A fully automatic dry immunological closed bipolar electrochemiluminescence analyzer and its application in immunoassay

Abstract

The invention discloses a fully automatic dry immune closed bipolar electrochemiluminescence analyzer, comprising a dry immune closed bipolar electrochemiluminescence chip and a closed bipolar electrochemiluminescence analyzer. The dry immune closed bipolar electrochemiluminescence chip comprises a shell and a fiber microfluidic chip. The shell comprises an upper cover and a lower cover. The upper cover is provided with a sample addition hole, a buffer addition hole, an observation window and an electrode contact area. The fiber microfluidic chip is arranged between the upper cover and the lower cover. The fiber microfluidic chip comprises a bottom plate, an electrode sheet, a detection sheet, a binding sheet and a sample addition sheet. The electrode sheet, the detection sheet, the binding sheet and the sample addition sheet are stacked on the bottom plate. The electrode sheet comprises a closed bipolar electrode and a pair of driving electrodes. The closed bipolar electrode comprises two anodes and a shared cathode. A reporting channel and a supporting channel are provided on the electrode sheet. The two anodes and the corresponding driving electrodes are located in the reporting channel. The shared cathode and the corresponding driving electrode are located in the supporting channel. The electrode sheet does not need to be folded.

Description

Technical Field

[0001] The present invention relates to the technical field of medical detection devices, in particular to a fully automatic dry immune closed bipolar electrochemiluminescence analyzer and its application in immune detection. Background Art

[0002] In recent years, bioanalytical technology has developed rapidly in different fields. Among them, immunoelectrochemiluminescence (ECL) technology is an advanced labeling immunoassay technology established on the basis of immunology and ECL technology. Its principle is to use electrochemiluminescence probes such as terpyridine ruthenium as markers without affecting the activity of antibodies (antigens). After binding with their corresponding antigens (antibodies), specific ECL reactions are triggered under electrical triggering, making the detection results stable and reliable, and the accuracy and precision are better than those of enzyme-linked immunosorbent assay technology. With its advantages of strong specificity, high sensitivity and wide detection range, the immunoelectrochemiluminescence technology has shown good application prospects in medical testing, food analysis, environmental testing, etc., and has gradually become one of the mainstream detection methods.

[0003] Microfluidic chip technology has great application prospects in immunoelectrochemiluminescence analysis and has gradually become a new analytical detection platform. This technology has the advantages of high throughput, easy operation, low cost and instant detection, and has aroused widespread research interest. At present, many studies are committed to combining certain detection methods with microfluidic chip technology to develop high-performance and widely used microfluidic chip-based detection technologies. Among them, the combination of dry chemical analysis technology and microfluidic chip technology can realize integrated, high-precision and instant biomarker detection by fixing the required reaction reagents on the microfluidic chip, which provides a new technical idea for dry immunoelectrochemiluminescence detection based on microfluidic chips.

[0004] At present, analyzers based on immunoelectrochemiluminescence technology have been on the market for many years, such as Roche's RocheElecsys electrochemiluminescence immunoassay and its supporting kit. The system uses terpyridine ruthenium as a labeling probe and magnetic beads as a reaction carrier, which can realize rapid immunoassay of multiple disease markers. However, this electrochemiluminescence immunoassay based on wet chemical analysis technology still faces some problems, such as being very expensive, bulky, and not portable; the detection process requires the addition of multiple reagents, cumbersome operations, and long analysis time, which not only limits its wide application in clinical testing, but also greatly limits its application in on-site, point of care testing (POCT).

[0005] Therefore, developing a portable, fool-proof, low-cost and fast-analysis fully automatic immunoelectrochemiluminescence analyzer has become an urgent problem to be solved by technicians in this field. Summary of the invention

[0006] The purpose of the present invention is to overcome the defects of the prior art and provide a fully automatic dry immune closed bipolar electrochemiluminescence analyzer, which is suitable for medical institutions and even personal use at home. At the same time, it has great application prospects in instant detection, can quickly and accurately obtain chip detection data, and has instant detection applications with high analytical performance.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A fully automatic dry immune closed bipolar electrochemiluminescence analyzer, comprising a dry immune closed bipolar electrochemiluminescence chip and a closed bipolar electrochemiluminescence analyzer, wherein the dry immune closed bipolar electrochemiluminescence chip comprises a housing and a fiber microfluidic chip, wherein the housing comprises an upper cover and a lower cover, wherein the upper cover is provided with a sample addition hole, a buffer addition hole, an observation window and an electrode contact area, and the fiber microfluidic chip is arranged between the upper cover and the lower cover;

[0009] The fiber microfluidic chip includes a bottom plate, an electrode sheet, a detection sheet, a binding sheet and a sample adding sheet, wherein the electrode sheet, the detection sheet, the binding sheet and the sample adding sheet are stacked on the bottom plate, the electrode sheet includes a closed bipolar electrode and a pair of driving electrodes, the closed bipolar electrode includes two anodes and a shared cathode, a reporting channel and a supporting channel are arranged on the electrode sheet, the two anodes and the corresponding driving electrodes are located in the reporting channel, and the shared cathode and the corresponding driving electrode are located in the supporting channel;

