different concentrations under a ternary battery method by using an electrochemical instrument, and making a linear curve, namely a standard curve of the electrochemical signals, by taking the log value of the concentration as an X axis and the corresponding current value as a Y axis.
Further, in the above method, simultaneous detection of the respective antigens can be achieved by modifying different antibodies with different electrochemical signals.
Further, the electrochemical signal is selected from ferrocene or quantum dots selected from ZnS, CdS, CdTe, CdSe or PbS and the like.
Furthermore, when an electrochemical signal is detected by using an electrochemical instrument, the used electrode is an electrode with the surface modified by dendritic gold and conductive polymer.
Specifically, the electrode with the surface modified with the dendritic gold and the conductive polymer means that the surface of the electrode is electroplated with the dendritic gold, and the surface of the dendritic gold is electroplated with the conductive polymer.
The conductive polymer is thinly electroplated on the surface of the dendritic gold, and the stability and the conductivity of the electroplated electrode can be greatly improved.
Further, the method for obtaining the electrode modified with the dendritic gold and the conductive polymer on the surface comprises the following steps: the surface of the electrode is modified with dendritic gold by an electroplating method, and then the surface of the dendritic gold is modified with conductive polymers by the electroplating method.
Further, the method for obtaining the electrode with the surface modified with the dendritic gold and the conductive polymer comprises the following steps:
(1) electroplating the surface of the electrode in a chloroauric acid and sodium sulfate solution by using a chronoamperometry to obtain dendritic gold;
(2) dissolving a conductive polymer monomer and tetrabutylammonium perchlorate in acetonitrile, introducing nitrogen, removing air in the solution, and electroplating a conductive polymer on the surface of the dendritic gold by using a cyclic voltammetry method to obtain an electrode with the surface modified with the dendritic gold and the conductive polymer.
Wherein, preferably, in the step (1), the concentration of the chloroauric acid is 30mM, and the concentration of the sodium sulfate solution is 0.1M Na2SO4。
Among them, the conductive polymer is preferably selected from ethylene dioxythiophene, thiophene acetic acid, pyrrole, and the like.
Further, the method for modifying different electrochemical signals on the antibody comprises the following steps: electrochemical signals (e.g., ferrocene, quantum dots, etc.) are incubated with specific antibodies in the presence of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.
The method can directly correspond to the quantity of the antigens according to the strength of the electrochemical signals, and realize full quantitative detection in the sample to be detected.
The invention also provides a method for electrochemically detecting the content of the antigen in the sample based on the immunohistochemical space, which comprises the following steps:
a. modifying an electrochemical signal to the antibody;
b. adding the antibody modified by the electrochemical signal into the tissue to be detected, incubating, and combining the antigen and the antibody;
c. removing tissues with the antigen content to be detected, and detecting the electrochemical signals of the unbound antibodies by using a differential pulse voltammetry method;
d. comparing the measured electrochemical signal with a standard curve of the electrochemical signal, and calculating to obtain the content of the antigen in the tissue of which the content of the antigen is to be measured;
e. extracting other partial region tissues, and repeating the detection method to obtain electrochemical signal values of different region tissues;
f. and converting electrochemical signal values obtained from different areas into a heat map, and combining the heat map with an HE (hematoxylin-eosin) map to realize spatial immunohistochemical electrochemical detection.
Further, in step f, converting the electrochemical signal values obtained from the different regions into a heat map is a conventional method for spatial detection, and visually expresses the content of the antigen in the tissues of the different regions according to the change of the color of the points in the heat map, wherein the darker the color of the points in the heat map with high expression of the antigen is, the lighter the color of the points in the heat map with low expression of the antigen is.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) different quantum dots are used for modifying different antibodies, so that the simultaneous detection of corresponding antigens can be realized;
(2) the electrochemical strength directly corresponds to the quantity of the antigen, so that full quantitative detection is realized;
(3) compared with immunohistochemistry, the electrochemical detection step is simple to operate and has low technical requirements on operators;
(4) the modified electrode can greatly improve the sensitivity and realize the trace detection of the antigen.
Drawings
FIG. 1: electrode modification flow diagram.
FIG. 2: scanning electron micrographs of dendritic gold in example 1.
FIG. 3: cyclic voltammetry and electrochemical impedance mapping after gold dendrite in example 1.
FIG. 4: standard curve of electrochemical signal.
FIG. 5: immunohistochemistry profile obtained after colorectal cancer tissue section treatment.
FIG. 6: and comparing the electrochemical signal of the unbound antibody with a standard curve to obtain the content of the antigen in the sample to be detected.
FIG. 7: ferrocene-modified antibody of example 1, used in a flow chart for electrochemical detection of tissue surface antigens.
FIG. 8: ferrocene-modified antibodies of example 3, used in a flow chart for electrochemical detection of cell surface antigens.
FIG. 9: the antibody modified by different quantum dots in example 4 is used in the electrochemical detection flow chart of cell surface antigen.
FIG. 10: the antibodies modified by different quantum dots in examples 5 and 6 are used in the electrochemical detection flow chart of the tissue surface antigen.
FIG. 11: a tissue slice processing method and an electrochemical detection flow chart thereof.
Detailed Description
Referring to fig. 1, the electrode with the surface modified with the dendritic gold and the conductive polymer means that the surface of the electrode is electroplated with the dendritic gold, and the surface of the dendritic gold is electroplated with the conductive polymer. The conductive polymer is thinly electroplated on the surface of the dendritic gold, and the stability and the conductivity of the electroplated electrode can be greatly improved.
Referring to fig. 1, the method for obtaining the electrode modified with dendritic gold and conductive polymer on the surface comprises:
(1) electroplating the surface of the electrode in a chloroauric acid and sodium sulfate solution by using a chronoamperometry to obtain dendritic gold;
(2) dissolving a conductive polymer monomer and tetrabutylammonium perchlorate in acetonitrile, introducing nitrogen, removing air in the solution, and electroplating a conductive polymer on the surface of the dendritic gold by using a cyclic voltammetry method to obtain an electrode with the surface modified with the dendritic gold and the conductive polymer.
Preferably, in step (1), the concentration of chloroauric acid is 30mM and the concentration of sodium sulfate solution is 0.1MNa2SO4. Preferably, the conductive polymer is selected from ethylene dioxythiophene, thiophene acetic acid, pyrrole, and the like.
The invention provides a method for detecting the content of an antigen in a sample based on immunohistochemical electrochemistry, which comprises the following steps: modifying an electrochemical signal on an antibody, mixing the antibody modified by the electrochemical signal with a sample with the antigen content to be detected, combining the antigen and the antibody, detecting the electrochemical signal of the antibody combined with the antigen or detecting the electrochemical signal of the antibody not combined with the antigen by using an electrochemical instrument, comparing the detected electrochemical signal with a standard curve of the electrochemical signal, and calculating to obtain the antigen content in the sample to be detected.
When the sample with the antigen content to be detected is the tissue with the antigen content to be detected, mixing the tissue with the antigen content to be detected with the antibody modified by the electrochemical signal, incubating, combining the antigen and the antibody, removing the tissue with the antigen content to be detected, detecting the electrochemical signal of the antibody which is not combined by using a differential pulse voltammetry, comparing the measured electrochemical signal with a standard curve of the electrochemical signal, and calculating to obtain the antigen content in the tissue with the antigen content to be detected.
When the sample with the antigen content to be detected is the cell with the antigen content to be detected, mixing the cell with the antigen content to be detected with the antibody modified by the electrochemical signal, incubating, combining the antigen and the antibody, centrifuging to remove the unbound antibody, detecting the electrochemical signal on the surface of the cell by using a differential pulse voltammetry, comparing the measured electrochemical signal with a standard curve of the electrochemical signal, and calculating to obtain the antigen content in the tissue with the antigen content to be detected.
When an electrochemical instrument is used for detecting an electrochemical signal of the antibody combined with the antigen, the detected electrochemical signal is compared with a standard curve of the electrochemical signal, the content of the antibody combined with the antigen is obtained through calculation, and the content of the antigen in the detection sample is obtained through calculation according to the content of the antibody combined with the antigen;
when an electrochemical instrument is used for detecting an electrochemical signal of the antibody which is not combined with the antigen, the detected electrochemical signal is compared with a standard curve of the electrochemical signal, the content of the antibody which is not combined with the antigen is calculated, the content of the antibody which is combined with the antigen is calculated according to the total amount of the antibody modified by the electrochemical signal before the antibody is mixed with the antigen sample to be detected, and the content of the antigen in the detected sample is calculated according to the content of the antibody which is combined with the antigen.
Wherein, the standard curve of the electrochemical signal is prepared by the following method:
and detecting electrochemical signals with different concentrations under a ternary battery method by using an electrochemical instrument, and making a linear curve, namely a standard curve of the electrochemical signals, by taking the log value of the concentration as an X axis and the corresponding current value as a Y axis.
In the above method, simultaneous detection of the corresponding antigens can be achieved by modifying different antibodies with different electrochemical signals.
Wherein the electrochemical signal is selected from ferrocene or quantum dots, and the quantum dots are selected from ZnS, CdS, CdTe, CdSe or PbS and the like.
The method for modifying the electrochemical signal to the antibody comprises the following steps: electrochemical signals (e.g., ferrocene, quantum dots, etc.) are incubated with specific antibodies in the presence of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.
The method can directly correspond to the quantity of the antigens according to the strength of the electrochemical signals, and realize full quantitative detection in the sample to be detected.
The invention also provides a method for electrochemically detecting the content of the antigen in the sample based on the immunohistochemical space, which comprises the following steps:
a. modifying an electrochemical signal to the antibody;
b. adding the antibody modified by the electrochemical signal into the tissue to be detected, incubating, and combining the antigen and the antibody;
c. removing tissues with the antigen content to be detected, and detecting the electrochemical signals of the unbound antibodies by using a differential pulse voltammetry method;
d. comparing the measured electrochemical signal with a standard curve of the electrochemical signal, and calculating to obtain the content of the antigen in the tissue of which the content of the antigen is to be measured;
e. extracting other partial region tissues, and repeating the detection method to obtain electrochemical signal values of different region tissues;
f. and converting electrochemical signal values obtained from different areas into a heat map, and combining the heat map with an HE (hematoxylin-eosin) map to realize spatial immunohistochemical electrochemical detection.
Further, in step f, converting the electrochemical signal values obtained from the different regions into a heat map is a conventional method for spatial detection, and visually expresses the content of the antigen in the tissues of the different regions according to the change of the color of the points in the heat map, wherein the darker the color of the points in the heat map with high expression of the antigen is, the lighter the color of the points in the heat map with low expression of the antigen is.
The invention is described in detail below with reference to the figures and specific embodiments.
Example 1
The electrode modification scheme is shown in FIG. 1, and 30mM HAuCl is taken4，0.1M Na2SO4In chloroauric acid and sodium sulfate solution, dendritic gold is obtained after the electrode surface is electroplated by using a chronoamperometry, and the morphology of the obtained dendritic gold is shown in fig. 2. The current resistance values of the electrodes before and after modification of the dendritic gold were compared by cyclic voltammetry and electrochemical impedance, as shown in fig. 3.
Dissolving a conductive polymer monomer (ethylene dioxythiophene in the embodiment) and tetrabutylammonium perchlorate in anhydrous acetonitrile, introducing nitrogen, removing air in the solution, and electroplating a conductive polymer on the surface of the dendritic gold by using a cyclic voltammetry method to obtain an electrode with the surface modified with the dendritic gold and the conductive polymer.
Wherein the concentration of the conductive polymer monomer and tetrabutylammonium perchlorate in acetonitrile is 7.5mM and 50mM, the voltage range of cyclic voltammetry is 0.65-1.7V, the scanning rate is 50mV/s, and the cycle number is 20 times.
The current values of electrochemical signals (ferrocene, 1 μ M,10 μ M,50 μ M,100 μ M,500 μ M,1mM) with different concentrations are detected by using differential pulse voltammetry, the log value of the concentration is taken as an X axis, the corresponding current intensity value is taken as a Y axis, and a linear curve is prepared, namely a standard curve of the electrochemical signals is shown in FIG. 4.
Colorectal cancer tissue sections were processed to obtain immunohistochemistry maps, as shown in fig. 5.
The electrochemical signal (ferrocene) was co-incubated with a specific antibody (Ki 67 in this example) in acetonitrile in the presence of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to give an electrochemical signal modified antibody.
Wherein the concentrations of the electrochemical signal (ferrocene), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in acetonitrile are respectively 1mM and 20mM.20mM, and the antibody dilution concentration ratio is 1: 100.
adding the antibody modified by the electrochemical signal into a glass slide of a paraffin section tissue to be detected, incubating, combining antigen and antibody, removing the glass slide, detecting the electrochemical signal of the antibody which is not combined in the detection solution by using a differential pulse voltammetry, and comparing with a standard curve to obtain the content of the antigen in the sample to be detected. As shown in fig. 6.
Wherein, the standard curve of the electrochemical signal is prepared by the following method: and detecting electrochemical signals with different concentrations under a ternary battery method by using an electrochemical instrument, and making a linear curve, namely a standard curve of the electrochemical signals, by taking the log value of the concentration as an X axis and the corresponding current intensity value as a Y axis.
The general flow of this embodiment is shown in fig. 7.
Example 2
30mM HAuCl was taken4，0.1M Na2SO4The dendritic gold is obtained after electroplating by using a chronoamperometry, conductive high molecular monomers such as ethylene dioxythiophene, thiophene acetic acid, pyrrole and the like and tetrabutyl ammonium perchlorate are dissolved in anhydrous acetonitrile, nitrogen is introduced, air in the solution is removed, and the high molecular is electroplated on the surface of the dendritic gold by using a cyclic voltammetry. Adding ferrocene and specific antibody into solution containing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimideIn vivo co-culture, then immersing the slide of the frozen section tissue to be detected in the solution, co-incubating, removing the slide, and detecting the electrochemical signal of the unbound antibody in the solution, the process is shown in FIG. 7.
Example 3
30mM HAuCl was taken4，0.1M Na2SO4And electroplating the surface of the electrode by using a chronoamperometry to obtain the dendritic gold. Dissolving conductive polymer monomers such as ethylene dioxythiophene, thiophene acetic acid, pyrrole and tetrabutylammonium perchlorate in acetonitrile, introducing nitrogen, removing air in the solution, and electroplating conductive polymer on the surface of the dendritic gold by using cyclic voltammetry to obtain the electrode with the surface modified with the dendritic gold and the conductive polymer. And (3) co-incubating the electrochemical signal (ferrocene) with the specific antibody in acetonitrile in the presence of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to obtain the antibody modified by the electrochemical signal. Adding the antibody modified by the electrochemical signal into a cell to be detected, incubating, combining antigen and antibody, centrifuging to remove the unbound antibody, detecting the electrochemical signal on the surface of the cell by using a differential pulse voltammetry method, and comparing the electrochemical signal in the sample to be detected with a standard curve of the electrochemical signal to obtain the content of the antigen in the sample to be detected. The flow is shown in fig. 8.
Example 4
30mM HAuCl was taken4，0.1M Na2SO4The dendritic gold is obtained after electroplating by using a chronoamperometry, conductive high molecular monomers such as ethylene dioxythiophene, thiophene acetic acid, pyrrole and the like and tetrabutyl ammonium perchlorate are dissolved in anhydrous acetonitrile, nitrogen is introduced, air in the solution is removed, and the high molecular is electroplated on the surface of the dendritic gold by using a cyclic voltammetry. Adding A ' quantum dots and specific antibodies A into a solution containing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide for co-culture, adding B ' quantum dots and specific antibodies B into a solution containing EDC and NHS for co-culture, and adding C ' quantum dots and specific antibodies C into a solution containing EDC and NHS for co-culture. Subsequently adding to the cells to be detected, incubating, centrifuging to remove unboundAntibody, cell surface electrochemical signal was detected using DPV, as shown in figure 9.
Example 5
30mM HAuCl was taken4，0.1M Na2SO4The dendritic gold is obtained after electroplating by using a chronoamperometry, conductive high molecular monomers such as ethylene dioxythiophene, thiophene acetic acid, pyrrole and the like and tetrabutyl ammonium perchlorate are dissolved in anhydrous acetonitrile, nitrogen is introduced, air in the solution is removed, and the high molecular is electroplated on the surface of the dendritic gold by using a cyclic voltammetry. Adding A ' quantum dots to a solution containing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to perform coculture with a specific antibody A, adding B ' quantum dots to a solution containing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to perform coculture with a specific antibody B, and adding C ' quantum dots to a solution containing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to perform coculture with a specific antibody C. And then, immersing a slide of a paraffin section tissue to be detected in a solution, incubating, removing the slide, detecting an electrochemical signal of an unbound antibody in the solution, extracting a part of regional tissue by using a needle, adding the antibody, incubating, detecting an electrochemical signal value, converting the electrochemical signal values obtained from different regions into a heat map, and combining the heat map with a picture obtained by a hematoxylin-eosin staining method to realize spatial immunohistochemical electrochemical detection, wherein the obtained picture can be compared with immunohistochemistry, and the flow is shown in fig. 10.
Example 6
30mM HAuCl was taken4，0.1M Na2SO4And electroplating by using a chronoamperometry to obtain dendritic gold, dissolving the conductive polymer monomer and tetrabutylammonium perchlorate in anhydrous acetonitrile, introducing nitrogen, removing air in the solution, and electroplating the polymer on the surface of the dendritic gold by using a cyclic voltammetry. Adding A 'quantum dot and specific antibody A into a solution containing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide for co-culture, and adding B' quantum dot and specific antibody A into a solution containing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimideAnd (3) co-culturing the antibody B, namely adding a C' quantum dot into a solution containing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to co-culture the specific antibody C. And then, immersing a slide of a frozen section tissue to be detected in a solution, incubating, removing the slide, detecting an electrochemical signal of an unbound antibody in the solution as shown in figure 10, extracting a part of regional tissue by using a needle, adding the antibody, incubating, detecting an electrochemical signal value, converting the electrochemical signal values obtained from different regions into a heat map, combining the heat map with a picture obtained by a hematoxylin-eosin staining method to realize space immunohistochemical electrochemical detection, and comparing the obtained picture with immunohistochemistry, wherein the flow is shown in figure 10.
In the above embodiment, the tissue slice processing method and the electrochemical detection flowchart thereof are shown in fig. 11:
1. paraffin section (IHC staining)
a. Preparation of Paraffin section
1) Fresh tissue that had just been removed was placed in an embedding cassette and fixed in 4% paraformaldehyde solution.
2) Dewatering with automatic dewatering machine.
IHC staining
1) Dewaxing: xylene 1(25min) → xylene 2(25 min).
2) Hydration: gradient alcoholic hydration, anhydrous ethanol 1(15min) → anhydrous ethanol 2(10min) → 95% ethanol (5min) → 90% ethanol (5min) → 80% ethanol (5 min).
3) Antigen retrieval:
high-temperature high-pressure repairing method: adding appropriate amount of citrate buffer solution into pressure cooker, placing into glass slide, and repairing for 5-20 min.
4) Blocking endogenous peroxidase activity:
place the slide in preheated 3% H2O2-methanol solution for endogenous peroxidase elimination at 37 ℃ for 30 min.
5) Sealing of
Tissue sections were loaded with 10% BSA and placed in a wet box at 37 ℃ for 1 h.
2. Frozen section (IHC staining)
a. Frozen section
1) Taking out fresh tissue, dehydrating sucrose solution, and embedding the tissue with OCT embedding medium.
2) The slice thickness was adjusted to 7 μm for slicing.
IHC staining
1) Acetone fixation and hydration: the sections were first fixed with acetone and then briefly hydrated with PBS.
2) Antigen retrieval:
high-temperature high-pressure repairing method: adding appropriate amount of citrate buffer solution into pressure cooker, placing into glass slide, and repairing for 5-20 min.
3) Blocking endogenous peroxidase activity:
place the slide in preheated 3% H2O2-methanol solution for endogenous peroxidase elimination at 37 ℃ for 30 min.
4) Sealing of
Tissue sections were loaded with 10% BSA and placed in a wet box at 37 ℃ for 1 h.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (7)
1. A method for detecting the content of antigen in a sample based on immunohistochemical electrochemistry is characterized by comprising the following steps: modifying an electrochemical signal on an antibody, mixing the antibody modified by the electrochemical signal with a sample with the antigen content to be detected, combining the antigen and the antibody, detecting the electrochemical signal of the antibody combined with the antigen or detecting the electrochemical signal of the antibody not combined with the antigen by using an electrochemical instrument, comparing the detected electrochemical signal with a standard curve of the electrochemical signal, and calculating to obtain the antigen content in the sample to be detected;
when the sample with the antigen content to be detected is the tissue with the antigen content to be detected, mixing the tissue with the antigen content to be detected with the antibody modified by the electrochemical signal, incubating, combining the antigen and the antibody, removing the tissue with the antigen content to be detected, detecting the electrochemical signal of the antibody which is not combined by using a differential pulse voltammetry, comparing the measured electrochemical signal with a standard curve of the electrochemical signal, and calculating to obtain the antigen content in the tissue with the antigen content to be detected;
when the sample with the antigen content to be detected is the cell with the antigen content to be detected, mixing the cell with the antigen content to be detected with the antibody modified by the electrochemical signal, incubating together, combining the antigen and the antibody, centrifuging to remove the unbound antibody, detecting the electrochemical signal on the surface of the cell by using a differential pulse voltammetry, comparing the measured electrochemical signal with a standard curve of the electrochemical signal, and calculating to obtain the antigen content in the tissue with the antigen content to be detected;
when an electrochemical instrument is used for detecting electrochemical signals, the used electrode is an electrode with the surface modified with dendritic gold and conductive macromolecules.
2. The method for immunohistochemical-based electrochemical detection of antigen content in a sample according to claim 1,
when an electrochemical instrument is used for detecting an electrochemical signal of the antibody combined with the antigen, the detected electrochemical signal is compared with a standard curve of the electrochemical signal, the content of the antibody combined with the antigen is obtained through calculation, and the content of the antigen in the detection sample is obtained through calculation according to the content of the antibody combined with the antigen;
when an electrochemical instrument is used for detecting an electrochemical signal of the antibody which is not combined with the antigen, the detected electrochemical signal is compared with a standard curve of the electrochemical signal, the content of the antibody which is not combined with the antigen is calculated, the content of the antibody which is combined with the antigen is calculated according to the total amount of the antibody modified by the electrochemical signal before the antibody is mixed with the antigen sample to be detected, and the content of the antigen in the detected sample is calculated according to the content of the antibody which is combined with the antigen.
3. The method for detecting the antigen content in a sample based on the immunohistochemical electrochemistry according to claim 1, wherein the standard curve of the electrochemical signal is prepared by the following method: and detecting electrochemical signals with different concentrations under a ternary battery method by using an electrochemical instrument, and making a linear curve, namely a standard curve of the electrochemical signals, by taking the log value of the concentration as an X axis and the corresponding current value as a Y axis.
4. The method for the immunohistochemical-based electrochemical detection of the antigen content of the sample according to claim 1, wherein the simultaneous detection of the corresponding antigens can be achieved by modifying different antibodies with different electrochemical signals.
5. The method for immunohistochemical-based electrochemical detection of antigen content in a sample according to claim 1, wherein said electrochemical signal is selected from ferrocene or quantum dots selected from ZnS, CdS, CdTe or PbS.
6. A method for electrochemically detecting the content of an antigen in a sample based on an immunohistochemical space is characterized by comprising the following steps of:
a. modifying an electrochemical signal to the antibody;
b. adding the antibody modified by the electrochemical signal into the tissue to be detected, incubating, and combining the antigen and the antibody;
c. removing tissues with the antigen content to be detected, and detecting the electrochemical signals of the unbound antibodies by using a differential pulse voltammetry method;
d. comparing the measured electrochemical signal with a standard curve of the electrochemical signal, and calculating to obtain the content of the antigen in the tissue of which the content of the antigen is to be measured;
e. extracting other partial region tissues, and repeating the detection method to obtain electrochemical signal values of different region tissues;
f. converting electrochemical signal values obtained from different areas into a heat map, and combining the heat map with an HE (hematoxylin-eosin) map to realize space immunohistochemical electrochemical detection;
when an electrochemical instrument is used for detecting electrochemical signals, the used electrode is an electrode with the surface modified with dendritic gold and conductive macromolecules.
7. The method for spatial electrochemical detection of antigen content in a sample based on immunohistochemistry according to claim 6, wherein in step f, the electrochemical signal values obtained from different regions are converted into heat maps, which are conventional methods for spatial detection, and the amount of antigen in different regions of tissue is visually expressed according to the change of color of the dots in the heat maps, and the darker the color of the dots in the heat maps where the antigen is highly expressed is, the lighter the color of the dots in the heat maps where the antigen is less expressed is.
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