CN114965447A - Full-automatic dry immune closed bipolar electrochemical luminescence analyzer and application thereof in immunoassay - Google Patents
Full-automatic dry immune closed bipolar electrochemical luminescence analyzer and application thereof in immunoassay Download PDFInfo
- Publication number
- CN114965447A CN114965447A CN202210518635.5A CN202210518635A CN114965447A CN 114965447 A CN114965447 A CN 114965447A CN 202210518635 A CN202210518635 A CN 202210518635A CN 114965447 A CN114965447 A CN 114965447A
- Authority
- CN
- China
- Prior art keywords
- closed bipolar
- sample
- sheet
- detection
- electrochemical luminescence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/5302—Apparatus specially adapted for immunological test procedures
- G01N33/5304—Reaction vessels, e.g. agglutination plates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/532—Production of labelled immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Plasma & Fusion (AREA)
- Electrochemistry (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a full-automatic dry-type immune closed bipolar electrochemical luminescence analyzer, which comprises a dry-type immune closed bipolar electrochemical luminescence chip and a closed bipolar electrochemical luminescence analyzer, wherein the dry-type immune closed bipolar electrochemical luminescence chip comprises a shell and a fiber micro-fluidic chip, the shell comprises an upper cover and a lower cover, the upper cover is provided with a sample adding hole, a buffer solution adding hole, an observation window and an electrode contact area, and the fiber micro-fluidic chip is arranged between the upper cover and the lower cover; the fiber microfluidic 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 a closed bipolar electrode and a pair of driving electrodes, the closed bipolar electrode comprises two anodes and a shared cathode, a report channel and a support channel are arranged on the electrode plate, the two anodes and the corresponding driving electrodes are located in the report channel, the shared cathode and the corresponding driving electrodes are located in the support channel, and the electrode plate does not need to be folded.
Description
Technical Field
The invention relates to the technical field of medical detection devices, in particular to a full-automatic dry immune closed bipolar electrochemiluminescence analyzer and application thereof in immune detection.
Background
In recent years, bioanalytical techniques have been rapidly developed in various fields. The principle of the method is that under the condition of not influencing the activity of an antibody (antigen), electrochemical luminescence probes such as terpyridyl ruthenium and the like are used as markers, and after the probes are combined with the corresponding antigen (antibody), specific electrochemical luminescence reaction is initiated under electric trigger, so that the detection result is stable and reliable, and the accuracy and precision are superior to those of the enzyme-linked immunosorbent assay technology. The immune electrochemical luminescence technology shows good application prospects in the aspects of medical detection, food analysis, environmental detection and the like by virtue of strong specificity, high sensitivity, wide detection range and the like, and gradually becomes one of mainstream detection methods.
The microfluidic chip technology has a great application prospect in the aspect of immune electrochemical luminescence analysis, and gradually becomes a novel analysis and detection platform. The technology has the advantages of high flux, easy operation, low cost, realization of instant detection and the like, and has attracted the wide research interest of people. At present, many researches are dedicated to combine a certain detection method with a microfluidic chip technology to develop a detection technology based on the microfluidic chip with high performance and wide application. The dry chemical analysis technology is combined with the micro-fluidic chip technology, and the required reaction reagent is fixed on the micro-fluidic chip, so that the integrated, high-precision and instant biomarker detection can be realized, and a new technical thought is provided for the dry-type immune electrochemical luminescence detection based on the micro-fluidic chip.
At present, analyzers based on the electrochemical luminescence technology are marketed for many years, for example, Roche Elecsys type electrochemical luminescence immunoassay analyzer and kit thereof of Roche company, the system of which adopts terpyridyl ruthenium as a labeled probe and magnetic beads as a reaction carrier, and can realize rapid immunoassay of various disease markers. However, such an electrochemiluminescence immunoassay analyzer based on wet chemical analysis technology still faces some problems, such as very high price, bulky volume, inability of portable detection; the detection process needs to add a plurality of reagents, is complicated in operation, has long analysis time and the like, and not only limits the wide application of the detection process in clinical detection, but also greatly limits the application of the detection process in Point of care (POCT).
Therefore, it is an urgent need to solve the problem for those skilled in the art to develop a portable, fool-proof, low-cost and fast-analysis fully-automatic immunochemistry luminescence analyzer.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a full-automatic dry immune closed bipolar electrochemiluminescence analyzer, is suitable for medical institutions and even families, has a great application prospect in instant detection, can quickly and accurately acquire chip detection data, and has instant detection application with high analysis performance.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a full-automatic dry-type immune closed bipolar electrochemical luminescence analyzer comprises a dry-type immune closed bipolar electrochemical luminescence chip and a closed bipolar electrochemical luminescence analyzer, wherein the dry-type immune closed bipolar electrochemical luminescence chip comprises a shell and a fiber micro-fluidic chip, the shell comprises an upper cover and a lower cover, the upper cover is provided with a sample adding hole, a buffer solution adding hole, an observation window and an electrode contact area, and the fiber micro-fluidic chip is arranged between the upper cover and the lower cover;
the fiber microfluidic 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 closed bipolar electrochemical luminescence analyzer comprises a chip automatic loading module, an imaging detection module, a data processing and instrument control module, an electrochemical reaction excitation module, a power supply module and a display, wherein the data processing and instrument control module is respectively connected with the display, the imaging detection module, the electrochemical reaction excitation module and the power supply module;
the chip automatic loading module is used for conveying the dry immune closed bipolar electrochemiluminescence chip to the position under the imaging detection module, the electrochemical reaction excitation module is used for exciting an object to be detected to generate electrochemiluminescence reaction and send out optical signals, the imaging detection module is used for converting the optical signals generated by electrochemiluminescence into electric signals and transmitting the electric signals to the data processing and instrument control module, the data processing and instrument control module is used for processing the received electric signals and transmitting results to the display, and the display is used for man-machine interaction, image preview and result display.