[0010] The closed bipolar electrochemiluminescence analyzer includes a chip automatic loading module, an imaging detection module, a data processing and instrument control module, an electrochemical reaction excitation module, a power module and a display, wherein the data processing and instrument control module are respectively connected to the display, the imaging detection module, the electrochemical reaction excitation module and the power module, and the power module is respectively connected to the display, the electrochemical reaction excitation module and the chip automatic loading module;

[0011] The chip automatic loading module is used to transport the dry immune closed bipolar electrochemiluminescence chip to the bottom of the imaging detection module. The electrochemical reaction excitation module is used to stimulate the object to be tested to undergo an electrochemiluminescence reaction and emit a light signal. The imaging detection module is used to convert the light signal generated by the electrochemiluminescence into an electrical signal and transmit it to the data processing and instrument control module. The data processing and instrument control module is used to process the received electrical signal and transmit the result to the display. The display is used for human-computer interaction, image preview and result display.

[0012] The closed bipolar electrode is distributed in an E shape, and the two anodes of the closed bipolar electrode are respectively a detection anode and a quality control anode.

[0013] Three hydrophilic channels are designed on the test sheet, two of which are hydrophilic channels for the flow of test samples, and one is a hydrophilic channel for the flow of buffer solution. The test sheet is stacked on the electrode sheet. The two hydrophilic channels for the flow of test samples overlap with the detection anode and quality control anode of the closed bipolar electrode one by one, and the hydrophilic channel for the flow of buffer solution overlaps with the shared cathode of the closed bipolar electrode.

[0014] The hydrophilic channel on the test sheet that overlaps with the test anode on the electrode sheet serves as the test area, and the hydrophilic channel on the test sheet that overlaps with the quality control anode on the electrode sheet serves as the quality control area. The positions of the test area and the quality control area correspond to the observation window of the upper cover.

[0015] Both the binding sheet and the sample adding sheet are designed with two hydrophilic channels which are distributed in parallel, one is the hydrophilic channel for the sample to be tested and the other is the hydrophilic channel for the buffer solution. The hydrophilic channel for the sample to be tested and the hydrophilic channel for the buffer solution of the binding sheet are respectively connected one-to-one with the hydrophilic channel for the sample to be tested and the hydrophilic channel for the buffer solution of the sample adding sheet, and are also connected correspondingly with the two hydrophilic channels for the sample to be tested and the hydrophilic channels for the buffer solution of the detection sheet.

[0016] The sample flow hydrophilic channel of the sample sheet corresponds to the sample addition hole of the upper cover, the buffer flow hydrophilic channel of the sample sheet corresponds to the buffer addition hole of the upper cover, and the driving electrode of the electrode sheet is correspondingly arranged with the electrode contact area of ​​the upper cover.

[0017] The labeled antibody coupled with the electrochemiluminescent probe and specifically combined with the biomarker to be detected is dried on the hydrophilic channel of the binding chip where the sample to be detected flows.

[0018] The electrochemiluminescent probe is a linear poly-lysine coupled terpyridine ruthenium derivative, and the amount of the labeled antibody is 4.5-7 μL, preferably 5 μL.

[0019] The detection area of ​​the detection sheet is modified with a fixed biomarker capture antibody, and the quality control area of ​​the detection sheet is modified with a fixed quality control capture antibody. The concentration of the biomarker capture antibody ranges from 150 to 250 μg / mL, preferably 200 μg / mL.

[0020] An application of a closed bipolar electrochemiluminescent chip in immunoassay of biomarkers, using the above-mentioned fully automatic dry immune closed bipolar electrochemiluminescent analyzer, comprises the following steps:

[0021] S1. The sample solution to be tested is dripped into the sample adding hole. The solution flows from the sample adding sheet to the detection sheet. The biomarker to be tested specifically binds with the labeled antibody on the binding sheet and the capture antibody on the detection sheet in sequence to form a "labeled antibody-biomarker-capture antibody" sandwich immune complex. Buffer is dripped into the buffer adding hole. The buffer flows to and fills the support channel of the electrode sheet;

[0022] S2, waiting for the immune response incubation time to end, adding buffer to the sample well to promote further binding of the biomarker with the labeled antibody and the capture antibody, and then washing the unbound substances on the detection sheet and filling the reporting channel of the electrode sheet;

[0023] S3, start the closed bipolar electrochemiluminescence analyzer, place the dry immune closed bipolar electrochemiluminescence chip on the chip automatic loading module, and the chip automatic loading module transports the dry immune closed bipolar electrochemiluminescence chip to the bottom of the imaging detection module;

[0024] S4, pressing the detection button on the closed bipolar electrochemiluminescence analyzer, the electrochemical excitation module starts and triggers the closed bipolar electrochemiluminescence reaction, the imaging detection module collects the electrochemiluminescence signal, converts it into an electrical signal and transmits it to the data processing and instrument control module;

[0025] S5. The data processing and instrument control module processes and analyzes the received electrical signals and transmits the results to the display. The user reads the test results on the display and further processes and analyzes them.

