CN217587030U - Dry-type immune closed bipolar electrochemical luminescence chip - Google Patents

Abstract

The utility model discloses a dry-type immune closed bipolar electrochemical luminescence chip, which comprises a shell, a fiber micro-fluidic chip and a transparent cover plate, wherein 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, the fiber micro-fluidic chip is arranged between the upper cover and the lower cover, and the transparent cover plate is arranged between the upper cover and the fiber micro-fluidic chip and compresses the fiber micro-fluidic chip; 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 positioned in the report channel, the shared cathode and the corresponding driving electrodes are positioned in the support channel, dry quantitative detection of biomarkers is convenient to realize, the operation is convenient and fast, and the sensitivity is high.

Description

Dry-type immune closed bipolar electrochemical luminescence chip

Technical Field

The utility model relates to a medical science detection device technical field, specifically speaking relates to a bipolar electrochemical luminescence chip of dry-type immune closed type.

Background

In recent years, bioanalytical techniques have been rapidly developed in various fields. The principle of the immunoassay is that electrochemical luminescence probes such as terpyridyl ruthenium and the like are used as markers under the condition of not influencing the activity of an antibody (antigen), and after the electrochemical luminescence probes are combined with the corresponding antigen (antibody), specific electrochemical luminescence reaction is initiated under electric triggering, so that the detection result is stable and reliable, and the accuracy and precision are superior to those of 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 extensive research interest. At present, many researches are dedicated to combine a certain detection method with a microfluidic chip technology and develop a microfluidic chip-based detection technology 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.

In the traditional liquid phase and wet chemical detection at present, the electrochemical luminescence chip material is generally an inorganic material or a polymer material, the electrode material is expensive, the processing equipment related to the electrochemical luminescence chip is expensive, the modification process of the electrochemical luminescence chip is complicated, the electrochemical luminescence chip is not easy to use quickly and conveniently, and the application scenes of the electrochemical luminescence chip are greatly limited.

Disclosure of Invention

An object of the utility model is to overcome prior art's defect, provide a bipolar electrochemical luminescence chip of dry-type immune closed type, can realize biomarker's dry-type quantitative determination, simple operation, sensitivity height.

In order to achieve the above object, the utility model adopts the following technical scheme:

a dry-type immune closed bipolar electrochemical luminescence chip comprises a shell, a fiber microfluidic chip and a transparent cover plate, wherein 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, the fiber microfluidic chip is arranged between the upper cover and the lower cover, and the transparent cover plate is arranged between the upper cover and the fiber microfluidic chip and compresses the fiber microfluidic chip;

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, and the shared cathode and the corresponding driving electrodes are located in the support channel.

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 is used as a detection area, and the hydrophilic channel overlapped with the quality control anode on the detection sheet is used as a quality control area.

The detection area and the quality control area correspond to the observation window of the upper cover, and the drive electrode of the electrode plate corresponds to the electrode contact area 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 flow hydrophilic channel of the sample adding sheet corresponds to the sample adding hole of the upper cover, and the buffer flow hydrophilic channel of the sample adding sheet corresponds to the buffer adding hole of 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 mug/mL, preferably 200 mug/mL.

Compared with the prior art, the utility model has the advantages and effects of it is following:

1. the utility model has the advantages that the electrode plates do not need to be folded on the chip structure, the chip manufacturing process is simplified, and the mass production is easy; 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.

2. The utility model discloses dry-type immunity closed type bipolar electrochemiluminescence chip only need dropwise need await measuring sample and buffer liquid alright obtain the testing result fast in 7min, detect more convenient, the time is shorter, is applicable to on-the-spot short-term test.