The closed bipolar electrodes are distributed in an E shape, and two anodes of the closed bipolar electrodes are respectively a detection anode and a quality control anode.
The detection sheet is provided with three hydrophilic channels, two hydrophilic channels are used for flowing of samples to be detected, one hydrophilic channel is used for flowing of buffer solution, the detection sheet is stacked on the electrode plate, the two hydrophilic channels used for flowing of the samples to be detected are correspondingly overlapped with the detection anode and the quality control anode of the closed bipolar electrode one by one, and the hydrophilic channels used for flowing of the buffer solution are correspondingly overlapped with the shared cathode of the closed bipolar electrode.
The hydrophilic channel overlapped with the detection anode on the detection sheet and the quality control anode on the electrode sheet is used as a detection area, the hydrophilic channel overlapped with the quality control anode on the detection sheet and the electrode sheet is used as a quality control area, and the positions of the detection area and the quality control area correspond to the observation window of the upper cover.
The combination piece and the sample adding piece are respectively provided with two hydrophilic channels which are distributed in parallel, one is a sample to be detected flowing hydrophilic channel, the other is a buffer solution flowing hydrophilic channel, the sample to be detected flowing hydrophilic channel and the buffer solution flowing hydrophilic channel of the combination piece are respectively communicated with the sample to be detected flowing hydrophilic channel and the buffer solution flowing hydrophilic channel of the sample adding piece in a one-to-one correspondence manner, and are correspondingly communicated with the two sample to be detected flowing hydrophilic channels and the buffer solution flowing hydrophilic channel of the detection piece.
The sample feeding chip has one flowing hydrophilic channel corresponding to the sample feeding hole in the upper cover, one buffering liquid flowing hydrophilic channel corresponding to the buffering liquid feeding hole in the upper cover, and one driving electrode set in the electrode contact area between the electrode plate and the upper cover.
The sample to be detected of the binding sheet flows on the hydrophilic channel and is dried with a labeled antibody which is coupled with the electrochemiluminescence probe and is specifically bound with the biomarker to be detected.
The electrochemical luminescence probe is a linear polylysine coupled terpyridyl ruthenium derivative, and the dosage of the labeled antibody is 4.5-7 mu L, preferably 5 mu L.
The detection area of the detection sheet is modified and fixed with a biomarker capture antibody, the quality control area of the detection sheet is modified and fixed with a quality control capture antibody, and the concentration range of the biomarker capture antibody is 150-250 mu g/mL, preferably 200 mu g/mL.
The application of the closed bipolar electrochemiluminescence chip in immunodetection of a biological marker adopts the full-automatic dry-type immune closed bipolar electrochemiluminescence analyzer, and comprises the following steps:
s1, dropwise adding a sample solution to be detected into the sample adding hole, allowing the solution to flow from the sample adding sheet to the detection sheet, allowing the biomarker to be detected to specifically bind with the labeled antibody on the binding sheet and the capture antibody on the detection sheet in sequence to form a sandwich immune complex of labeled antibody-biomarker-capture antibody, dropwise adding a buffer solution into the buffer solution adding hole, and allowing the buffer solution to flow to and fill the support channel of the electrode sheet;
s2, after the immunoreaction incubation time is over, dripping buffer solution into the sample adding hole to promote the further combination of the biomarker, the labeled antibody and the capture antibody, and then flushing the uncombined substances on the detection sheet and filling the report channel of the electrode sheet;
s3, starting the closed bipolar electrochemical luminescence analyzer, placing the dry immune closed bipolar electrochemical luminescence chip on an automatic chip loading module, and conveying the dry immune closed bipolar electrochemical luminescence chip to the position under the imaging detection module by the automatic chip loading module;
s4, pressing a detection button on the closed bipolar electrochemical luminescence analyzer, starting and triggering the closed bipolar electrochemical luminescence reaction by the electrochemical excitation module, and converting an electrochemical luminescence signal acquired by the imaging detection module into an electric signal and transmitting the electric signal to the data processing and instrument control module;
and S5, the data processing and instrument control module processes and analyzes the received electric signal and transmits the result to the display, and the user reads the detection result on the display and further processes and analyzes the detection result.
Wherein the incubation time of the immunoreaction is 2.5-5min, and the driving voltage of the closed bipolar electrochemiluminescence reaction is 8.5V-9.5V, preferably 9V.
Compared with the prior art, the invention has the following advantages and effects:
1. the invention firstly invents a full-automatic dry-type immune closed bipolar electrochemical luminescence analyzer, and overcomes the defects that the existing electrochemical luminescence analyzer based on wet chemical analysis technology needs to add different reaction reagents in multiple steps, is complex in operation, long in detection time, heavy in volume, high in price and the like.
2. The full-automatic dry immune closed bipolar electrochemical luminescence analyzer comprises the steps of automatic chip in and out, automatic signal acquisition, automatic data analysis and storage and automatic result display, and can realize the full-automatic detection process of sample in/out.
3. According to the automatic chip loading module, automatic sample introduction of the chips is completed by moving on the rail, manual chip insertion is replaced, the chips are not required to be manually loaded one by one, and user experience and satisfaction are improved.