[0026] The incubation time of the immune reaction is 2.5-5 min, and the driving voltage of the closed bipolar electrochemiluminescence reaction is 8.5V-9.5V, preferably 9V.

[0027] Compared with the prior art, the present invention has the following advantages and effects:

[0028] 1. The present invention is the first to invent a fully automatic, dry, immune closed bipolar electrochemiluminescence analyzer, which overcomes the shortcomings of existing electrochemiluminescence analyzers based on wet chemical analysis technology, such as the need for multiple steps to add different reaction reagents, complex operation, long detection time, bulky size and high price.

[0029] 2. The "full automatic" of the fully automatic dry immune closed bipolar electrochemiluminescence analyzer of the present invention includes automatic chip entry and exit, automatic signal acquisition, automatic analysis and storage of data, and automatic display of results, which can realize a fully automatic detection process of sample entry / result output.

[0030] 3. The chip automatic loading module of the present invention completes the automatic injection of chips by moving on the track, replacing the manual insertion of chips. There is no need to manually load the chips one by one, thus improving the user experience and satisfaction.

[0031] 4. In the chip structure of the present invention, the electrode sheet does not need to be folded, which simplifies the chip manufacturing process and is easy to mass produce. The electrochemiluminescent probe of linear poly-lysine coupled to a terpyridine ruthenium derivative is used to cascade amplify the electrochemiluminescent signal, achieving higher sensitivity without any modification of the electrode sheet, and realizing ultrasensitive detection of biomarkers.

[0032] 5. The fully automatic dry immune closed bipolar electrochemiluminescence analyzer of the present invention can quickly obtain the test results within 7 minutes by only adding the sample to be tested and the buffer solution; compared with the traditional electrochemiluminescence analyzer, the detection is more convenient and the time is shorter, and it is suitable for on-site rapid detection.

[0033] 6. The fully automatic dry immune closed bipolar electrochemiluminescence analyzer of the present invention can realize rapid quantitative and ultrasensitive detection of different biomarkers, which is beneficial to the early diagnosis, treatment and prognosis of different diseases, such as cancer markers, acute myocardial infarction markers, inflammation markers, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a schematic diagram of the structural decomposition of a dry immune closed bipolar electrochemiluminescent chip;

[0035] Figure 2 It is a schematic diagram of the overall structure of a dry immune closed bipolar electrochemiluminescent chip;

[0036] Figure 3 This is a schematic diagram of the structural decomposition of the fiber microfluidic chip;

[0037] Figure 4 It is the overall structural schematic diagram of the fiber microfluidic chip;

[0038] Figure 5 It is a schematic diagram of the structure of a fully automatic bipolar electrochemiluminescence analyzer;

[0039] Figure 6 This is a graph showing the relationship between the electrochemical luminescence intensity and the driving voltage.

[0040] Figure 7 This is a graph showing the relationship between the electrochemiluminescence intensity and the volume of the labeled antibody.

[0041] Figure 8 Figure 2 is a graph showing the relationship between the electrochemiluminescence intensity value and the capture antibody concentration.

[0042] Fig. 9 This is a graph showing the relationship between the electrochemiluminescence intensity and the immune reaction incubation time.

[0043] Fig.10This is the analytical curve diagram for detecting different concentrations of cardiac troponin cTnI, and the inset is the linear fitting curve diagram of the data.

[0044] Fig.11 Schematic diagram of selectivity evaluation when detecting cardiac troponin cTnI.

[0045] Description of Figure Numbers:

[0046] 1-upper cover, 2-transparent cover plate, 3-fiber microfluidic chip, 4-lower cover, 5-sample addition hole, 6-buffer addition hole, 7-observation window, 8-electrode contact area, 9-sample addition sheet, 10-binding sheet, 11-detection sheet, 12-electrode sheet, 13-bottom plate, 14-closed bipolar electrode, 15, 16-driving electrode, 17-two anodes of closed bipolar electrode, 18-shared cathode, 19-reporting channel, 20-support channel, 21-chip automatic loading module, 22-imaging detection module, 23-data processing and instrument control module, 24-electrochemical reaction excitation module, 25-power module, 26-display. DETAILED DESCRIPTION

[0047] The fully automatic dry immune closed bipolar electrochemiluminescence analyzer of the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0048] The invention discloses a fully automatic dry immune closed bipolar electrochemiluminescence analyzer, comprising a dry immune closed bipolar electrochemiluminescence chip and a closed bipolar electrochemiluminescence analyzer. The dry immune closed bipolar electrochemiluminescence chip generates a specific electrochemiluminescence signal by electrical triggering, and the closed bipolar electrochemiluminescence analyzer acquires, collects and analyzes the electrochemiluminescence signal and displays the detection result to a user.

[0049] The structure and composition of the dry immuno-closed bipolar electrochemiluminescent chip are as follows Figures 1 to 4 As shown, the dry immune closed bipolar electrochemical luminescence chip includes a housing, a transparent cover plate 2 and a fiber microfluidic chip 3. The housing includes an upper cover 1 and a lower cover 4, and the upper cover 1 is provided with a sample injection hole 5, a buffer sample injection hole 6, an observation window 7 and an electrode contact area 8. The fiber microfluidic chip 3 is arranged between the upper cover 1 and the lower cover 4, and the transparent cover plate 2 is arranged between the upper cover 1 and the fiber microfluidic chip 3 and presses the fiber microfluidic chip 3, and the transparent cover plate 2 prevents the fiber microfluidic chip 3 from directly contacting the outside world.

[0050] The fiber microfluidic chip 3 includes a sample adding sheet 9, a binding sheet 10, a detection sheet 11, an electrode sheet 12 and a bottom plate 13. The sample adding sheet 9, the binding sheet 10, the detection sheet 11 and the electrode sheet 12 are sequentially stacked on the bottom plate 13, and each part overlaps by 2 mm.

[0051] The electrode sheet 12 includes a closed bipolar electrode 14 and a pair of driving electrodes 15, 16 arranged in an E shape. The closed bipolar electrode 14 arranged in an E shape includes two anodes 17 and a shared cathode 18, and the two anodes 17 are a quality control anode and a detection anode, respectively. The two anodes 17 of the closed bipolar electrode are adjacently arranged and have the same shape. A reporting channel 19 and a supporting channel 20 are provided on the electrode sheet 12, and the two anodes 17 and the corresponding driving electrodes 15 are located in the reporting channel 19, and the shared cathode 18 and the corresponding driving electrode 16 are located in the supporting channel 20.

[0052] The driving electrodes 15 and 16 of the electrode sheet 12 correspond to the electrode contact area 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 sheet 12 is not modified in any way, and the immunoelectrochemical luminescence reaction of the bipolar electrode can be triggered by powering on.

[0053] The detection sheet 11 is designed with three hydrophilic channels, two of which are hydrophilic channels for the sample to be tested, and the other is a hydrophilic channel for the buffer solution. The two hydrophilic channels for the sample to be tested are adjacent and symmetrically distributed in a "square" shape. The detection sheet 11 is stacked on the electrode sheet 12, and the two hydrophilic channels for the sample to be tested overlap with the two anodes 17 of the closed bipolar electrode in a one-to-one correspondence, and the other hydrophilic channel for the buffer solution overlaps with the shared cathode 18 of the closed bipolar electrode.

[0054] The hydrophilic channel for the sample to be tested that overlaps with the detection anode on the detection sheet 11 is the detection zone (T zone), and the hydrophilic channel for the sample to be tested that overlaps with the quality control anode on the detection sheet 11 is the quality control zone (C zone). The significance of such a design is that after the electrode sheet 12 is connected to a power source, its detection anode and quality control anode can trigger the immunoelectrochemical luminescence reaction of the T zone and C zone of the detection sheet 11.

[0055] The positions of the T zone and the C zone of the detection sheet 11 correspond to the observation window 7 of the upper cover 1. The T zone is used to detect the sample to be tested, and the C zone is used to monitor the chip quality. During the immunoassay, if the C zone does not emit light, it indicates that the test result is meaningless. The T zone of the detection sheet 11 is modified with a fixed 0.2 mg / mL capture antibody of cardiac troponin cTnI (Product Code: D4160MA01-MA, OriGene Company). The C zone of the detection sheet 11 is modified with a fixed quality control capture antibody (goat anti-chicken IgY).

[0056] The binding sheet 10 and the sample adding sheet 9 are both designed with two hydrophilic channels, which are distributed in parallel, one for the sample to be tested and the other for the buffer. The sample to be tested and the buffer flow hydrophilic channels of the binding sheet 10 are connected to the sample to be tested and the buffer flow hydrophilic channels of the sample adding sheet 9, respectively, one by one, and are connected to the two sample to be tested and the buffer flow hydrophilic channels of the detection sheet 11; the significance of such design is to construct a good sample to be tested and the buffer flow channels.

[0057] The hydrophilic channel of the sample adding sheet 9 for the sample to be tested to flow corresponds to the sample adding hole 5 of the upper cover 1 , and the other hydrophilic channel corresponds to the buffer solution adding hole 6 of the upper cover 1 .

[0058] The labeled antibody (Product Code: B9085MA06-MA, OriGene Company) coupled with an electrochemiluminescent probe and specifically bound to the biomarker cardiac troponin cTnI to be tested is dried on the hydrophilic channel of the sample to be tested of the binding sheet 10. The electrochemiluminescent probe is preferably a linear poly-lysine coupled terpyridine ruthenium derivative, which can realize the cascade amplification of the immunoelectrochemiluminescent signal and greatly improve the sensitivity of the detection.

[0059] The detection principle of dry closed bipolar immunoelectrochemiluminescence is based on sandwich-type immune reaction and electrochemiluminescence system based on linear poly-lysine coupled terpyridine ruthenium derivative. The structure of the dry closed bipolar immunoelectrochemiluminescence chip has been further improved, making the chip of the present invention easier to manufacture and simpler to operate. The electrochemiluminescence signal of the dry closed bipolar immunoelectrochemiluminescence chip is triggered, acquired and analyzed by a fully automatic bipolar electrochemiluminescence analyzer.

[0060] The structure of the closed bipolar electrochemiluminescence analyzer is as follows Figure 5 As shown, the closed bipolar electrochemiluminescence analyzer includes 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 supply module 25 and a display 26. The data processing and instrument control module 23 is respectively connected to the display 26, the imaging detection module 22, the electrochemical reaction excitation module 24 and the rechargeable power supply module 25, and the rechargeable power supply module 25 is respectively connected to the display 26, the electrochemical reaction excitation module 24 and the chip automatic loading module 21.

[0061] The chip automatic loading module 21 is controlled by a motor, which can realize the automatic entry and exit of the dry immune closed bipolar electrochemical luminescent chip. The dry immune closed bipolar electrochemical luminescent chip is transported to the bottom of the imaging detection module 22 through the motion track to ensure that the luminous area of ​​the dry immune closed bipolar electrochemical luminescent chip is within the detection field of view. The imaging detection module 22 is a CMOS camera, which is used to obtain the electrochemical luminescence signal of the dry immune closed bipolar electrochemical luminescent chip and convert it into an electrical signal, which is transmitted to the data processing and instrument control module 23. The data processing and instrument control module 23 receives, processes and stores the above electrical signals, and finally transmits the test results to the display 26. The display 26 is a touchable liquid crystal display screen for human-computer interaction, image preview, and result display. The electrochemical reaction excitation module 24 provides a DC power supply for the electrochemical luminescence reaction to trigger electrochemical luminescence. The voltage of the DC power supply is adjustable and can be adjusted according to the actual required voltage. The rechargeable power supply module 25 supplies power to the above modules and has a strong endurance.

[0062] The fully automatic closed bipolar electrochemiluminescence analyzer also includes a data transmission function. The data processing and instrument control module 23 transmits the detection data to the client through wifi, Bluetooth, USB, etc., so that users can view historical detection results and further analyze them. The fully automatic dry immune closed bipolar electrochemiluminescence analyzer of the present invention can realize the detection of different disease biomarkers.

[0063] The method for making the immune dry closed bipolar electrochemiluminescent chip of the present invention comprises the following steps:

[0064] (1) Configuration design: Adobe Illustrator CS6 drawing software was used to design the configurations of the sample adding sheet 9, the binding sheet 10, the detection sheet 11, and the electrode sheet 12 of the fiber microfluidic chip 3, thereby customizing the polyester fiber channel mesh and the electrode mesh, and Solid Works drawing software was used to design the configuration of the shell.

[0065] (2) Printing of chip shell: Use a 3D printer to print the structure of the shell, including an upper cover 1 and a lower cover 4.

[0066] (3) Fiber microfluidic chip processing: The hydrophobic dams of the sample sheet 9, the binding sheet 10, and the detection sheet 11 are made of screen-printed ink, the front closed bipolar electrode 14 and the driving electrodes 15, 16 of the electrode sheet 12 are made of screen-printed carbon paste, and the hydrophobic dam on the back is made of screen-printed solid crayon.

[0067] (4) Fiber microfluidic chip modification

[0068] After the binding sheet 10 is treated with the binding sheet pretreatment solution, the labeled antibody against cardiac troponin cTnI is dripped into the sample flow hydrophilic channel of the binding sheet 10 and vacuum dried at 37°C.

[0069] The T region and C region of the detection sheet 11 are modified and fixed with anti-cardiac troponin cTnI capture antibody and quality control region capture antibody respectively, and the fixation method is covalent bonding of chitosan-glutaraldehyde chemical bonds.

[0070] (5) Chip assembly

[0071] like Figure 4 As shown, the fiber microfluidic chip 3 is first stacked on the bottom plate 13 by the electrode sheet 12, the detection sheet 11, the binding sheet 10, and the sample sheet 9, and each part overlaps 2mm. Then, the transparent cover plate 2 and the assembled fiber microfluidic chip 3 are placed in the housing to complete the assembly of the dry immune closed bipolar electrochemical luminescence chip.

[0072] The assembled dry immune closed bipolar electrochemiluminescence chip can be placed in the chip automatic loading module 21 of the closed bipolar electrochemiluminescence analyzer for sample detection.

[0073] Application Example 1

[0074] Taking the detection of cardiac troponin cTnI as an example, the detection process of the fully automatic dry immunobipolar electrochemiluminescence analyzer of the present invention is as follows:

[0075] (1) The sample solution containing cardiac troponin cTnI to be tested is dripped into the sample well 5, and the solution flows from the sample well 9 to the detection sheet 11. The sample cardiac troponin cTnI to be tested is specifically bound to the dried labeled antibody on the binding sheet 10 and the modified and fixed capture antibody on the detection sheet 11, and finally forms a sandwich immune complex of "labeled antibody-cardiac troponin cTnI-capture antibody". At the same time, a buffer solution is dripped into the buffer well 6, and the solution flows to and fills the support channel 20.

[0076] (2) Wait for 3 minutes and add buffer to the sample well 5. This will promote the rebinding of cardiac troponin cTnI and its corresponding antibody, and wash away the unbound substances in the T and C regions of the detection sheet 11 and fill the reporting channel 19.

[0077] (3) Start the fully automatic closed bipolar electrochemiluminescence analyzer, set the CMOS camera parameters (such as exposure time 300 ms, gain 16, etc.), and open the detection program for standby use.

[0078] (4) The chip is placed on the chip automatic loading module 21, and the chip is transported to the bottom of the detection module 22 via the track.

[0079] (5) Click the detection button on the instrument, the electrochemical reaction excitation module 24 is activated, and the electrochemical luminescence reaction of the chip is electrically triggered to generate a specific electrochemical luminescence signal.

[0080] (6) The electrochemical luminescence signal generated above is collected by the imaging detection module 22 and analyzed by the data processing and instrument control module 23.

[0081] (7) Subsequently, the analyzed test results are transmitted to the display 26, and the user can directly read and further save and analyze them. The test results are imported into the Origin software for processing and analysis, and the data relationship between the electrochemical luminescence intensity value and a certain parameter can be obtained (each data point is calculated using 5 repeated experiments).

[0082] The relationship between the driving voltage and the electrochemiluminescence intensity value is tested by taking a sample solution containing 1 ng / mL cardiac troponin cTnI and the fully automatic closed bipolar electrochemiluminescence analyzer of the present invention as an example.

[0083] The test results are as follows Figure 6 As shown, it can be seen that the electrochemical luminescence intensity value gradually increases with the driving voltage from 6V to 9V. However, when the driving voltage is further increased to 9.5V, the electrochemical luminescence intensity value decreases. The reason may be that the background reaction (such as water oxidation) is excited at a high driving voltage, thereby interfering with the generation of the electrochemical luminescence signal. Therefore, the driving voltage is preferably 9V, and the acceptable range is 8.5-9.5V.

[0084] Application Example 2

[0085] Several important factors affecting the electrochemiluminescence intensity value in Application Example 1 (labeled antibody volume, capture antibody concentration, immune reaction incubation time) were optimized:

[0086] 1) Optimal volume of labeled antibody

[0087] 1. The concentration of cardiac troponin cTnI to be tested is 1 ng / mL, the volume of cardiac troponin cTnI is 30 μL, the driving voltage is 9 V, the volume of labeled antibody is to be determined, the concentration of capture antibody is 100 μg / mL, and the immune reaction incubation time is 3 min.

[0088] 2. Set up several experimental groups: The volume of the labeled antibody was set to several different values ​​(3 μL, 4 μL, 4.5 μL, 5 μL, 6 μL, 7 μL).

[0089] 3. The detection operation and analysis process are the same as in Application Example 1. The experimental results are as follows: Figure 7 shown.

[0090] It can be seen from the experimental results that the electrochemiluminescence intensity value increases with the increase of the labeled antibody volume, and the electrochemiluminescence intensity value does not increase significantly when it is greater than 5μL. The reason for this phenomenon may be that when the labeled antibody volume is greater than 5μL, the number of molecules of the sandwich immune complex formed gradually saturates. Therefore, the labeled antibody volume is preferably 5μL, and the acceptable range is 4.5-7μL.

[0091] 2) Optimal capture antibody concentration

[0092] 1. The concentration of cardiac troponin cTnI to be tested is 1 ng / mL, the volume of cardiac troponin cTnI is 30 μL, the driving voltage is 9 V, the volume of the labeled antibody is 5 μL, the concentration of the capture antibody is to be determined, and the immune reaction incubation time is 3 min.

[0093] 2. Set up several experimental groups: The capture antibody concentration was set to several different values ​​(50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL).

[0094] 3. The detection operation and analysis process are the same as in Application Example 1. The experimental results are as follows: Figure 8 shown.

[0095] It can be seen from the experimental results that the electrochemiluminescence intensity value first increases and then decreases with the increase of the capture antibody concentration. The possible reason for this phenomenon is that the high concentration of capture antibody fixed in the detection area produces a steric effect, which hinders the electron transfer between the electrochemiluminescence probe and the electrode. Based on this fact, the capture antibody concentration is preferably 200μg / mL, and the acceptable range is 150-250μg / mL.

[0096] 3) Optimal immune response incubation time

[0097] 1. The concentration of cardiac troponin cTnI to be tested is 1 ng / mL, the volume of cardiac troponin cTnI is 30 μL, the driving voltage is 9 V, the volume of the labeled antibody is 5 μL, the concentration of the capture antibody is 200 μg / mL, and the immune reaction incubation time is to be determined.

[0098] 2. Set up several experimental groups: the immune response incubation time was set to several different values ​​(1min, 2min, 2.5min, 3min, 4min, 5min, etc.).

[0099] 3. The detection operation and analysis process are the same as in Application Example 1. The experimental results are as follows: Fig. 9 shown.

