CN113884481B - Dry bipolar electrochemical luminescence chip and application thereof in immunodetection - Google Patents
Dry bipolar electrochemical luminescence chip and application thereof in immunodetection Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
Abstract
The invention discloses a dry bipolar electrochemiluminescence chip and application thereof in immunodetection, wherein the chip comprises a bottom plate, an electrode plate, a detection sheet, a combination sheet and a sample adding sheet; the electrode plate comprises an integrated bipolar electrode and a driving electrode, the integrated bipolar electrode is provided with two anodes distributed in a Y shape, and one bipolar electrode anode is correspondingly placed with the driving electrode and is called a detection anode; the electrode plate is folded between the report channel and the support channel; hydrophilic channels distributed in a Y shape are designed on the detection sheet; the detection sheet is stacked on the electrode sheet, so that the hydrophilic channel is overlapped with the anode of the integrated bipolar electrode; the hydrophilic channel overlapping the detection anode on the electrode plate serves as a detection zone. The patent firstly invents a dry-type immune CBP-ECL chip, and the chip overcomes the defects of complex operation and multi-step solution addition of the existing immune CBP-ECL chip by integrating reaction reagent components on the chip.
Description
Technical Field
The invention belongs to the field of design and detection of microfluidic chips, and particularly relates to a dry bipolar electrochemical luminescence chip and application thereof in immunodetection.
Background
In recent years, due to the characteristics that a bipolar electrode (BPE) has a simple structure and is convenient to manufacture, and can simultaneously perform an oxidation-reduction reaction on two electrodes of the BPE under the trigger of an external electric field, the BPE has been successfully applied to the aspects of electrochemical synthesis, electrochemical luminescence sensing and the like, and becomes a research hotspot.
BPEs are classified into open BPEs (O-BPEs) and closed BPEs (C-BPEs). The cathode and the anode of the O-BPE are in the same micro-channel, and the reactions at the two poles are easy to influence each other. In addition, the current not only flows through the BPE, but also through the solution, the current efficiency is greatly reduced, and the required driving voltage is large. Different from O-BPE, the cathode and the anode of C-BPE are respectively positioned in different reaction solutions, thereby effectively solving the defect of O-BPE, not only avoiding the cross contamination of different reaction solutions, but also having the current efficiency of almost 100 percent and greatly expanding the application range of the C-BPE.
Bipolar electrochemiluminescence (BPE-ECL) is a combination of BPE and electrochemiluminescence, which offers the potential for further development in biomarker hypersensitivity detection in biochemical analysis. The BPE-ECL has the obvious advantages of simple operation, low background signal, no use of radioactive isotope, realization of high-throughput analysis and the like, and gradually becomes an effective detection means of various biomarkers. Among them, the microfluidic chip combining C-BPE and ECL (CBP-ECL) has good analytical performance, and is successfully applied to detecting small molecules, proteins, cells and the like, but still faces some problems. For example, the chip material is generally an inorganic material or a polymer material, and the electrode material is expensive; the processing equipment involved with the chip is expensive; the chip modification process is complicated; is not easy to use quickly and conveniently. These all greatly limit the application scenarios of the CBP-ECL microfluidic chip.
Dry chemical analysis is a broad class of analytical detection techniques based on wet chemical analysis. More and more detection technologies are applied to the field of dry chemical detection, for example, immunochromatography is an immunoreagent dry sheet based on specific binding of antigen and antibody, such as enzyme label, fluorescent label, gold nanoparticle label, and the like. At present, the immunochromatography technology in the market has the advantages of low price, convenient use, rapid detection, no need of expensive instruments and the like, and is a conventional in-vitro diagnosis method. The technology can effectively overcome the defects of the traditional CBP-ECL microfluidic chip, but is generally difficult to quantitatively detect. The detection of some biomarkers (cardiac troponin, creatine kinase isoenzyme, myoglobin, etc.) still does not meet the need for quantitative detection.
In conclusion, the novel, rapid, simple and convenient microfluidic chip is suitable for hospitals and families, and can effectively overcome the defects of the traditional CBP-ECL and immunochromatographic test strip, and the technical problem to be solved by technical personnel in the field is urgently needed.