4. In the chip structure, the electrode plate does not need to be folded, so that the chip manufacturing process is simplified, and the chip structure is easy for mass production; the electrochemical luminescence probe of the linear polylysine coupled terpyridyl ruthenium derivative is used for cascade amplification of electrochemical luminescence signals, higher sensitivity is achieved under the condition that no modification is made on an electrode slice, and hypersensitive detection of biomarkers can be achieved.
5. The full-automatic dry immune closed bipolar electrochemiluminescence analyzer can quickly obtain a detection result within 7min only by dripping a sample to be detected and a buffer solution; compared with the traditional electrochemical luminescence analyzer, the electrochemical luminescence analyzer is more convenient and faster to detect, has shorter time and is suitable for on-site rapid detection.
6. The full-automatic dry immune closed bipolar electrochemiluminescence analyzer can realize rapid quantification and hypersensitive detection on different biomarkers, and is beneficial to early diagnosis, treatment and prognosis of different diseases, such as cancer markers, acute myocardial infarction markers, inflammation markers and the like.
Drawings
FIG. 1 is a schematic structural decomposition diagram of a dry immune closed bipolar electrochemiluminescence chip;
FIG. 2 is a schematic diagram of the whole structure of a dry immune closed bipolar electrochemiluminescence chip;
FIG. 3 is an exploded view of the fiber microfluidic chip;
FIG. 4 is a schematic diagram of the whole structure of the fiber microfluidic chip;
FIG. 5 is a schematic view of a fully automatic bipolar electrochemical luminescence analyzer;
FIG. 6 is a graph of the electrochemiluminescence intensity value versus the driving voltage.
FIG. 7 is a graph of electrochemiluminescence intensity values as a function of labeled antibody volume.
FIG. 8 is a graph of electrochemiluminescence intensity values versus capture antibody concentration.
FIG. 9 is a graph of electrochemiluminescence intensity values versus incubation time for immunoreactions.
Fig. 10 is an analysis graph of the detection of different concentrations of cardiac troponin cTnI, and an interpolation graph is a data linear fitting graph.
FIG. 11 is a diagram showing the evaluation of selectivity in detecting cardiac troponin cTnI.
The reference numbers illustrate:
1-upper cover, 2-transparent cover plate, 3-fiber microfluidic chip, 4-lower cover, 5-sample adding hole, 6-buffer adding hole, 7-observation window, 8-electrode contact area, 9-sample adding sheet, 10-combination sheet, 11-detection sheet, 12-electrode sheet, 13-bottom plate, 14-closed bipolar electrode, 15, 16-driving electrode, 17-two anodes of the closed bipolar electrode, 18-shared cathode, 19-reporting channel, 20-supporting channel, 21-chip automatic loading module, 22-imaging detection module, 23-data processing and instrument control module, 24-electrochemical reaction excitation module, 25-power supply module and 26-display.
Detailed Description
The full-automatic dry immune closed bipolar electrochemical luminescence analyzer is further described with reference to the accompanying drawings and specific embodiments.
The invention discloses a full-automatic dry-type immune closed bipolar electrochemiluminescence analyzer, which comprises a dry-type immune closed bipolar electrochemiluminescence chip and a closed bipolar electrochemiluminescence analyzer, wherein the dry-type immune closed bipolar electrochemiluminescence chip is electrically triggered to generate a specific electrochemiluminescence signal, and the closed bipolar electrochemiluminescence analyzer acquires, collects and analyzes the electrochemiluminescence signal and displays a detection result to a user.
The structure and composition of the dry immune closed bipolar electrochemical luminescence chip are shown in fig. 1 to fig. 4, and the dry immune closed bipolar electrochemical luminescence chip comprises a shell, a transparent cover plate 2 and a fiber microfluidic chip 3. The shell includes upper cover 1 and lower cover 4, and upper cover 1 is equipped with application of sample hole 5, buffer solution application of sample hole 6, observation window 7 and electrode contact area 8. The fiber microfluidic chip 3 is arranged between the upper cover 1 and the lower cover 4, the transparent cover plate 2 is arranged between the upper cover 1 and the fiber microfluidic chip 3 and compresses the fiber microfluidic chip 3, and the transparent cover plate 2 prevents the fiber microfluidic chip 3 from directly contacting with the outside.
The fiber micro-fluidic chip 3 comprises a sample adding sheet 9, a combination sheet 10, a detection sheet 11, an electrode sheet 12 and a bottom plate 13, wherein the sample adding sheet 9, the combination sheet 10, the detection sheet 11 and the electrode sheet 12 are sequentially overlapped on the bottom plate 13, and the overlapping position of each part is overlapped by 2 mm.
The electrode sheet 12 includes a closed bipolar electrode 14 and a pair of drive electrodes 15, 16 distributed in an E-shape. The closed bipolar electrode 14 distributed in an E shape comprises two anodes 17 and a shared cathode 18, wherein the two anodes 17 are respectively a quality control anode and a detection anode. The two closed bipolar electrodes 17 are adjacently distributed and have the same shape. The electrode plate 12 is provided with a report channel 19 and a support channel 20, the two anodes 17 and the corresponding driving electrodes 15 are located in the report channel 19, and the shared cathode 18 and the corresponding driving electrodes 16 are located in the support channel 20.
The drive electrodes 15, 16 of the electrode sheet 12 correspond to the electrode contact regions 8 of the upper cover 1. The substrate material of the electrode sheet 12 is cotton cloth or paper. The closed bipolar electrode 14 of the electrode plate 12 is not modified, and the photoelectrochemistry luminescence reaction of the bipolar electrode can be triggered by electrifying.