[0100] It can be seen from the experimental results that as the incubation time of the immune response increases, the electrochemiluminescence intensity value increases accordingly; when the incubation time increases from 3 minutes to 5 minutes, the electrochemiluminescence intensity value tends to be stable. The possible reason for this phenomenon is that after the immune response ends, the number of sandwich immune complexes tends to be stable, resulting in no significant increase in the electrochemiluminescence intensity value. Therefore, the immune response incubation time is preferably 3 minutes, and the acceptable range is 2.5-5 minutes.

[0101] Application Example 4

[0102] Based on the optimized conditions found in Application Example 1 and Application Example 2, the myocardial troponin cTnI was detected using a fully automatic dry immune closed bipolar electrochemiluminescence analyzer.

[0103] 1. Use optimized parameters: cardiac troponin cTnI sample volume is 30μL, driving voltage is 9V, labeled antibody volume is 5μL, capture antibody concentration is 200μg / mL, and immune reaction incubation time is 3min.

[0104] 2. Set up several experimental groups: the concentration of the cardiac troponin cTnI to be tested is set to several different values ​​(100 ng / mL, 50 ng / mL, 10 ng / mL, 1 ng / mL, 0.1 ng / mL, 10 pg / mL, 1 pg / mL, 0.1 pg / mL, 0).

[0105] 3. The detection operation and analysis process are the same as in Application Example 1. The experimental results are as follows: Fig.10 shown.

[0106] From the experimental results, it can be seen that the electrochemiluminescence intensity value increases with the increase of cardiac troponin cTnI concentration. The electrochemiluminescence intensity value (expressed by Y) and the logarithm of cardiac troponin cTnI concentration (expressed by X) have a certain linear relationship, and the linear equation can be expressed as Y = 10.614 + 1.712X (R 2 =0.991, n=5). The background signal without cardiac troponin cTnI plus three times its standard deviation was used as the electrochemiluminescence intensity value, and the detection limit of cardiac troponin cTnI of this method was calculated to be 0.0141 pg / mL. Fig.10 It can also be seen that this method can achieve ultrasensitive quantitative detection of cardiac troponin cTnI in a wide range and has good linearity.

[0107] Application Example 5

[0108] A selective experiment for the detection of cardiac troponin cTnI was carried out using some optimized conditions explored in Application Example 1 and Application Example 2.

[0109] 1. Use optimized parameters: cardiac troponin cTnI sample volume is 30μL, driving voltage is 9V, labeled antibody volume is 5μL, capture antibody concentration is 200μg / mL, and immune reaction incubation time is 3min.

[0110] 2. Set up several interference experimental groups: cardiac troponin cTnI, hemoglobin (HGB), C-reactive protein (CRP), bovine serum albumin (BSA), mixed sample (Mix, a mixture of the first four proteins) and blank control (Blank, PBS buffer). The concentrations of cardiac troponin cTnI and interfering proteins were both 10 ng / mL.

[0111] 3. The detection operation and analysis process are the same as in Application Example 1. The experimental results are as follows: Fig.11 shown.

[0112] It can be seen from the experimental results that compared with the cardiac troponin cTnI group, the electrochemiluminescence intensity value of the mixed sample group containing cardiac troponin cTnI is almost unaffected, and the difference between the two is not large. The electrochemiluminescence intensity values ​​of the hemoglobin HGB, C-reactive protein CRP, and bovine serum albumin BSA interference experimental groups are almost the same as the electrochemiluminescence intensity values ​​of the blank control group Blank. The above results show that the electrochemiluminescence signal only responds to the target cardiac troponin cTnI. Therefore, the method of the present invention can achieve specific detection of cardiac troponin cTnI.

[0113] The above description is a detailed description of the preferred feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes completed under the technical spirit disclosed by the present invention should fall within the patent scope covered by the present invention.