3. The dry-type immune closed bipolar electrochemiluminescence chip can realize rapid quantification and hypersensitive detection of different biomarkers, and is favorable for 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 view showing the structural decomposition of a dry immuno-closed bipolar electrochemiluminescence chip;

FIG. 2 is a schematic diagram of the whole structure of a dry immune closed bipolar electrochemical luminescence 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 diagram of a fully automatic bipolar electrochemical luminescence analyzer;

FIG. 6 is a graph of the relationship between the electrochemiluminescence intensity and 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 indicate:

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 following description will further explain the dry immuno-closed bipolar electrochemiluminescence chip of the present invention with reference to the accompanying drawings and specific embodiments.

The utility model discloses a bipolar electrochemiluminescence chip of dry-type immune closed type, the structure and the constitution of the bipolar electrochemiluminescence chip of dry-type immune closed type are as shown in fig. 1 to fig. 4, and the bipolar electrochemiluminescence chip of dry-type immune closed type includes shell, transparent cover 2 and fibre micro-fluidic chip 3. The shell comprises an upper cover 1 and a lower cover 4, wherein the upper cover 1 is provided with a sample adding hole 5, a buffer solution sample adding 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, 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 microfluidic 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 stacked on the bottom plate 13, and the stacking positions of all parts are overlapped by 2mm.

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 a quality control anode and a detection anode respectively. 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 positioned in the report channel 19, and the shared cathode 18 and the corresponding driving electrodes 16 are positioned 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 can trigger the immune electrochemical luminescence reaction of the bipolar electrode when electrified without any modification.

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 plate 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 plate 11.

The positions of the T area and the C area of the detection piece 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 detection sheet 11 is modified and fixed with a 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, oriGenecompany) which is coupled with an electrochemiluminescence probe and is specifically bonded with cardiac troponin cTnI of a 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 bipolar immunochemistry electro-luminescence chip is further improved, so that the chip of the utility model is simpler in manufacture and simpler in operation. 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 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 is triggered by electricity to generate a specific electrochemical luminescence signal, and the closed bipolar electrochemical luminescence analyzer acquires, collects and analyzes the electrochemical luminescence signal and displays a detection result for a user.

The structure of the closed bipolar electrochemical luminescence analyzer is shown in fig. 5, the closed bipolar electrochemical luminescence analyzer includes a chip auto-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 display 26 is respectively connected with the data processing and instrument control module 23, the imaging detection module 22 and the electrochemical reaction excitation module 24, and the rechargeable power module 25 is respectively connected with the data processing and instrument control module 23, the electrochemical reaction excitation module 24 and the chip auto-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 utility model discloses dry-type immunity closed bipolar electrochemical luminescence chip can realize the detection to different disease biomarkers.

The utility model discloses a manufacturing method of dry-type immunity closed type bipolar electrochemical luminescence chip, including following step:

(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 SolidWorks 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 fiber microfluidic chip 3 is first stacked on a bottom plate 13 by an electrode sheet 12, a detection sheet 11, a combination sheet 10, and a sample addition sheet 9 in sequence, and each stacked position is overlapped by 2mm. 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 the cardiac troponin cTnI as an example, the detection process of the full-automatic dry-type immune bipolar electrochemiluminescence analyzer of the utility model 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 rebinding of the cardiac troponin cTnI to its corresponding antibody, and washes away the 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 type 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 (3) importing the detection result into Origin software for processing and analysis, and obtaining a data relation between the electrochemiluminescence intensity value and a certain parameter (each data point is obtained by adopting 5 repeated experiments for calculation).

The sample solution that awaits measuring that now contains 1ng/mL cardiac troponin cTnI and the utility model discloses a relation between driving voltage and the electrochemiluminescence intensity value is tested for example to full-automatic closed bipolar electrochemical luminescence analyzer of formula.

The test results are shown in fig. 6, and it can be seen that: the electrochemiluminescence intensity value is gradually increased with the increase of the driving voltage 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 adding 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 3min.

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 addition 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 3min.

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. This may be caused by steric hindrance of the high concentration capture antibody immobilized in the detection region, which may hinder electron transfer between the electrochemiluminescent probe and the electrode. In view of this fact, the capture antibody concentration is preferably 200. Mu.g/mL, with an acceptable range of 150-250. 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 (1 min, 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 of the immunoreaction is preferably 3min, and the acceptable range is 2.5-5min.

Application example 4

The detection of cardiac troponin cTnI was carried out using a dry immuno-closed bipolar electrochemiluminescence chip under the 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 3min.

2. Several experimental groups were set up: the cTnI concentration of the cardiac troponin to be tested was set at several different values (100 ng/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: electrochemical luminescence intensity value following heartThe concentration of the troponin cTnI is increased. 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 Y =10.614+1.712X (R =10.614 +1.2X 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 0.0141pg/mL. It can also be seen from fig. 10 that the method can realize a wide range of myocardial troponin cTnI hypersensitive 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 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 3min.

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 of the first 4 proteins), and Blank control (Blank, PBS buffer). Both the cardiac troponin cTnI and the interfering protein concentration were 10ng/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 only responds to the target cardiac troponin cTnI. Therefore, the method of the utility model can realize the specific detection of the cardiac troponin cTnI.

The above description is for the detailed description of the preferred possible embodiments of the present invention, but the embodiments are not intended to limit the scope of the present invention, and all equivalent variations or modifications made under the technical spirit of the present invention shall fall within the scope of the present invention.

Claims (10)

1. A dry-type immune closed bipolar electrochemical luminescence chip is characterized by comprising a shell, a fiber microfluidic chip and a transparent cover plate, wherein the shell comprises an upper cover and a lower cover, the upper cover is provided with a sample adding hole, a buffer adding hole, an observation window and an electrode contact area, the fiber microfluidic chip is arranged between the upper cover and the lower cover, and the transparent cover plate is arranged between the upper cover and the fiber microfluidic chip and tightly presses the fiber microfluidic chip;

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, and the shared cathode and the corresponding driving electrodes are located in the support channel.

2. The dry-type immune closed bipolar electrochemiluminescence chip 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 dry-type immuno closed bipolar electrochemiluminescence chip of claim 2, wherein the detection sheet is designed with three hydrophilic channels, two are sample flow hydrophilic channels to be detected, and one is buffer flow hydrophilic channel, the detection sheet is stacked on the electrode sheet, the two sample flow hydrophilic channels to be detected are overlapped with the detection anode and the quality control anode of the closed bipolar electrode in a one-to-one correspondence, and the buffer flow hydrophilic channel is overlapped with the shared cathode of the closed bipolar electrode in a one-to-one correspondence.

4. The dry-type immuno-closed bipolar electrochemiluminescence chip of claim 3, wherein the hydrophilic channel of the test strip overlapping with the test anode of the electrode strip is used as a detection zone, and the hydrophilic channel of the test strip overlapping with the quality control anode of the electrode strip is used as a quality control zone.

5. The dry immuno-occlusive bipolar electrochemiluminescence chip of claim 4, wherein the detection zone and the quality control zone are located corresponding to the viewing window of the top cover, and the driving electrode of the electrode sheet is located corresponding to the electrode contact area of the top cover.

6. The dry-type immuno-closed bipolar electrochemiluminescence chip of claim 3, 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 channel of the detection pad.

7. The dry-type immuno-closed bipolar electrochemiluminescence chip of claim 6, wherein the sample-flow hydrophilic channel of the sample application sheet corresponds to the sample application well of the top cover, and the buffer-flow hydrophilic channel of the sample application sheet corresponds to the buffer application well of the top cover.

8. The dry-type immune closed bipolar electrochemiluminescence chip as claimed in claim 3, wherein the sample flowing 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 detected.

9. The dry-type reclosable bipolar electrochemiluminescence chip of claim 8, 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.

10. The dry immuno-closed bipolar electrochemiluminescence chip of claim 3, 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 150-250 μ g/mL.

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