The detection sheet 11 is designed with three hydrophilic channels, two of which are hydrophilic channels for the flow of the sample to be detected, and the other is hydrophilic channel for the flow of the buffer solution. The two sample flow hydrophilic channels to be detected are in the shape of right-angle rulers which are symmetrically distributed adjacently. The detection piece 11 is laminated on the electrode piece 12, two sample flow hydrophilic channels to be detected are correspondingly overlapped with two anodes 17 of the closed bipolar electrode one by one, and the other buffer flow hydrophilic channel is correspondingly overlapped with a shared cathode 18 of the closed bipolar electrode.
The sample flow hydrophilic channel to be detected on the detection sheet 11, which overlaps with the detection anode, is a detection area (T area), and the sample flow hydrophilic channel to be detected on the detection sheet 11, which overlaps with the quality control anode, is a quality control area (C area). The significance of the design is that after the electrode slice 12 is connected with a power supply, the detection anode and the quality control anode can trigger the immune electrochemical luminescence reaction of the T area and the C area of the detection slice 11.
The positions of a T area and a C area of the detection sheet 11 correspond to the observation window 7 of the upper cover 1, the T area is used for detecting a sample to be detected, and the C area is used for monitoring the quality of a chip. When the C area does not emit light during immunoassay, the detection result of the time is meaningless. The T region of the test piece 11 was modified and immobilized with a capture antibody of 0.2mg/mL cardiac troponin cTnI (Product Code: D4160MA01-MA, OriGene Company). The C region of the test piece 11 is modified with a fixed quality control capture antibody (goat anti-chicken IgY).
The combination piece 10 and the sample adding piece 9 are both designed with two hydrophilic channels which are distributed in parallel, one is a sample flow hydrophilic channel to be detected, and the other is a buffer flow hydrophilic channel. The sample flowing hydrophilic channel to be detected and the buffer flowing hydrophilic channel of the combination piece 10 are respectively communicated with the sample flowing hydrophilic channel to be detected and the buffer flowing hydrophilic channel of the sample adding piece 9 in a one-to-one correspondence manner, and are simultaneously communicated with the two sample flowing hydrophilic channels to be detected and the buffer flowing hydrophilic channel of the detection piece 11 in a corresponding manner; the significance of the design is to construct a good sample flow channel to be detected and a buffer flow channel.
The sample to be tested of the sample adding piece 9 flows through a hydrophilic channel corresponding to the sample adding hole 5 of the upper cover 1, and the other hydrophilic channel corresponds to the buffer solution sample adding hole 6 of the upper cover 1.
The sample flowing hydrophilic channel of the bonding sheet 10 is dried with a labeled antibody (Product Code: B9085MA06-MA, OriGene Company) which is coupled with an electrochemiluminescence probe and is specifically bonded with the cardiac troponin cTnI of the biomarker to be detected. The electrochemical luminescence probe is preferably a linear polylysine coupled terpyridyl ruthenium derivative, can realize the cascade amplification of an immune electrochemical luminescence signal, and greatly improves the detection sensitivity.
The detection principle of dry closed bipolar immune electrochemiluminescence is based on sandwich type immunoreaction and an electrochemiluminescence system based on linear polylysine coupled terpyridyl ruthenium derivative. The structure of the dry closed type bipolar immune electrochemical luminescence chip is further improved, so that the chip is simpler to manufacture and simpler to operate. The electrochemical luminescence signal of the dry closed bipolar immune electrochemical luminescence chip is triggered, obtained and analyzed by a full-automatic bipolar electrochemical luminescence analyzer.
The structure of the closed bipolar electrochemical luminescence analyzer is shown in fig. 5, the closed bipolar electrochemical luminescence analyzer comprises a chip automatic loading module 21, an imaging detection module 22, a data processing and instrument control module 23, an electrochemical reaction excitation module 24, a rechargeable power module 25 and a display 26, the data processing and instrument control module 23 is respectively connected with the display 26, the imaging detection module 22, the electrochemical reaction excitation module 24 and the rechargeable power module 25, and the rechargeable power module 25 is respectively connected with the display 26, the electrochemical reaction excitation module 24 and the chip automatic loading module 21.
The automatic chip loading module 21 is controlled by a motor, so that the dry-type immune closed bipolar electrochemical luminescence chip can be automatically fed in and out, and the dry-type immune closed bipolar electrochemical luminescence chip is conveyed to the position under the imaging detection module 22 through the motion track, so that the luminous area of the dry-type immune closed bipolar electrochemical luminescence chip is ensured to be in the detection visual field. The imaging detection module 22 is a CMOS camera, and is configured to acquire an electrochemiluminescence signal of the dry-type immune closed bipolar electrochemiluminescence chip, convert the electrochemiluminescence signal into an electrical signal, and transmit the electrical signal to the data processing and instrument control module 23. The data processing and instrument control module 23 receives, processes and stores the electrical signals, and finally transmits the detection result to the display 26. The display 26 is a touchable liquid crystal display for human-computer interaction, image preview, and result display. The electrochemical reaction excitation module 24 provides a dc power source for the electrochemiluminescence reaction to trigger electrochemiluminescence. The voltage of the direct current power supply is adjustable and can be adjusted according to the actually required voltage. The rechargeable power module 25 supplies power to the modules, and the cruising ability is strong.