Claims

1. A fully automatic dry immune closed bipolar electrochemiluminescence analyzer, characterized in that: It comprises a dry immune closed bipolar electrochemiluminescent chip and a closed bipolar electrochemiluminescent analyzer. The dry immune closed bipolar electrochemiluminescent chip comprises a shell, a transparent cover plate and a fiber microfluidic chip. The shell comprises an upper cover and a lower cover. The upper cover is provided with a sample addition hole, a buffer addition hole, an observation window and an electrode contact area. The fiber microfluidic chip is arranged between the upper cover and the lower cover. The transparent cover plate is arranged between the upper cover and the fiber microfluidic chip and presses the fiber microfluidic chip. The fiber microfluidic chip includes a bottom plate, an electrode sheet, a detection sheet, a binding sheet and a sample adding sheet, wherein the electrode sheet, the detection sheet, the binding sheet and the sample adding sheet are stacked on the bottom plate, the electrode sheet includes a closed bipolar electrode and a pair of driving electrodes distributed in an E shape, the closed bipolar electrode includes two anodes and a shared cathode, the two anodes are a detection anode and a quality control anode respectively, the two anodes are adjacently distributed and have the same shape, a reporting channel and a supporting channel are provided on the electrode sheet, the two anodes and the corresponding driving electrodes are located in the reporting channel, and the shared cathode and the corresponding driving electrode are located in the supporting channel; The closed bipolar electrochemiluminescence analyzer includes a chip automatic loading module, an imaging detection module, a data processing and instrument control module, an electrochemical reaction excitation module, a power module and a display, wherein the data processing and instrument control module are respectively connected to the display, the imaging detection module, the electrochemical reaction excitation module and the power module, and the power module is respectively connected to the display, the electrochemical reaction excitation module and the chip automatic loading module; The chip automatic loading module is used to transport the dry immune closed bipolar electrochemiluminescent chip to the bottom of the imaging detection module. The electrochemical reaction excitation module is used to excite the object to be tested to undergo an electrochemiluminescent reaction and emit a light signal. The imaging detection module is used to convert the light signal generated by the electrochemiluminescence into an electrical signal and transmit it to the data processing and instrument control module. The data processing and instrument control module is used to process the received electrical signal and transmit the result to the display. The display is used for human-computer interaction, image preview and result display. The test sheet is designed with three hydrophilic channels, two of which are for the flow of the sample to be tested, and one is for the flow of the buffer solution. The test sheet is stacked on the electrode sheet, and the two hydrophilic channels for the flow of the sample to be tested are in the shape of a "square" that is adjacent and symmetrically distributed. The two hydrophilic channels for the flow of the sample to be tested overlap with the detection anode and the quality control anode of the closed bipolar electrode one by one, and the hydrophilic channel for the flow of the buffer solution overlaps with the shared cathode of the closed bipolar electrode. The binding sheet and the sample adding sheet are both designed with two hydrophilic channels which are distributed in parallel, one is the hydrophilic channel for the sample to be tested and the other is the hydrophilic channel for the buffer solution. The hydrophilic channel for the sample to be tested and the hydrophilic channel for the buffer solution of the binding sheet are respectively connected with the hydrophilic channel for the sample to be tested and the hydrophilic channel for the buffer solution of the sample adding sheet in a one-to-one correspondence, and are also connected with the two hydrophilic channels for the sample to be tested and the hydrophilic channels for the buffer solution of the detection sheet in a corresponding manner. The labeled antibody coupled with electrochemiluminescent probe and specifically combined with the biomarker to be tested is dried on the hydrophilic channel of the binding sheet where the sample to be tested flows. The electrochemiluminescent probe is a linear poly-lysine coupled terpyridine ruthenium derivative.

2. The fully automatic dry immune closed bipolar electrochemiluminescence analyzer according to claim 1, characterized in that: The hydrophilic channel on the test sheet that overlaps with the test anode on the electrode sheet serves as the test area, and the hydrophilic channel on the test sheet that overlaps with the quality control anode on the electrode sheet serves as the quality control area. The positions of the test area and the quality control area correspond to the observation window of the upper cover.

3. The fully automatic dry immune closed bipolar electrochemiluminescence analyzer according to claim 1, characterized in that: The sample flow hydrophilic channel of the sample sheet corresponds to the sample addition hole of the upper cover, the buffer flow hydrophilic channel of the sample sheet corresponds to the buffer addition hole of the upper cover, and the driving electrode of the electrode sheet is correspondingly arranged with the electrode contact area of ​​the upper cover.

4. The fully automatic dry immune closed bipolar electrochemiluminescence analyzer according to claim 1, characterized in that: The detection area of ​​the test piece is modified with a fixed biomarker capture antibody, and the quality control area of ​​the test piece is modified with a fixed quality control capture antibody. The concentration range of the biomarker capture antibody is 150-250 μg / mL.

5. Application of a closed bipolar electrochemiluminescent chip in immunoassay of biomarkers, characterized in that: The fully automatic dry immune closed bipolar electrochemiluminescence analyzer according to any one of claims 1 to 4 is used, comprising the following steps: S1. The sample solution to be tested is dripped into the sample adding hole. The solution flows from the sample adding sheet to the detection sheet. The biomarker to be tested specifically binds with the labeled antibody on the binding sheet and the capture antibody on the detection sheet in sequence to form a "labeled antibody-biomarker-capture antibody" sandwich immune complex. Buffer is dripped into the buffer adding hole. The buffer flows to and fills the support channel of the electrode sheet; S2, waiting for the immune response incubation time to end, adding buffer to the sample well to promote further binding of the biomarker with the labeled antibody and the capture antibody, and then washing the unbound substances on the detection sheet and filling the reporting channel of the electrode sheet; S3, start the closed bipolar electrochemiluminescence analyzer, place the dry immune closed bipolar electrochemiluminescence chip on the chip automatic loading module, and the chip automatic loading module transports the dry immune closed bipolar electrochemiluminescence chip to the bottom of the imaging detection module; S4, pressing the detection button on the closed bipolar electrochemiluminescence analyzer, the electrochemical excitation module starts and triggers the closed bipolar electrochemiluminescence reaction, the imaging detection module collects the electrochemiluminescence signal, converts it into an electrical signal and transmits it to the data processing and instrument control module; S5, the data processing and instrument control module processes and analyzes the received electrical signal and transmits the result to the display, and the user reads the test result on the display and further processes and analyzes it; Among them, the incubation time of the immune reaction is 2.5-5 min, and the driving voltage of the closed bipolar electrochemiluminescence reaction is 8.5 V-9.5 V.

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