Disclosure of Invention
The invention aims to provide a dry bipolar electrochemiluminescence chip and application thereof in immunoassay, the chip integrates reaction reagent components on the chip, and can realize dry quantitative detection of biomarkers; the detection method is rapid, convenient to operate and high in sensitivity.
The purpose of the invention is realized by the following technical scheme:
a dry-type bipolar electrochemical luminescence chip comprises a bottom plate, an electrode plate, a detection sheet, a combination sheet and a sample adding sheet, wherein the electrode plate, the detection sheet, the combination sheet and the sample adding sheet are stacked on the bottom plate;
the electrode plate comprises an integrated bipolar electrode and a driving electrode, the anode of the bipolar electrode and the corresponding driving electrode are positioned in the report channel, and the cathode of the bipolar electrode and the corresponding driving electrode are positioned in the support channel; the electrode plate is used for being communicated with an external circuit to trigger CBP-ECL reaction, and the report channel is used for absorbing redundant reaction solution on the detection plate on one hand and can be used for being communicated with a CBP current loop on the other hand;
the integrated bipolar electrode is provided with two anodes distributed in a Y shape, wherein one bipolar electrode anode is correspondingly placed with the driving electrode and is called a detection anode; the other bipolar electrode anode is independently placed and is called a quality control anode;
the two bipolar electrode anodes are symmetrically distributed and have the same length;
the electrode plate is folded between the report channel and the support channel, after the electrode plate is folded, the report channel faces upwards (namely faces to the detection plate), and the support channel faces downwards (namely faces to the bottom plate); after being folded, the two channels are basically overlapped but not communicated;
the electrode plate is a paper-based or cotton-based substrate;
the length of the reporting channel on the electrode sheet is 9-11mm, preferably 10 mm.
Hydrophilic channels distributed in a Y shape are designed on the detection sheet; the detection sheet is stacked on the electrode sheet, so that the hydrophilic channel is overlapped with the anode of the integrated bipolar electrode; the hydrophilic channel overlapped with the detection anode on the electrode plate is used as a detection area (T area), and the hydrophilic channel overlapped with the quality control anode on the electrode plate is used as a quality control area (C area); the region T is used for detecting the biomarker, and the region C plays a role in controlling the quality of the chip; the significance of the design is that after the electrode slice is communicated with an external power supply, the detection anode and the quality control anode of the electrode slice can electrically trigger ECL luminescent markers on the T area and the C area of the corresponding detection slice; meanwhile, the parallel distribution of the T area and the C area of the detection piece can overcome the problems encountered by the front-back distribution of the T line and the C line of the traditional test strip.
The combination piece and the sample adding piece are provided with hydrophilic channels which are communicated with the hydrophilic channels on the detection piece;
the bottom plate is provided with an opening at the position corresponding to the electrode plate supporting channel, so that buffer solution can be conveniently added into the supporting channel.
A labeled antibody which is used for modifying and coupling ECL luminescent substances and corresponds to the biomarkers is fixed on the hydrophilic channel of the binding sheet, and the dosage of the labeled antibody is 6-8 mu L, preferably 6 mu L;
the ECL luminescent material is preferably a terpyridyl ruthenium derivative.
Fixing a modified biomarker coated antibody in a detection area of the detection sheet; preferably, the concentration of the coating antibody of the biomarker is 60-100. mu.g/mL, particularly preferably 100. mu.g/mL.
The quality control region is fixed with a modified quality control region coating antibody (such as goat anti-chicken IgY), and the coating antibody is a secondary antibody irrelevant to the biomarker and plays a role in quality control.
The bipolar electrode cathode of the electrode plate can promote the electron transfer efficiency and provide an efficient surface area by modifying the nano material, thereby providing a stronger ECL signal and higher detection sensitivity;
the nano material can be graphene quantum dot-gold nanoparticle compound (GQD-AuNPs), multi-walled carbon nanotube or gold nanoparticle and the like;
preferably, the modified Chitosan (CS) is firstly dripped on the bipolar electrode cathode of the electrode plate, and then the modified graphene quantum dot-gold nanoparticle composite (GQD-AuNPs) is dripped; further preferably, the amount of GQDs-AuNPs to be applied by drop coating is 2 to 6. mu.L, particularly preferably 6. mu.L.
The dry bipolar electrochemiluminescence chip can be used for carrying out immunodetection on the biomarker;
the immunoassay comprises the following steps:
dripping a sample solution to be detected containing a specific biomarker on the sample adding sheet of the chip, wherein the solution flows through the binding sheet and is bound with a corresponding labeled antibody of the biomarker on the binding sheet; further flowing to a detection sheet, and combining with the biomarker coated antibody on the detection sheet to form a 'labeled antibody-biomarker-coated antibody' immune sandwich type compound; after the immune reaction of the biological marker is finished, dropping buffer solution into the sample adding sheet, on one hand, filling the report channel with solution, and on the other hand, removing the marker antibody which is not combined in the detection area; subsequently, buffer solution is added into the supporting channel of the electrode plate; finally, a driving electrode of the electrode plate is communicated with an external direct-current power supply to trigger a CBP-ECL reaction, and an ECL signal is acquired by a CCD camera so as to perform qualitative and quantitative detection on the biological marker;
the driving voltage for the CBP-ECL reaction is 6.5-8.5V, preferably 8.5V.
The incubation time of the immune reaction is 3-5min, preferably 3 min.
The biomarker is one of cardiac troponin I (cTn I), C-reactive protein (CRP), alanine Aminotransferase (ALT), Ig E, carcinoembryonic antigen (CEA), prostate antigen (PSA) or Bovine Serum Albumin (BSA);
the buffer was Phosphate Buffered Saline (PBS).
Compared with the prior art, the invention has the following advantages and effects:
1. the patent firstly invents a dry-type and immune CBP-ECL chip, and the chip overcomes the defects of complex operation and multi-step solution addition of the existing immune CBP-ECL chip by integrating reaction reagent components on the chip.
2. The chip of the invention takes paper and cotton cloth as main substrate materials, the two materials have good flexibility, no pollution and low cost, the problem of high cost of glass fiber, NC membrane and the like in the market is solved, and the practicability and universality of the chip are further improved.
3. The present invention uses an intramolecular co-reactant to excite ECL signals. Compared with the existing CBP-ECL, the chip provided by the invention does not need to drop the co-reaction reagent again when in use, so that the influence of the micro-environment of the co-reaction reagent solution is avoided, the ECL detection is more accurate, and the operation is simpler.
4. The invention realizes quantitative detection and even hypersensitive detection of biomarkers by two signal amplification modes of intramolecular self-enhanced ECL luminescent markers and modification of C-BPE cathode nano materials, and effectively overcomes the defect that the traditional immunochromatography test strip is difficult to quantify generally.
5. The chip is simple and convenient to operate, can be used for instrument analysis only by dripping the solution of the sample to be detected and the buffer solution, does not need to be operated by professional personnel, and is suitable for on-site instant detection.
6. The invention takes about 7min from sample adding to signal acquisition, and can realize rapid quantitative detection.
7. The dry-type immune CBP-ECL chip can realize quantitative detection of different biomarkers essentially, and has important significance in early diagnosis and prevention of cardiovascular diseases, cancers and the like.
8. The chip manufacturing method is simple, environment-friendly, low in cost and easy to realize batch production.
Drawings
FIG. 1 is a schematic diagram (front side) of the chip of the present invention;
FIG. 2 is a schematic diagram of the backside of a chip according to the present invention;
FIG. 3 is an exploded view of the chip of the present invention;
FIG. 4 is a schematic view of the structure of the electrode plate of the chip of the present invention;
the device comprises a sample adding sheet 1, a binding sheet 2, a detection sheet 3, an electrode sheet 4, a bottom plate 5, a bottom plate opening 6, a driving electrode 7, a hydrophobic wax dam 8, a bipolar electrode anode 9, a reporting channel 10, a folding line 11, a bipolar electrode cathode 12 and a supporting channel 13.
FIG. 5 is a graph of ECL intensity values versus reporting channel length.
FIG. 6 is a graph of ECL intensity values versus driving voltage.
FIG. 7 is a graph of ECL intensity values as a function of labeled antibody amount.
FIG. 8 is a graph of ECL intensity values versus concentration of coated antibody.
FIG. 9 is a graph of ECL intensity values versus incubation time.
FIG. 10 is a graph of ECL intensity values as a function of quantum dot usage on a C-BPE cathode.
Fig. 11 is an analysis graph for detecting different concentrations of cTn I (the inset is a linear fit graph of the data).
FIG. 12 is a selectivity assessment for the detection of cTn I.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
A dry bipolar electrochemical luminescence chip is shown in figures 1-4, and comprises a sample adding sheet 1, a combination sheet 2, a detection sheet 3, an electrode sheet 4 and a bottom plate 5.
The electrode plate 4 (figure 4) comprises an integrated bipolar electrode and a driving electrode 7, a bipolar electrode anode 9 and a corresponding driving electrode are positioned in a report channel 10, and a bipolar electrode cathode 12 and a corresponding driving electrode are positioned in a support channel 13; the electrode plate is used for being communicated with an external circuit to trigger CBP-ECL reaction, and the report channel is used for absorbing redundant reaction solution on the detection plate on one hand and can be used for being communicated with a CBP current loop on the other hand;
the integrated bipolar electrode is provided with two anodes 9 distributed in a Y shape, wherein one bipolar electrode anode is correspondingly placed with the driving electrode and is called a detection anode; the other bipolar electrode anode is called a quality control anode; the two bipolar electrode anodes are symmetrically distributed in parallel and have the same length;
the electrode plate 4 is folded between the report channel 10 and the support channel 13, after the folding, the report channel 10 faces upwards (i.e. faces the detection sheet), and the support channel 13 faces downwards (i.e. faces the bottom plate); after being folded, the two channels are basically overlapped but not communicated; the electrode plate 4 is a folding cloth-based C-BPE, the cloth-based carrier adopts cotton cloth, and the cloth-based carrier is bonded by hydrophobic and sticky double-sided adhesive during folding.
The C-BPE cathode 12 is modified by dropping 2.5mg/mL CS with adhesion on the surface, and then dropping 1mg/mL GQD-AuNPs (prepared by Biosensors and Bioelectronics,2019,130: 55-64).
The detection sheet 3 is provided with a detection area (T area) and a quality control area (C area) which are surrounded by a hydrophobic wax dam and are distributed in parallel, and the detection sheet 3 is overlapped on the electrode plate 4, so that the hydrophilic channel is overlapped with the anode 9 of the integrated bipolar electrode; the hydrophilic channel overlapped with the detection anode on the electrode plate is used as a detection area (T area), and the hydrophilic channel overlapped with the quality control anode on the electrode plate is used as a quality control area (C area); the T region and the C region of the detection sheet 3 are respectively modified and fixed with 0.1mg/mL coating antibody (Product Code: D4160MA01-MA, OriGene Company) of cardiac troponin I (cTn I) and 0.1mg/mL coating antibody of quality control region (goat anti-chicken IgY, B108, Kittai Biotech, Hangzhou) by means of chemical bond covalent bonding.
The combination piece 2 and the sample adding piece 1 are composed of a hydrophilic reaction channel surrounded by a hydrophobic wax dam, wherein two ends of the hydrophilic channel of the combination piece 2 are respectively communicated with the sample adding piece 1 and the detection piece 3. As shown in fig. 3, the entire chip is formed by sequentially stacking a base plate 5, an electrode sheet 4, a detection sheet 3, a bonding sheet 2, and a sample application sheet 1.
The hydrophilic channel of the bonding sheet 2 is dripped with 0.1mg/mL solution of a cardiac troponin I (cTn I) labeled antibody (Product Code: B9085MA06-MA, OriGene Company) coupled with a terpyridyl ruthenium derivative (refer to Talanta,2014,129: 219-226), and dried at 37 ℃ in a vacuum environment;
the bottom plate 5 is a PVC thin plate with glue on one side, and the sample sheet 1, the combination sheet 2, the detection sheet 3 and the electrode sheet 4 are superposed on the upper layer of the bottom plate; the bottom plate 5 is a substrate for stacking, fixing and supporting the chip; the bottom plate is provided with an opening 6 at the position corresponding to the electrode plate supporting channel, so that buffer solution can be conveniently added into the supporting channel.
The electrode plate 4 is used for being communicated with an external circuit to trigger CBP-ECL reaction, and the anode report channel 10 is used for absorbing redundant reaction solution on the detection sheet 3 on one hand and can be used for being communicated with a CBP current loop on the other hand; the T area and the C area on the detection sheet 3 are respectively used for modifying the coating antibody for fixing the biomarker and the coating antibody for the quality control area so as to monitor the concentration of the biomarker and the feasibility of the chip; the binding sheet 2 is used for drying the labeled antibody labeled with the ECL luminescent substance, one end of the binding sheet is communicated with the detection sheet 3, and the other end of the binding sheet is communicated with the sample adding sheet 1, so that a good sample flow channel to be detected is constructed; the sample adding sheet 1 is used for dropwise adding a sample to be detected, can carry out primary filtration on the sample to be detected, quickly absorbs the sample solution to be detected and flows to the combining sheet 2.
Example 2
The manufacturing method of the dry bipolar electrochemical luminescence chip comprises the following steps:
1) configuration design: the configuration of the sample application sheet 1, the binding sheet 2, the detection sheet 3 and the electrode sheet 4 is designed by using Adobe Illustrator CS6 drawing software, so as to customize the 300-mesh polyester micro-channel mesh plate and the electrode mesh plate.
The sample adding sheet, the combination sheet, the detection sheet and the electrode sheet can be made of paper or cloth fiber materials.
2) Chip printing: the front hydrophobic wax dam of the sample adding sheet 1, the combination sheet 2 and the detection sheet 3 is manufactured by printing a micro-channel screen plate by using a wax screen printer (such as CN 112192941A). The electrode sheet 4 was made by printing an electrode screen plate on the front side of the C-BPE and the driving electrode using a screen printer (model CW 3050, available from doguan caiwei printing machinery), and the hydrophobic wax dam 8 on the back side was made by the above-mentioned wax screen printer.
The electrode sheet 4 is folded inward between the report channel 10 and the support channel 13, and is bonded by a hydrophobic and adhesive double-sided adhesive so that the report channel and the support channel are overlapped with each other while not communicating with each other, thereby constructing the electrode sheet 4 of a folded cloth chip.
3) Chip decoration
After the binding sheet 2 is pretreated, the labeled antibody is dripped into the hydrophilic channel of the binding sheet 2, and vacuum drying is carried out for 1h at 37 ℃.
And the T area and the C area of the detection sheet 3 respectively modify and fix the coating antibody of the biomarker and the coating antibody of the quality control area in a CS-glutaraldehyde chemical bond covalent bonding mode.
The C-BPE cathode is modified, CS with adhesiveness is firstly dripped on the surface of the C-BPE cathode, and then GQD-AuNPs are dripped on the surface of the C-BPE cathode.
4) Chip assembly
As shown in FIG. 3, the entire chip is formed by stacking a base plate 5, an electrode sheet 4, a detection sheet 3, a bonding sheet 2, and a sample application sheet 1 in this order, and the stacking positions of the respective portions are overlapped by 2 mm.
Example 3
The application of the dry bipolar electrochemiluminescence chip in the detection of cTn I is as follows:
1) dropwise adding a solution containing cTn I to be detected on the sample adding sheet 1, enabling the solution to rapidly flow through the binding sheet 2 and be bound with the labeled antibody on the binding sheet, further flowing to the detection sheet 3, and being bound with the specific affinity coating antibody on the detection sheet to form a labeled antibody-cTn I-coating antibody immune sandwich type compound;
2) after waiting for several minutes, after the immunoreaction of the sample solution to be detected is finished, a buffer solution is dripped into the sample adding sheet, so that the report channel 10 is filled with the solution on one hand, and the unbound labeled antibody in the detection area is removed on the other hand. Subsequently, a buffer solution is added to the support channel 13 of the electrode sheet 4.
3) The driving electrode 7 of the electrode plate 4 is connected to a set output direct current power supply through a lead of the alligator clip, and ECL reaction can be triggered by electrifying;
4) putting the chip into a dark box, setting parameters of a CCD camera, aligning a lens to a detection sheet area of the chip, and displaying a clear image at a computer end; the CCD automatic photographing function and the direct-current power switch are sequentially started, the ECL reaction is triggered, and the CCD collects images;
5) the image collected by the CCD is further analyzed by an image automatic processing program of MatLab software at the computer end, so that the average gray value of the image is obtained; the average gray value of the picture is multiplied by the pixel points of the light-emitting area to obtain the number of photons of the light-emitting area, the obtained data is imported into Origin software for further processing and analysis, and the data relation between the ECL intensity value and a certain parameter can be obtained (each data point is obtained by adopting 5 times of repeated experiments).
Taking a sample solution to be tested containing 34ng/mL cTn I and the chip of the invention as an example, the relationship between the report channel length and the ECL luminescence intensity value when the driving voltage is 6.5V is tested.
The test results are shown in fig. 5, and it can be seen that: when the report channel width was fixed at 14mm, the ECL luminescence intensity value gradually increased as the report channel length was changed from 17mm to 10mm, and when the length was further decreased to 9mm, the luminescence intensity value decreased. Therefore, an acceptable range for the length of the reporting channel is 9-11mm, preferably 10 mm.
Example 4
Several important factors (driving voltage, labeled antibody dosage, coated antibody concentration, incubation time (i.e., immunoreaction time), GQDs-AuNPs dosage) affecting the ECL intensity values in example 3 were optimized:
a) preferred drive voltage
1. The concentration of cTn I to be detected is 34ng/mL, the length of a report channel is 10mm, the driving voltage is undetermined, the dosage of the labeled antibody is 5 muL, the concentration of the coated antibody is 100 mug/mL, the incubation time is 3min, and the dosage of GQDs-AuNPs is 5 muL.
2. Several experimental groups were set up: the drive voltage is set to several different values (5V, 6V, 6.5V, 7V, 8V, 8.5V, 9V).
3. Chip testing procedure example 3, the test results are shown in fig. 6.
As can be seen from the experimental results, the ECL intensity value gradually increases as the driving voltage increases from 5V to 8.5V; when the driving voltage was further increased to 9V, the ECL intensity value tended to decrease. Possible causes of the reduction in ECL intensity values are: background reactions (e.g., oxidation of water) occur at high drive voltages, which chemically and physically interfere with ECL emission. Therefore, an acceptable range of the driving voltage is 6.5-8.5V, preferably 8.5V.
b) Preferably the amount of labeled antibody used
1. The concentration of cTn I to be detected is 34ng/mL, the length of a report channel is 10mm, the driving voltage is 8.5V, the dosage of the labeled antibody is undetermined, the concentration of the coating antibody is 100 mu g/mL, the incubation time is 3min, and the dosage of GQDs-AuNPs is 5 mu L.
2. Several experimental groups were set up: the volume of the amount of labeled antibody was set to several different values (4. mu.L, 5. mu.L, 5.5. mu.L, 6. mu.L, 7. mu.L, 8. mu.L).
3. Chip test procedure example 3, the test results are shown in fig. 7.
As can be seen from the experimental results, the ECL intensity value gradually increased as the labeled antibody volume increased from 4. mu.L to 6. mu.L; as the labeled antibody volume was further increased to 8 μ L, the ECL intensity values increased slowly. Possible causes of this phenomenon are: at a certain target concentration, the number of molecules of the labeled antibody gradually saturates. Therefore, an acceptable range for the volume of labeled antibody is 6-8. mu.L, preferably 6. mu.L.
c) Preferred concentration of coating antibody
1. The concentration of cTn I to be detected is 34ng/mL, the length of a report channel is 10mm, the driving voltage is 8.5V, the dosage of the labeled antibody is 6 muL, the concentration of the coated antibody is undetermined, the incubation time is 3min, and the dosage of GQDs-AuNPs is 5 muL.
2. Several experimental groups were set up: the concentration of the coating antibody was set at several different values (40. mu.g/mL, 60. mu.g/mL, 80. mu.g/mL, 100. mu.g/mL, 120. mu.g/mL).
3. Chip testing procedure example 3, the test results are shown in fig. 8.
As can be seen from the experimental results, the ECL intensity value gradually increased as the concentration of the coated antibody increased from 40. mu.g/mL to 100. mu.g/mL; when the concentration of the coating antibody was further 120. mu.g/mL, the ECL intensity value tended to decrease. Possible causes of this phenomenon are: too many coated antibody molecules block electron transfer between the ECL luminescent reagent and the electrode, thereby reducing ECL signal. Therefore, the acceptable range of the concentration of the coating antibody is 60 to 100. mu.g/mL, preferably 100. mu.g/mL.
d) Preferred incubation time
1. The concentration of cTn I to be detected is 34ng/mL, the length of a report channel is 10mm, the driving voltage is 8.5V, the volume of the labeled antibody is 6 muL, the concentration of the coated antibody is 100 mug/mL, the incubation time is to be determined, and the dosage of GQDs-AuNPs is 5 muL.
2. Several experimental groups were set up: the immunoreaction incubation time was set at several different values (0min, 1min, 2min, 3min, 4min, 5 min).
3. Chip testing procedure example 3, the test results are shown in fig. 9.
From the experimental results, it can be seen that the ECL intensity value gradually increases when the immunoreaction incubation time increases from 0min to 3 min; the ECL intensity values gradually subsided as the incubation time increased from 3min to 5 min. Possible causes of this phenomenon are: the sandwich complex of "labeled antibody-cTn I-coated antibody" is no longer produced after the immune reaction is completed, resulting in almost no increase in ECL intensity values. Therefore, the acceptable range of the incubation time for the immunoreaction is 3-5min, preferably 3 min.
e) The preferred dosage of GQDs-AuNPs
1. The concentration of cTn I to be detected is 3.4ng/mL, the length of a report channel is 10mm, the driving voltage is 8.5V, the volume of a labeled antibody is 6 mu L, the concentration of a coated antibody is 100 mu g/mL, the incubation time is 3min, and the dosage of GQDs-AuNPs is undetermined.
2. Several experimental groups were set up: the volume of quantum dot dose was set to several different values (0. mu.L, 2. mu.L, 4. mu.L, 6. mu.L, 8. mu.L, 10. mu.L).
3. Chip test procedure example 3, the test results are shown in fig. 10.
As can be seen from the experimental results, the ECL intensity value gradually increases when the volume of GQDs-AuNPs increases from 0 μ L to 6 μ L; the ECL intensity values gradually decreased as the GQDs-AuNPs volume increased from 6. mu.L to 10. mu.L. Possible causes of this phenomenon are: the excessive GQDs-AuNPs can be seriously gathered on the surface of the electrode, so that the electrocatalysis of the GQDs-AuNPs and the binding capacity of the GQDs-AuNPs on the electrode are hindered, and the ECL strength value is reduced. Therefore, the acceptable volume range of GQDs-AuNPs is 2-6. mu.L, preferably 6. mu.L.
Example 5
cTn I detection was performed using the chip of example 1 under the optimized conditions found in example 4
The length of a report channel is 10mm, the driving voltage is 8.5V, the dosage of the labeled antibody is 6 mu L, the concentration of the coated antibody is 100 mu g/mL, the incubation time is 3min, and the dosage of GQDs-AuNPs is 6 mu L.
Several experimental groups were set up: cTn I concentrations were set at several different values (0ng/mL, 0.001ng/mL, 0.01ng/mL, 0.1ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 25ng/mL, 50ng/mL, 100 ng/mL).
The chip testing process is the same as example 3, and the test results are shown in FIG. 11.
As can be seen from the experimental results, the ECL intensity values increased with increasing cTn I concentration when the cTn I concentration was in the range of 0.001-100 ng/mL. The ECL intensity value (expressed by Y) and the cTn I concentration logarithm value (expressed by X) have good linear relation, and the linear equation can be expressed as Y-20.695X +74.408(R20.9925, n 5), detection limit is estimated to be 0.4427 pg/mL.
The calculation method of the detection limit comprises the following steps: y isL＝Yb+3SbWherein Y isbRepresents the mean ECL intensity value, S, for the blank controlbStandard deviations on blank control (5 replicates) were used to determine YLAnd calculating the corresponding cTn I concentration as the detection limit.
Example 6
Selective experiments for chip cTn I detection with optimized conditions found in example 4
A plurality of interference experimental groups are set: cTn I, C-reactive protein (CRP), alanine Aminotransferase (ALT), Ig E, carcinoembryonic antigen (CEA), prostate antigen (PSA), Bovine Serum Albumin (BSA), pooled samples (Mix, pooled of the first 7 proteins) and Blank control (Blank) (PBS buffer), with cTn I at a concentration of 1ng/mL and other interfering proteins at a concentration of 10 ng/mL.
The chip testing process is the same as that of example 3, and the test results are shown in FIG. 12.
From the experimental results, it can be seen that the reduction of ECL intensity values is very significant in CRP, ALT, Ig E, CEA, PSA, BSA interference experimental group compared to cTn I group, and is not much different from blank control. Furthermore, the ECL intensity values in the case of a mixture of cTn I and six interfering substances were almost identical to those of the cTn I group. Therefore, the chip can realize good detection of cTn I.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A dry-type bipolar electrochemical luminescence chip is characterized by comprising a bottom plate, an electrode plate, a detection sheet, a combination sheet and a sample adding sheet, wherein the electrode plate, the detection sheet, the combination sheet and the sample adding sheet are sequentially stacked on the bottom plate;
the electrode plate comprises an integrated bipolar electrode and a driving electrode, the anode of the bipolar electrode and the corresponding driving electrode are positioned in the report channel, and the cathode of the bipolar electrode and the corresponding driving electrode are positioned in the support channel;
the integrated bipolar electrode is provided with two anodes distributed in a Y shape, wherein one bipolar electrode anode is correspondingly placed with the driving electrode and is called a detection anode; the other bipolar electrode anode is independently placed and is called a quality control anode;
the electrode plate is folded between the report channel and the support channel; after folding, the report channel faces upwards, the support channel faces downwards, and the two channels are overlapped but not communicated;
hydrophilic channels distributed in a Y shape are designed on the detection sheet; the detection sheet is stacked on the electrode sheet, so that the hydrophilic channel is overlapped with the anode of the integrated bipolar electrode; the hydrophilic channel overlapped with the detection anode on the electrode plate is used as a detection area, and the hydrophilic channel overlapped with the quality control anode on the electrode plate is used as a quality control area;
the combination piece and the sample adding piece are provided with hydrophilic channels which are communicated with the hydrophilic channels on the detection piece;
a labeled antibody which is coupled with ECL luminescent substances and corresponds to a biomarker is fixed and modified on the hydrophilic channel of the binding sheet;
fixing a modified biomarker coated antibody in a detection area of the detection sheet; the quality control region is fixed with a modified quality control region coated antibody.
2. The chip of claim 1, wherein: the bottom plate is provided with an opening at the position corresponding to the electrode plate supporting channel.
3. The chip of claim 1, wherein: the two bipolar electrode anodes of the integrated bipolar electrode are symmetrically distributed and have the same length.
4. The chip of claim 1, wherein: the bipolar electrode cathode of the electrode plate is used for modifying the nano material.
5. The chip of claim 1, wherein: the electrode slice is a paper base or cotton cloth base substrate.
6. The chip of claim 1, wherein:
the length of the reporting channel on the electrode sheet is 9-11 mm;
the dosage of the labeled antibody which is coupled with the ECL luminescent substance and corresponds to the biomarker is 6-8 mu L;
the concentration of the biomarker-coated antibody is 60-100. mu.g/mL.
7. Use of the dry bipolar electrochemiluminescence chip according to any of claims 1 to 6 for immunodetection of a biomarker.
8. Use according to claim 7, characterized in that it comprises the following steps:
dripping a sample solution to be detected containing a biomarker on a sample adding sheet of the chip, and dripping a buffer solution on the sample adding sheet after the immune reaction of the biomarker is finished; subsequently, buffer solution is added into the supporting channel of the electrode plate; and finally, a driving electrode of the electrode plate is communicated with an external direct-current power supply to trigger a CBP-ECL reaction, and an ECL signal is acquired by a CCD camera so as to perform qualitative and quantitative detection on the biological marker.
9. Use according to claim 7, characterized in that: the biomarker is one of cardiac troponin I, C-reactive protein, glutamic-pyruvic transaminase, Ig E, carcinoembryonic antigen, prostate antigen or bovine serum albumin.
10. Use according to claim 8, characterized in that:
the incubation time of the immune reaction is 3-5 min;
the buffer solution is phosphate buffer solution;
the driving voltage of the CBP-ECL reaction is 6.5-8.5V.
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