The full-automatic closed bipolar electrochemiluminescence analyzer further comprises a data transmission function, the data processing and instrument control module 23 transmits detection data to the client side through wifi, Bluetooth, USB and other modes, and a user can conveniently check historical detection results and further analyze the historical detection results. The full-automatic dry immune closed bipolar electrochemiluminescence analyzer can realize detection of different disease biomarkers.
The manufacturing method of the immune dry type closed bipolar electrochemical luminescence chip comprises the following steps:
(1) configuration design: the configurations of a sample adding sheet 9, a combination sheet 10, a detection sheet 11 and an electrode sheet 12 of the fiber microfluidic chip 3 are designed by using Adobe Illustrator CS6 drawing software, so that a polyester fiber channel screen plate and an electrode screen plate are customized, and the configuration of a shell is designed by using Solid Works drawing software.
(2) Printing the chip shell: the structure of the housing is printed using a 3D printer, including an upper cover 1 and a lower cover 4.
(3) Processing the fiber microfluidic chip: the sample adding sheet 9, the combination sheet 10 and the hydrophobic dam of the detection sheet 11 are made of screen printing ink, the front closed bipolar electrode 14 and the driving electrodes 15 and 16 of the electrode sheet 12 are made of screen printing carbon paste, and the hydrophobic dam on the back is made of screen printing solid crayon.
(4) Fiber microfluidic chip modification
After the binding sheet 10 is treated by the binding sheet pretreatment solution, a labeled antibody of anti-cardiac troponin cTnI is dripped into a flowing hydrophilic channel of a sample to be detected of the binding sheet 10, and vacuum drying is carried out at 37 ℃.
The T area and the C area of the detection sheet 11 are respectively modified and fixed with a capture antibody of anti-cardiac troponin cTnI and a capture antibody of a quality control area, and the fixation mode is chitosan-glutaraldehyde chemical bond covalent bonding.
(5) Chip assembly
As shown in fig. 4, the microfluidic fiber chip 3 is first stacked on a bottom plate 13 by an electrode sheet 12, a detection sheet 11, a bonding sheet 10, and a sample addition sheet 9 in sequence, and each stacked position is overlapped by 2 mm. Subsequently, the transparent cover plate 2 and the assembled fiber microfluidic chip 3 are placed in a housing, thereby completing the assembly of the dry immune closed type bipolar electrochemical luminescence chip.
The assembled dry immune closed bipolar electrochemiluminescence chip can be placed on the automatic chip loading module 21 of the closed bipolar electrochemiluminescence analyzer for sample detection.
Application example 1
Taking the detection of cardiac troponin cTnI as an example, the detection process of the full-automatic dry-type immune bipolar electrochemiluminescence analyzer is as follows:
(1) dropwise adding a to-be-detected sample solution containing the cardiac troponin cTnI into the sample adding hole 5, allowing the solution to flow from the sample adding sheet 9 to the detection sheet 11, and specifically binding the cardiac troponin cTnI of the to-be-detected sample with the dried labeled antibody on the binding sheet 10 and the modified and fixed capture antibody on the detection sheet 11 in sequence to finally form the labeled antibody-cardiac troponin cTnI-capture antibody sandwich type immune complex. At the same time, a buffer solution is dropped into the buffer addition well 6, and the solution flows to and fills the support channel 20.
(2) After waiting for 3 minutes, a buffer solution is dropped into the well 5, which promotes the re-binding of the cardiac troponin cTnI to its corresponding antibody, and washes away unbound substances in the T-and C-domains of the test piece 11, and fills the reporter channel 19.
(3) Starting the full-automatic closed bipolar electrochemical luminescence analyzer, setting parameters (such as exposure time 300ms, gain 16 and the like) of the CMOS camera, and opening a detection program for standby.
(4) The chip is placed in the chip automatic loading module 21, and the chip is transported to a position right below the detection module 22 by the rail.
(5) Clicking the detection button on the instrument, the electrochemical reaction excitation module 24 is started, and the electrochemical luminescence reaction of the chip is electrically triggered to generate a specific electrochemical luminescence signal.
(6) The generated electrochemical luminescence signal is collected by the imaging detection module 22 and analyzed by the data processing and instrument control module 23.
(7) The results of the analysis are then transmitted to the display 26 where they can be read directly by the user and further stored and analyzed. And (4) introducing the detection result into Origin software for processing and analysis, and obtaining the data relation between the electrochemical luminescence intensity value and a certain parameter (each data point is obtained by adopting 5 times of repeated experiments).
Taking a sample solution to be tested containing 1ng/mL cardiac troponin cTnI and the full-automatic closed bipolar electrochemical luminescence analyzer of the invention as examples to test the relationship between the driving voltage and the electrochemical luminescence intensity value.
The test results are shown in fig. 6, and it can be seen that: the electrochemiluminescence intensity value is gradually increased as the driving voltage is increased from 6V to 9V. However, when the driving voltage is further increased to 9.5V, the electrochemiluminescence intensity value is decreased. The reason for this may be that background reactions (such as water oxidation) are excited at high driving voltages, thereby interfering with the generation of the electrochemiluminescent signal. Therefore, the driving voltage is preferably 9V, and an acceptable range is 8.5-9.5V.
Application example 2
Several important factors (labeled antibody volume, capture antibody concentration, immunoreaction incubation time) affecting the electrochemiluminescence intensity values in application example 1 were optimized:
1) preferred volume of labeled antibody
1. The concentration of the cardiac troponin cTnI to be detected is 1ng/mL, the sample addition volume of the cardiac troponin cTnI is 30 muL, the driving voltage is 9V, the volume of the labeled antibody is undetermined, the concentration of the capture antibody is 100 mug/mL, and the immunoreaction incubation time is 3 min.
2. Several experimental groups were set up: the labeled antibody volume was set at several different values (3. mu.L, 4. mu.L, 4.5. mu.L, 5. mu.L, 6. mu.L, 7. mu.L).
3. The detection operation and analysis process were the same as in application example 1, and the experimental results are shown in FIG. 7.
From the experimental results it can be seen that: the electrochemiluminescence intensity value is increased along with the increase of the volume of the labeled antibody, and the electrochemiluminescence intensity value is not obviously increased when the volume is more than 5 mu L. This may occur because the molecular weight of the sandwich immune complex formed gradually saturates when the labeled antibody volume is greater than 5 μ L. Therefore, the labeled antibody volume is preferably 5. mu.L, and an acceptable range is 4.5-7. mu.L.
2) Preferred capture antibody concentration
1. The concentration of the cardiac troponin cTnI to be detected is 1ng/mL, the sample adding volume of the cardiac troponin cTnI is 30 muL, the driving voltage is 9V, the volume of the labeled antibody is 5 muL, the concentration of the capture antibody is undetermined, and the immunoreaction incubation time is 3 min.
2. Several experimental groups were set up: the capture antibody concentration was set at several different values (50. mu.g/mL, 100. mu.g/mL, 150. mu.g/mL, 200. mu.g/mL, 250. mu.g/mL, 300. mu.g/mL).
3. The detection operation and analysis process were the same as in application example 1, and the experimental results are shown in FIG. 8.
From the experimental results it can be seen that: the electrochemiluminescence intensity values increase first and then decrease as the concentration of capture antibody increases. The possible reason for this is that the high concentration capture antibody immobilized in the detection zone is modified to generate steric hindrance effect, which hinders the electron transfer between the electrochemiluminescence probe and the electrode. Based on this fact, the capture antibody concentration is preferably 200. mu.g/mL, and the acceptable range is 150. mu.g/mL and 250. mu.g/mL.
3) Preferred immunoreaction incubation times
1. The concentration of the cardiac troponin cTnI to be detected is 1ng/mL, the sample adding volume of the cardiac troponin cTnI is 30 muL, the driving voltage is 9V, the volume of the labeled antibody is 5 muL, the concentration of the capture antibody is 200 mug/mL, and the immunoreaction incubation time is undetermined.
2. Several experimental groups were set up: the immunoreaction incubation time was set at several different values (1min, 2min, 2.5min, 3min, 4min, 5 min).
3. The detection operation and analysis process were the same as in application example 1, and the experimental results are shown in FIG. 9.
From the experimental results it can be seen that: along with the increase of the incubation time of the immunoreaction, the electrochemical luminescence intensity value is enhanced; when the incubation time is increased from 3min to 5min, the electrochemiluminescence intensity value tends to be stable. This may be due to the fact that the sandwich immune complex tends to stabilize after the immune response is over, resulting in no significant increase in the electrochemiluminescence intensity values. Therefore, the incubation time for the immunoreaction is preferably 3min, and an acceptable range is 2.5-5 min.
Application example 4
The cardiac troponin cTnI was detected using a fully automatic dry immune closed bipolar electrochemiluminescence analyzer under optimized conditions found in application example 1 and application example 2.
1. And (3) adopting optimized parameters: the sample volume of the cardiac troponin cTnI is 30 mu L, the driving voltage is 9V, the volume of the labeled antibody is 5 mu L, the concentration of the capture antibody is 200 mu g/mL, and the immunoreaction incubation time is 3 min.
2. Several experimental groups were set up: the cTnI concentration of the cardiac troponin to be tested was set at several different values (100ng/mL, 50ng/mL, 10ng/mL, 1ng/mL, 0.1ng/mL, 10pg/mL, 1pg/mL, 0.1pg/mL, 0).
3. The detection operation and analysis process were the same as in application example 1, and the experimental results are shown in FIG. 10.
From the experimental results it can be seen that: the electrochemiluminescence intensity value increases with the increase of the concentration of cardiac troponin cTnI. The electrochemical luminescence intensity value (expressed by Y) and the logarithm of the concentration of the cardiac troponin cTnI (expressed by X) are in a certain linear relation, and the linear equation can be expressed as that Y is 10.614+1.712X (R is equal to 10.614 ×) (R is equal to X) 2 0.991, n 5). The background signal without the cardiac troponin cTnI and the three-fold standard deviation are taken as electrochemical luminescence intensity values, and the detection limit of the cardiac troponin cTnI in the method is calculated to be 0.0141 pg/mL. As can also be seen from fig. 10, the method can realize a wide range of myocardial troponin cTnI hypersensitivity quantitative detection with good linearity.
Application example 5
The selective experiment for detecting cardiac troponin cTnI was performed under some optimized conditions found in application example 1 and application example 2.
1. And (3) adopting optimized parameters: the sample adding volume of the cardiac troponin cTnI is 30 mu L, the driving voltage is 9V, the volume of the labeled antibody is 5 mu L, the concentration of the capture antibody is 200 mu g/mL, and the immunoreaction incubation time is 3 min.
2. A plurality of interference experimental groups are set: cardiac troponin cTnI, Hemoglobin (HGB), C-reactive protein (CRP), Bovine Serum Albumin (BSA), pooled samples (Mix, pooled first 4 proteins) and Blank control (Blank, PBS buffer). Both the cardiac troponin cTnI and the interfering protein concentration were 10 ng/mL.
3. The detection operation and analysis process were the same as in application example 1, and the results of the experiment are shown in FIG. 11.
From the experimental results it can be seen that: compared with the cardiac troponin cTnI group, the electrochemiluminescence intensity value of the mixed sample group containing the cardiac troponin cTnI is hardly influenced, and the difference between the electrochemiluminescence intensity value and the electrochemiluminescence intensity value is small. The electrochemical luminescence intensity values of the interference experiment groups of the hemoglobin HGB, the C-reactive protein CRP and the bovine serum albumin BSA are almost the same as the electrochemical luminescence intensity value of the Blank control group Blank. The above results indicate that the electrochemiluminescence signal responds only to the target cardiac troponin cTnI. Therefore, the method can realize the specific detection of the cardiac troponin cTnI.
The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.
Claims (10)
1. A full-automatic dry-type immune closed bipolar electrochemical luminescence analyzer is characterized by comprising a dry-type immune closed bipolar electrochemical luminescence chip and a closed bipolar electrochemical luminescence analyzer, wherein the dry-type immune closed bipolar electrochemical luminescence chip comprises a shell and a fiber micro-fluidic chip, the shell comprises an upper cover and a lower cover, the upper cover is provided with a sample adding hole, a buffer solution adding hole, an observation window and an electrode contact area, and the fiber micro-fluidic chip is arranged between the upper cover and the lower cover;
the fiber microfluidic 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 closed bipolar electrochemical luminescence analyzer comprises a chip automatic loading module, an imaging detection module, a data processing and instrument control module, an electrochemical reaction excitation module, a power supply module and a display, wherein the data processing and instrument control module is respectively connected with the display, the imaging detection module, the electrochemical reaction excitation module and the power supply module;
the chip automatic loading module is used for conveying the dry immune closed bipolar electrochemiluminescence chip to the position under the imaging detection module, the electrochemical reaction excitation module is used for exciting an object to be detected to generate electrochemiluminescence reaction and send out optical signals, the imaging detection module is used for converting the optical signals generated by electrochemiluminescence into electric signals and transmitting the electric signals to the data processing and instrument control module, the data processing and instrument control module is used for processing the received electric signals and transmitting results to the display, and the display is used for man-machine interaction, image preview and result display.
2. The fully automatic dry-immune closed bipolar electrochemiluminescence analyzer of claim 1, wherein the closed bipolar electrodes are distributed in an E-shape, and the two anodes of the closed bipolar electrodes are a detection anode and a quality control anode, respectively.
3. The fully automatic dry-type immuno-closed bipolar electrochemical luminescence analyzer of claim 2, wherein the detecting plate is designed with three hydrophilic channels, two are the sample flow hydrophilic channels to be tested, and one is the buffer flow hydrophilic channel, the detecting plate is stacked on the electrode plate, the two sample flow hydrophilic channels to be tested are overlapped with the detecting anode and the quality control anode of the closed bipolar electrode in a one-to-one correspondence manner, and the buffer flow hydrophilic channel is overlapped with the shared cathode of the closed bipolar electrode in a one-to-one correspondence manner.
4. The fully automatic dry-type immuno-closed bipolar electrochemical luminescence analyzer of claim 3, wherein the hydrophilic channel on the test strip overlapping the test anode on the electrode strip is used as a detection zone, the hydrophilic channel on the test strip overlapping the quality control anode on the electrode strip is used as a quality control zone, and the positions of the detection zone and the quality control zone correspond to the viewing window of the upper cover.
5. The fully automatic dry-type immuno-closed bipolar electrochemiluminescence analyzer of claim 4, wherein the bonding pad and the sample loading pad are designed with two hydrophilic channels in parallel, one is a sample-to-be-tested flow hydrophilic channel and the other is a buffer flow hydrophilic channel, the sample-to-be-tested flow hydrophilic channel and the buffer flow hydrophilic channel of the bonding pad are respectively in one-to-one communication with the sample-to-be-tested flow hydrophilic channel and the buffer flow hydrophilic channel of the sample loading pad, and are in communication with the two sample-to-be-tested flow hydrophilic channels and the buffer flow hydrophilic channels of the test pad.
6. The fully automatic dry immuno-closed bipolar electrochemiluminescence analyzer of claim 5, wherein the hydrophilic flow path of the sample to be measured of the sample application plate corresponds to the sample application hole of the upper cover, the hydrophilic flow path of the buffer of the sample application plate corresponds to the buffer application hole of the upper cover, and the driving electrode of the electrode pad is disposed in correspondence with the electrode contact area of the upper cover.
7. The fully automatic dry-type immuno-closed bipolar electrochemical luminescence analyzer of claim 5, wherein the sample-to-be-tested flow hydrophilic channel of the binding sheet is dried with a labeled antibody coupled with the electrochemiluminescence probe and specifically binding with the biomarker to be tested.
8. The fully automatic dry-immune closed bipolar electrochemiluminescence analyzer of claim 7, wherein the electrochemiluminescence probe is a linear polylysine coupled terpyridyl ruthenium derivative, and the amount of the labeled antibody is 4.5-7 μ L.
9. The fully automatic dry immuno-closed bipolar electrochemiluminescence analyzer of claim 8, wherein the detection zone of the test strip is modified by immobilized biomarker capture antibody, the quality control zone of the test strip is modified by immobilized quality control capture antibody, and the concentration of the biomarker capture antibody is in the range of 150-.
10. The application of a closed bipolar electrochemiluminescence chip in immunodetection of a biological marker is characterized in that a full-automatic dry-type immune closed bipolar electrochemiluminescence analyzer of any one of the claims 1 to 9 is adopted, and the method comprises the following steps:
s1, dropwise adding a sample solution to be detected into the sample adding hole, allowing the solution to flow from the sample adding sheet to the detection sheet, allowing the biomarker to be detected to specifically bind with the labeled antibody on the binding sheet and the capture antibody on the detection sheet in sequence to form a sandwich immune complex of labeled antibody-biomarker-capture antibody, dropwise adding a buffer solution into the buffer solution adding hole, and allowing the buffer solution to flow to and fill the support channel of the electrode sheet;
s2, after the immunoreaction incubation time is over, dripping buffer solution into the sample adding hole to promote the further combination of the biomarker, the labeled antibody and the capture antibody, and then flushing the uncombined substances on the detection sheet and filling the report channel of the electrode sheet;
s3, starting the closed bipolar electrochemical luminescence analyzer, placing the dry immune closed bipolar electrochemical luminescence chip on an automatic chip loading module, and conveying the dry immune closed bipolar electrochemical luminescence chip to the position under the imaging detection module by the automatic chip loading module;
s4, pressing a detection button on the closed bipolar electrochemical luminescence analyzer, starting and triggering the closed bipolar electrochemical luminescence reaction by the electrochemical excitation module, and converting an electrochemical luminescence signal acquired by the imaging detection module into an electric signal and transmitting the electric signal to the data processing and instrument control module;
s5, the data processing and instrument control module processes and analyzes the received electric signal and transmits the result to the display, and the user reads the detection result on the display and further processes and analyzes the result;
wherein the incubation time of the immunoreaction is 2.5-5min, and the driving voltage of the closed bipolar electrochemiluminescence reaction is 8.5V-9.5V.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210518635.5A CN114965447A (en) | 2022-05-13 | 2022-05-13 | Full-automatic dry immune closed bipolar electrochemical luminescence analyzer and application thereof in immunoassay |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210518635.5A CN114965447A (en) | 2022-05-13 | 2022-05-13 | Full-automatic dry immune closed bipolar electrochemical luminescence analyzer and application thereof in immunoassay |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114965447A true CN114965447A (en) | 2022-08-30 |
Family
ID=82983259
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210518635.5A Pending CN114965447A (en) | 2022-05-13 | 2022-05-13 | Full-automatic dry immune closed bipolar electrochemical luminescence analyzer and application thereof in immunoassay |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114965447A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117288941A (en) * | 2023-08-18 | 2023-12-26 | 华南师范大学 | Method for rapidly synthesizing self-enhanced electrochemiluminescence probe in water phase based on lysine and application thereof |
-
2022
- 2022-05-13 CN CN202210518635.5A patent/CN114965447A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117288941A (en) * | 2023-08-18 | 2023-12-26 | 华南师范大学 | Method for rapidly synthesizing self-enhanced electrochemiluminescence probe in water phase based on lysine and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | Battery-triggered microfluidic paper-based multiplex electrochemiluminescence immunodevice based on potential-resolution strategy | |
| Munge et al. | Multiplex immunosensor arrays for electrochemical detection of cancer biomarker proteins | |
| US5149629A (en) | Coulometric assay system | |
| CN102507953B (en) | Preparation method of electrochemistry immunosensor for determining alpha fetoprotein | |
| CN1781022A (en) | Membrane strip biosensor system for point-of-care testing | |
| CN113884481B (en) | Dry bipolar electrochemical luminescence chip and application thereof in immunodetection | |
| EP0441875B1 (en) | Enhanced electrochemiluminescence | |
| Wang et al. | Rechargeable battery-triggered electrochemiluminescence detection on microfluidic origami immunodevice based on two electrodes | |
| CN110441295B (en) | Ferritin-based packaging Ir (ppy)3Preparation method of biosensor | |
| KR20140012933A (en) | Integrated carbon electrode chips for the electric excitation of lanthanide chelates, and analytical methods using these chips | |
| CN114965447A (en) | Full-automatic dry immune closed bipolar electrochemical luminescence analyzer and application thereof in immunoassay | |
| US8524504B2 (en) | Sensor | |
| Faatz et al. | Automated Immunoassays for the detection of biomarkers in body fluids | |
| CN217587030U (en) | Dry-type immune closed bipolar electrochemical luminescence chip | |
| CN113533743A (en) | Biological analysis method for detecting human VEGF-A concentration by using chemical light emission method | |
| US8920718B2 (en) | Methods and devices to generate luminescence from integrated electrode chips by cathodic and bipolar pulses | |
| Kim et al. | Highly sensitive rapid test with chemiluminescent signal bands | |
| US20210116408A1 (en) | Improved Electrode for Electrochemical Device | |
| CN114011480B (en) | Electrochemiluminescence microfluidic detection chip and kit for protein detection | |
| Wang et al. | Individually addressable electrode array for multianalyte electrochemiluminescent immunoassay based on a sequential triggering strategy | |
| Norfun et al. | Electrochemical detection of human interleukin-15 using a graphene oxide-modified screen-printed carbon electrode | |
| JPH03504043A (en) | Device for multiple simultaneous measurements of electrochemiluminescence phenomena | |
| KR20140110795A (en) | Biosensor strips using redox cycling | |
| CN115389760A (en) | Detection reagent for immunoassay test strip | |
| Zheng et al. | based microfluidic immunoassay for electrochemical detection of B-type natriuretic peptide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |