Microfluidic analysis device and method for electrochemical luminescence immunoassay detection

Abstract

The invention relates to a microfluidic analysis device and method for electrochemical luminescence immunoassay detection, the device comprises a chip body and a heater, the chip body comprises a functional area, a capillary channel, an air connection channel, a detection chamber, an air pressure chamber and a three-electrode sensor, a fluid flowing unit with a height difference and a capillary structure is constructed on the basis of the chip body, every two adjacent functional areas are communicated through a corresponding air connecting channel, the capillary main channel is communicated with the detection cavity, the capillary main channel is provided with a plurality of branch pipe sections which are communicated with the corresponding functional areas one by one, and the branch pipe sections are communicated with the detection cavity. The capillary auxiliary channel enables the detection chamber to be communicated with the air pressure chamber, the three-electrode sensor is arranged in the detection chamber, and the heater is arranged on the back face of the air pressure chamber. According to the invention, dependence on an injection pump or an external mechanical clamp is eliminated, accurate control and automatic operation of liquid in the micro-fluidic chip are realized, and high-sensitivity biomolecule detection is completed on the micro-fluidic chip.

Description

Technical Field

[0001] The invention belongs to the field of microfluidic technology and bioanalysis, and specifically relates to a microfluidic analysis device and method for electrochemiluminescence immunoassay detection. Background Art

[0002] Electrochemiluminescence detection is a highly sensitive and selective analytical technique that is widely used in biosensing, environmental monitoring, and clinical diagnosis. However, traditional electrochemiluminescence detection systems often rely on large instruments and complex operating procedures, resulting in high system detection time and detection costs, which in turn limits the promotion and application of electrochemiluminescence in rapid on-site detection. In the process of biological analysis, precise control of reagent and sample dosage is crucial, especially in electrochemiluminescence immunoassay, where the precise requirements for reagent dosage are more stringent. Therefore, achieving precise management of reagent and sample dosage is a key step to improve analytical accuracy and reliability. Microfluidics, as a technology for precise control and manipulation of microscale fluids, provides the possibility of realizing integrated bioanalysis systems. By integrating multiple analytical functional areas on a microfluidic chip, rapid sample processing, reaction, separation, and detection can be achieved. However, commonly used microfluidic analysis systems mostly use manual operation or external precision syringe pumps to drive fluids, which not only increases the complexity of the system, but also limits its convenience in on-site application. Summary of the invention

[0003] In view of the above-mentioned deficiencies in the prior art, the present invention provides a microfluidic analysis device and method for electrochemiluminescence immunoassay detection.

[0004] The objective of the present invention is achieved through the following technical solutions:

[0005] According to the first aspect of the present specification, there is provided a microfluidic analysis device for electrochemiluminescence immunoassay detection, comprising:

[0006] The chip body comprises at least two functional areas, a capillary channel, a plurality of air connection channels, a detection chamber, an air pressure chamber and a three-electrode sensor, wherein two adjacent functional areas are connected through corresponding air connection channels, the capillary channel comprises a capillary main channel and a capillary sub-channel, the capillary main channel is connected with the detection chamber, the capillary main channel has a plurality of branch sections connected one by one with the corresponding functional areas, the capillary sub-channel connects the detection chamber with the air pressure chamber, all functional areas, capillary channels, all air connection channels, detection chambers and air pressure chambers together constitute a fluid flow unit with a height difference and a capillary structure, and the three-electrode sensor is arranged in the detection chamber and is communicatively connected with an external control circuit;

[0007] A heater is disposed on the back side of the air pressure chamber in the chip body;

[0008] When the air pressure chamber is in a non-negative pressure state, the fluid in each functional area fills the entire functional area in sequence due to the height difference of the fluid flow unit and the capillary structure, and the fluid flowing in from the inlet of the capillary main channel follows the path of the capillary main channel and flows into the detection chamber and the capillary sub-channel in sequence, and is blocked by the capillary structure at the end of the capillary sub-channel and ends at the junction of the capillary sub-channel and the air pressure chamber;

[0009] When the air pressure chamber is in a negative pressure state due to heating by the heater, the fluids in all functional areas flow into the detection chamber and the air pressure chamber in sequence through the capillary channel due to the negative pressure environment formed by the air pressure chamber. After the fluid in one functional area is emptied, the fluid in another adjacent functional area connected through the air connection channel begins to be discharged.

[0010] In certain embodiments, all functional areas include: an incubation chamber, a buffer chamber, and the plurality of air connection channels include a first air connection channel and a second air connection channel;

[0011] The incubation chamber, the buffer chamber and the air pressure chamber are connected to the detection chamber through a capillary channel, the buffer chamber is connected to the incubation chamber through a corresponding air connecting channel, and the incubation chamber can be connected to the outside through the corresponding air connecting channel. The incubation chamber, the corresponding air connecting channel and the air pressure chamber together constitute a pressure difference transmission structure, so that the pressure difference transmission order is: air pressure chamber, first air connecting channel, incubation chamber, second air connecting channel, buffer chamber.

[0012] In some embodiments, the chip body includes a first chip layer as a substrate, a second chip layer attached to the front side of the first chip layer, and a third chip layer attached to the front side of the second chip layer;

[0013] The second chip layer is provided with the incubation chamber, the buffer chamber, all air connection channels, all capillary channels, the detection chamber and the air pressure chamber, and the third chip layer is connected with the incubation chamber, the buffer chamber, the air pressure chamber and the capillary channel in the second chip layer.

[0014] In certain embodiments, the third chip layer has a first injection port, a second injection port, a third injection port, a first air hole and a second air hole. The first injection port, the second injection port, the third injection port, the first air hole and the second air hole on the third chip layer are respectively aligned with the corresponding incubation chamber, the buffer chamber, all capillary channels, the air pressure chamber and the first air connection channel on the second chip layer to construct a fluid channel.

[0015] In certain embodiments, the fluids input into the incubation chamber, the buffer chamber, and all capillary main channels on the second chip layer are procalcitonin sandwich complex solution, tri-n-propylamine buffer, and PBS buffer, respectively.

[0016] In certain embodiments, the three-electrode sensor employs an electrochemiluminescent signal sensor or an optical sensor.

[0017] In some embodiments, the first chip layer is made of glass, and the second chip layer and the third chip layer are both made of methyl methacrylate.

[0018] In certain embodiments, the air pressure chamber has a waste liquid collection area.

[0019] According to the second aspect of this specification, a microfluidic analysis method for electrochemiluminescence immunoassay detection, which is applicable to a microfluidic analysis device for electrochemiluminescence immunoassay detection, comprises the following steps:

[0020] First, a specific immune sandwich complex sample of labeled antibody-test marker-capture antibody, i.e., a procalcitonin sandwich complex solution, is constructed; the procalcitonin sandwich complex solution, tri-n-propylamine buffer, and PBS buffer are respectively injected into the incubation chamber, the buffer chamber, and the capillary main channel through the chip body, wherein the incubation chamber and the buffer chamber belong to the functional area, and the reagents will automatically fill the corresponding microfluidic area step by step along the corresponding flow path, wherein the reagent injection is in order, and the injection of the next level of reagents must wait until the current level of reagents completely fills its corresponding microfluidic area before it can start, and after the reagents fill the corresponding functional area, the PBS buffer reagent injected from the entrance of the capillary main channel will follow the path of the capillary main channel and flow into the detection chamber and the capillary side channel in sequence, and will be blocked by the capillary structure and terminate at the junction of the capillary side channel and the air pressure chamber;

[0021] Then, the heater is started to heat the gas in the air pressure chamber, so that the air pressure chamber is closed from the outside and a negative pressure environment is formed inside;

[0022] The negative pressure of the gas acts on the PBS solution in the capillary side channel through the gas-liquid contact surface at the interface between the gas pressure chamber and the capillary side channel. According to the almost incompressible property of the liquid, the negative pressure of the gas will pull the liquid toward the direction of the gas pressure chamber; the PBS solution stored in the capillary main channel will also flow toward the direction of the gas pressure chamber. According to the Bernoulli principle of fluid, when the fluid accelerates in the pipe or channel, the increase in its speed will cause the hydrostatic pressure of the fluid to decrease accordingly. At this time, the negative pressure of the gas in the gas pressure chamber is transmitted in the form of liquid negative pressure along the capillary main channel to each microchannel area along the way;

[0023] The fluids in all functional areas flow into the detection chamber and the air pressure chamber in turn through the capillary channel due to the negative pressure environment formed by the air pressure chamber. During this process, the procalcitonin sandwich complex solution comes to the detection chamber through the capillary main channel, and will be specifically recognized and captured by the streptavidin modified in the coating layer attached to the working electrode in the detection chamber; then the tri-n-propylamine buffer will enter the detection chamber from the capillary main channel to complete the flushing of the detection chamber, and provide electrochemiluminescence co-reaction conditions for the terpyridine ruthenium labeled antibody, and apply a pulse signal to the working electrode in the detection chamber through an external circuit, so that the detection chamber generates a luminescence signal reflecting the presence and content of the molecule to be tested, and transmits the signal to the external circuit through a three-pole sensor to achieve electrochemiluminescence detection;

[0024] Finally, after the fluid in one functional area is emptied due to negative pressure, the fluid in another adjacent functional area connected through the air connection channel begins to be discharged and is finally discharged into the air pressure chamber.

[0025] The beneficial effects of the present invention are:

[0026] (1) The microfluidic analysis device used for electrochemiluminescence immunoassay detection of the present invention realizes the deterministic release of reagents stored in a series of liquid storage chambers, wherein the release of reagents in the functional area of ​​the current level depends on whether the reagents in the functional area of ​​the previous level are completely emptied; when the functional area of ​​the current level is emptied, it will trigger the release of the next level microfluidic area (liquid storage chamber). Secondly, by utilizing the gas pressure difference, the present invention can realize the self-driving of the detection process. By releasing part of the heat energy accumulated by the gas in the air pressure chamber, a negative pressure difference relative to the atmospheric pressure will be generated in the air pressure chamber. The negative pressure will be transmitted to the microfluidic area discharged in series in the form of hydraulic pressure through the capillary channel and the air connection channel to control the propagation of the liquid chain reaction.

[0027] (2) The present invention adopts an integrated design based on air thermodynamics and capillary action to achieve precise control and automated operation of the liquid in the microfluidic chip, without the need for an external syringe pump or other complex mechanical components, and gets rid of the dependence on the syringe pump or external mechanical fixture. The analytical chip body is provided with key functional areas such as an incubation chamber, a detection chamber and an air pressure chamber. The microfluidic biochip integrates multiple functional areas and has an integrated detection function, which not only simplifies the operating process, reduces the cost and volume of the system, but also improves the stability and reliability of the entire device, and is particularly suitable for rapid on-site detection and emergency response. In addition, the present invention also combines electrochemiluminescence detection technology, which can achieve high-sensitivity biomolecule detection on the microfluidic chip, which is particularly suitable for rapid on-site detection and emergency response. By optimizing the chip design and the electrochemiluminescence system, the present invention can achieve accurate detection of target analytes as low as picomolar levels, providing a new and efficient detection method for disease diagnosis, environmental monitoring and food safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0029] Figure 1 An exploded view of a microfluidic biochip for detecting PCT procalcitonin human serum small molecule protein provided in an embodiment of the present invention.

[0030] Figure 2 A schematic structural diagram of a first chip layer provided in an embodiment of the present invention.

[0031] Figure 3 A schematic structural diagram of a second chip layer provided in an embodiment of the present invention.

[0032] Figure 4 A front view of a second chip layer provided in an embodiment of the present invention.

[0033] Figure 5 for Figure 4 Cross-sectional view along the A-A direction.

[0034] Figure 6 for Figure 4 Cross-sectional view along the B-B direction.

[0035] Figure 7 A schematic structural diagram of a third chip layer provided in an embodiment of the present invention.

[0036] Figure 8A schematic diagram of the structure of a three-electrode sensor provided in an embodiment of the present invention.

[0037] Fig. 9 A schematic structural diagram of a micro heater provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0038] In order to better understand the technical solution of the present application, the embodiments of the present application are described in detail below with reference to the accompanying drawings.

[0039] In some embodiments, Figure 1 As shown, a microfluidic analysis device for electrochemiluminescence immunoassay detection is provided, and the microfluidic analysis device for electrochemiluminescence immunoassay detection includes a chip body and a micro heater.

[0040] The chip body includes at least two functional areas, all capillary channels, several air connection channels, a detection chamber 27, an air pressure chamber 21 and a three-electrode sensor 4, wherein two adjacent functional areas are connected through corresponding air connection channels, the capillary channel includes a capillary main channel 26 and a capillary sub-channel 28, the capillary main channel 26 is connected with the detection chamber 27, the capillary main channel 26 has a plurality of branch sections connected one by one with the corresponding functional areas, the capillary sub-channel connects the detection chamber 27 with the air pressure chamber 21, and all functional areas, all capillary channels, all air connection channels, the detection chamber 27 and the air pressure chamber together constitute a fluid flow unit with a height difference and a capillary structure;

[0041] In some embodiments, all functional areas include: an incubation chamber 23, a buffer chamber 25, and several air connecting channels include a first air connecting channel 22 and a second air connecting channel 24; the incubation chamber 23, the buffer chamber 25 and the air pressure chamber 21 are connected to the detection chamber 27 through a capillary sub-channel 28, the buffer chamber 25 is connected to the incubation chamber 23 through the second air connecting channel 24, and the incubation chamber 23 is connected to the outside through the first air connecting channel 22 (externally connected to the second air hole 35). The incubation chamber 23, the corresponding air connecting channel and the air pressure chamber 21 together constitute a pressure difference transmission structure, so that the pressure difference transmission order is: air pressure chamber 21, first air connecting channel 22, incubation chamber 23, second air connecting channel 24, buffer chamber 25. Among them, the air pressure chamber 21, the incubation chamber 23, the buffer chamber 25 and the detection chamber 27 are all arranged at intervals, and the incubation chamber 23 and the buffer chamber 25 have an inlet and an outlet at both ends along their length directions, respectively. In addition, the incubation chamber 23 is connected with the buffer chamber 25 through the second air connecting channel 24, and both ends of the second air connecting channel 24 are connected between the inlet and the outlet of the incubation chamber 23 and the buffer chamber 25. The incubation chamber 23 is connected with the first air connecting channel 22, and the capillary main channel 26 includes a main section and two branch sections, one end of the main section has an inlet and the other end is connected to the inlet of the detection chamber 27, one end of the two branch sections is connected to the main section and the other end is connected to the outlet of the incubation chamber 23 and the buffer chamber 25 respectively; the outlet of the detection chamber 27 is connected with the air pressure chamber 21 through the capillary sub-channel 28.

[0042] In some embodiments, the chip body includes a first chip layer 1 as a substrate, a second chip layer 2 attached to the front side of the first chip layer 1, and a third chip layer 3 for top packaging and attached to the front side of the second chip layer 2. Figure 3 and Figure 4 The incubation chamber 23, the buffer chamber 25, all the air connection channels, the capillary channel, the detection chamber 27 and the air pressure chamber 21 are arranged on the third chip layer 3, and the third chip layer 3 is connected with the incubation chamber 23, the buffer chamber 25, the air pressure chamber 21 and the capillary channel in the second chip layer 2. In some embodiments, the material of the first chip layer 1 is glass, and the materials of the second chip layer 2 and the third chip layer 3 are both methyl methacrylate.

[0043] In some embodiments, the air pressure chamber 21 has a waste liquid collection area.

[0044] In some embodiments, the third chip layer 3 (eg Figure 7The first sample injection port 31, the second sample injection port 32, the third sample injection port 33, the first air hole 34 and the second air hole 35 on the third chip layer 3 are respectively aligned with the inlet end of the trunk section of the incubation chamber 23, the buffer chamber 25, the capillary main channel 26, the air pressure chamber 21 and the first air connecting channel 22 on the corresponding second chip layer 2 to construct a fluid channel.

[0045] The three-electrode sensor 4 is disposed in the detection chamber 27 and is in communication connection with an external control circuit, such as Figure 8 As shown, the three-electrode sensor 4 is composed of a counter electrode 42, a counter electrode lead 43, a working electrode 44, a working electrode lead 45, a reference electrode 46 and a reference electrode lead 47. The three-electrode sensor 4 is composed of a counter electrode 42, a counter electrode lead 43, a working electrode 44, a working electrode lead 45, a reference electrode 46 and a reference electrode lead 47. The substrate of the circular working electrode 44 is a carbon ink layer 441, and a specially modified streptavidin coating layer 442 thereon is used to capture the labeled procalcitonin small molecule protein. By applying a pulse signal to the three-electrode system, the labeled small molecule protein emits light, thereby linking the concentration of the small molecule protein to be measured with the light intensity signal. In some embodiments, the first chip layer 1 (such as Figure 2 The detection chamber 27 includes a circular reaction chamber, in which the three-electrode sensor 4 attached to the first chip layer 1 is arranged. The three-electrode sensor 4 can collect and detect the reaction signal of the detection chamber 27. In some embodiments, the three-electrode sensor 4 adopts an electrochemical luminescence signal sensor or an optical sensor.

[0046] The heater 5 is arranged on the back side of the air pressure chamber in the chip body and is connected to an external switch circuit, such as Fig. 9 As shown, the heater 5 is composed of a heater mask 51, a heating wire 52 and a heater base 53, wherein the heating wire 52 is designed to be a platinum wire in the shape of a tree root, and its positive electrode interface 521 and negative electrode interface 522 are connected to an external circuit to generate heat. The heater mask 51 and the heater base 53 are both made of polyimide, which can effectively transfer the heat generated by the heating wire to the air pressure chamber to achieve rapid heating.

[0047] In some embodiments, the fluids input into the incubation chamber 23, the buffer chamber 25, and the capillary main channel 26 on the second chip layer 2 are procalcitonin sandwich complex solution, tri-n-propylamine buffer, and PBS buffer, respectively.

[0048] In this embodiment, a gas plug valve is provided at the first air hole of the air pressure chamber 21 to control the flow of gas. When the valve is in an open state, the gas in the air pressure chamber 21 can escape to the outside; when the valve is in a closed state, the gas in the air pressure chamber 21 can only flow in the flow channel of the analysis chip.

[0049] This embodiment also provides a microfluidic analysis method for detecting procalcitonin concentration, which is suitable for a microfluidic analysis device for electrochemiluminescence immunoassay detection, comprising the following steps:

[0050] First, a specific immune sandwich complex sample of labeled antibody-detectable marker-capture antibody is constructed, i.e., a procalcitonin sandwich complex solution; in some embodiments, a procalcitonin small molecule protein sample to be detected is mixed with a labeled antibody and a capture antibody, and incubated at room temperature for 9 minutes to form a specific immune sandwich complex sample of labeled antibody-detectable marker-capture antibody;

[0051] The procalcitonin sandwich complex solution, tri-n-propylamine (TPA) buffer, and PBS buffer are respectively injected into the incubation chamber 23, the buffer chamber 25, and the capillary main channel 26 through the first injection port 31, the second injection port 32, and the third injection port 33 of the chip body. Among them, the incubation chamber 23 and the buffer chamber 25 belong to the functional area, such as Figure 5 As shown, the first vertical section 201 shows the cross-sectional design of the incubation chamber 23 in the microfluidic analysis device. Due to the height potential difference and capillary action, the reagent will automatically fill the corresponding microchannel area step by step along the corresponding flow path, wherein the reagent injection is sequential (the areas where the reagent is injected are the incubation chamber 23, the buffer chamber 25 and the capillary main channel 26 in sequence), and the injection of the next level of reagents can only be started after the current level of reagents completely fills its corresponding microchannel area, and after the reagents fill the corresponding functional area, the PBS buffer reagent injected from the entrance of the capillary main channel will follow the path of the capillary main channel and flow into the detection chamber and the capillary side channel in sequence, and be blocked by the capillary structure and terminate at the junction of the capillary side channel and the air pressure chamber; in addition, the role of the PBS solution is to soak the capillary main channel to prevent the generation of bubbles during the liquid chain reaction;

[0052] The microfluidic analysis device can accurately control the filling of reagents to ensure that there is no overflow, thanks to the bottleneck structure designed at each connection of the liquid storage chamber (for example, the incubation chamber 23, the buffer chamber 25). When the liquid storage chamber is filled with reagents, the first stop valve interface 231, the first fluid interface 232, the second stop valve interface 233, the third stop valve interface 251 and the second fluid interface 252 will be transformed into stop valves, and the reagents will be kept at the stop valves within a certain pressure threshold. Figure 6As shown, the second vertical section 202 shows the cross-sectional design of the stop valve structure between the incubation chamber 23 and the first air connection channel 22. Specifically, the first stop valve interface 231 in the middle of the stop valve structure is a shallow flow channel, and the incubation chamber 23 side is relatively deep, and the first air connection channel 22 side is deeper. According to the principle of capillary action, the reagent first enters the first stop valve interface 231 from the incubation chamber 23. When the reagent attempts to further flow into the first air connection channel 22, due to the sudden expansion of the hydrophilic capillary cross section, the capillary pressure at the liquid / gas interface increases, and this pressure change prevents the reagent from flowing further at the microchannel connection. In addition, the filled incubation chamber 23 is also regarded as the "liquid plug" between the first air connection channel 22, the second air connection channel 24 and the capillary main channel 26, which plays an important role in the subsequent liquid chain reaction. When PBS buffer is injected from the third injection port 33 and infiltrates the capillary main channel 26, the liquid / gas interface at the second fluid interface 252 and the first fluid interface 232 will be transformed into a liquid / liquid interface in sequence, but the pressure on both sides of the stop valve is still within the pressure threshold range, and the reagent in the liquid storage chamber will not flow into the capillary main channel 26 through the second fluid interface 252 and the first fluid interface 232;

[0053] Then, the heater 5 is started (in one embodiment, the heater 5 is a micro heater) to heat the gas in the air pressure chamber. The gas expands due to the heat and escapes to the outside through the first air hole 34. After a period of time, the heater 5 completes the heating cycle and stops working, and the first air hole 34 is closed, so that the air pressure chamber is closed from the outside and a negative pressure environment (about 0.65 to 0.78 standard atmospheric pressure) is formed inside; this negative pressure immediately triggers the start of the liquid chain reaction.

[0054] The negative pressure of the gas acts on the PBS solution in the capillary side channel 28 through the gas-liquid contact surface at the interface between the gas pressure chamber 21 and the capillary side channel 28. According to the almost incompressible property of the liquid, the negative pressure of the gas will pull the liquid toward the direction of the gas pressure chamber 21; the PBS solution stored in the capillary main channel 26 will also flow toward the gas pressure chamber 21. According to the Bernoulli principle of fluid, when the fluid accelerates in the pipeline or flow channel, the increase in its speed will cause the hydrostatic pressure of the fluid to decrease accordingly. At this time, the negative pressure of the gas in the gas pressure chamber 21 is transmitted in the form of liquid negative pressure along the capillary main channel to each microchannel area along the way, effectively controlling the propagation of the liquid chain reaction;

[0055] When the liquid negative pressure reaches the first fluid interface 232 along the capillary main channel 26, the procalcitonin sandwich mixture solution as a "liquid plug" in the incubation chamber 23 will break through the pressure threshold of the stop valve at the connection of the first air connection channel 22 under the combined action of the liquid negative pressure and the atmospheric pressure at the first stop valve interface 231, and the incubation chamber 23 begins to release. The procalcitonin sandwich mixture liquid as a "liquid plug" is pulled into the capillary main channel 26 and pulled toward the air pressure chamber 21. When the incubation chamber 23 is emptied, the "liquid plug" disappears, and the air link connected to the outside world passes through the second stop valve interface 233 and extends to the second air connection channel 24, and the atmospheric pressure directly acts on the third stop valve interface 251. At the same time, the liquid negative pressure reaches the second fluid interface 252 along the capillary main channel 26. In addition, the atmospheric pressure will not break through the first fluid interface 231 and enter through the capillary main channel 26. It can be seen from the cross-sectional structure of the incubation chamber 23 shown in the first vertical cross section 201 that the opening of the first fluid interface 231 is shallower and narrower. According to the principle of capillary action, the first fluid interface 231 has a smaller cross-sectional area and a higher flow resistance. This smaller capillary resistance and shape than the stop valve interface will enhance the capillary pressure at the new liquid / gas interface, effectively preventing air from flowing back into the capillary main channel 26 after the incubation chamber 23 is emptied.

[0056] When the negative pressure of the liquid reaches the second fluid interface 252, the tri-n-propylamine buffer solution as a "liquid plug" in the buffer chamber 25 will break through the pressure threshold of the stop valve at the connection of the second air connection channel 24 under the combined effect of the negative pressure of the liquid and the atmospheric pressure at the third stop valve interface 251, and the buffer chamber 25 begins to release. The tri-n-propylamine buffer solution as a "liquid plug" is pulled into the capillary main channel 26 and pulled toward the direction of the air pressure chamber 21. When the buffer chamber 25 is emptied, the "liquid plug" disappears, and the air link connected to the outside world will extend to the second fluid interface 252. Similarly, the second fluid interface 252 can effectively prevent air from flowing back into the capillary main channel 26 after the buffer chamber 25 is emptied.

[0057] Next, the atmospheric pressure will directly act on the liquid / gas interface at the third injection port 33, pushing the PBS buffer in the remaining capillary main channel to flow toward the air pressure chamber 21 until the pressure of the gas in the air pressure chamber 21 approaches or returns to the atmospheric pressure during the accumulation of waste liquid, and the liquid chain reaction stops.

[0058] The fluids in all functional areas flow into the detection chamber 27 and the pressure chamber 21 in turn through the capillary channel due to the negative pressure environment formed by the pressure chamber. During this process, the procalcitonin sandwich complex solution comes to the detection chamber 27 through the capillary main channel 26, and will be specifically recognized and captured by the streptavidin modified in the coating layer 442 attached to the working electrode 44 in the detection chamber 27; then the tri-n-propylamine buffer will enter the detection chamber 27 from the capillary main channel 26 to complete the flushing of the detection chamber 27 and provide a buffer for the terpyridine The ruthenium-labeled antibody provides electrochemiluminescence co-reaction conditions, and a pulse signal is applied to the working electrode 44 in the detection chamber 27 through an external circuit (the three-electrode sensor is electrically connected to the external circuit through the counter electrode lead 43, the working electrode lead 45 and the reference electrode lead 47). The external circuit applies a pulse signal to the detection chamber 27 through the three-electrode system, so that the detection chamber 27 generates a luminescent signal reflecting the presence and content of the molecule to be detected and transmits the signal to the external circuit through the three-electrode sensor 4, thereby realizing electrochemiluminescence detection;

[0059] Finally, after the fluid in one functional area is emptied due to negative pressure, the fluid in another adjacent functional area connected through the air connection channel begins to be discharged, and is finally discharged into the air pressure chamber 21;

[0060] Specifically, the negative pressure generated by the air pressure chamber 21 is transmitted to the incubation chamber 23 through the first air connection channel 22. The air-liquid contact surface between the first air connection channel 22 and the incubation chamber 23 is affected by the transmitted air negative pressure, and the relative hydraulic pressure of the solution in the incubation chamber 23 increases, first breaking through the hydraulic restraint of the PBS solution in the capillary main channel 26, and the sandwich complex sample liquid enters the capillary main channel 26. When the solution in the incubation chamber 23 is emptied, the capillary valve between the incubation chamber 23 and the second air connection channel 24 is opened, and the air negative pressure will continue to be transmitted to the second air connection channel 24, and the negative pressure will be transmitted to the buffer chamber 25; the process of the air negative pressure acting on the tri-n-propylamine buffer in the buffer chamber 25 is the same as the process of the negative pressure acting on the incubation chamber, and the tri-n-propylamine buffer will then break through the hydraulic restraint of the PBS solution and enter the capillary main channel 26 until it is completely emptied.

[0061] Among them, the PBS solution, sandwich complex sample liquid and tri-n-propylamine buffer will become waste liquid after passing through the detection chamber 27, and enter the air pressure chamber 21 through the capillary side channel 28; at this time, based on the design characteristics of the microfluidic chip, after the liquid chain reaction is started, the air pressure chamber 21 will also assume the function of collecting waste liquid to ensure the cleanliness and efficiency of the entire detection process. When the waste liquid gradually accumulates in the air pressure chamber 21, the gas pressure in the air pressure chamber 21 approaches or returns to the atmospheric pressure, and the liquid chain reaction stops immediately.

[0062] The above are only preferred embodiments of one or more embodiments of this specification, and are not intended to limit one or more embodiments of this specification. Any modifications made within the spirit and principles of one or more embodiments of this specification are not intended to limit this specification.

Claims

1. A microfluidic analysis device for electrochemiluminescence immunoassay detection, characterized in that: include: The chip body comprises at least two functional areas, a capillary channel, a plurality of air connection channels, a detection chamber, an air pressure chamber and a three-electrode sensor, wherein two adjacent functional areas are connected through corresponding air connection channels, the capillary channel comprises a capillary main channel and a capillary sub-channel, the capillary main channel is connected with the detection chamber, the capillary main channel has a plurality of branch sections connected one by one with the corresponding functional areas, the capillary sub-channel connects the detection chamber with the air pressure chamber, all functional areas, capillary channels, all air connection channels, detection chambers and air pressure chambers together constitute a fluid flow unit with a height difference and a capillary structure, and the three-electrode sensor is arranged in the detection chamber and is communicatively connected with an external control circuit; A heater is disposed on the back side of the air pressure chamber in the chip body; When the air pressure chamber is in a non-negative pressure state, the fluid in each functional area fills the entire functional area in sequence due to the height difference of the fluid flow unit and the capillary structure, and the fluid flowing in from the inlet of the capillary main channel follows the path of the capillary main channel and flows into the detection chamber and the capillary sub-channel in sequence, and is blocked by the capillary structure at the end of the capillary sub-channel and ends at the junction of the capillary sub-channel and the air pressure chamber; When the air pressure chamber is in a negative pressure state due to heating by the heater, the fluids in all functional areas flow into the detection chamber and the air pressure chamber in sequence through the capillary channel due to the negative pressure environment formed by the air pressure chamber. After the fluid in one functional area is emptied, the fluid in another adjacent functional area connected through the air connection channel begins to be discharged.

2. The microfluidic analysis device for electrochemiluminescence immunoassay detection according to claim 1, characterized in that: All functional areas include: an incubation chamber, a buffer chamber, and a plurality of air connection channels include a first air connection channel and a second air connection channel; The incubation chamber, the buffer chamber and the air pressure chamber are connected to the detection chamber through a capillary channel, the buffer chamber is connected to the incubation chamber through a corresponding air connecting channel, and the incubation chamber can be connected to the outside through the corresponding air connecting channel. The incubation chamber, the corresponding air connecting channel and the air pressure chamber together constitute a pressure difference transmission structure, so that the pressure difference transmission order is: air pressure chamber, first air connecting channel, incubation chamber, second air connecting channel, buffer chamber.

3. The microfluidic analysis device for electrochemiluminescence immunoassay detection according to claim 2, characterized in that: The chip body comprises a first chip layer as a substrate, a second chip layer attached to the front side of the first chip layer, and a third chip layer attached to the front side of the second chip layer; The second chip layer is provided with the incubation chamber, the buffer chamber, all air connection channels, the capillary channel, the detection chamber and the air pressure chamber, and the third chip layer is connected with the incubation chamber, the buffer chamber, the air pressure chamber and the capillary channel in the second chip layer.

4. The microfluidic analysis device for electrochemiluminescence immunoassay detection according to claim 3, characterized in that: The third chip layer has a first injection port, a second injection port, a third injection port, a first pore, and a second pore. The first injection port, the second injection port, the third injection port, the first pore, and the second pore on the third chip layer are respectively aligned with the corresponding incubation chamber, the buffer chamber, all capillary channels, the air pressure chamber, and the first air connection channel on the second chip layer to construct a fluid channel.

5. The microfluidic analysis device for electrochemiluminescence immunoassay detection according to claim 3, characterized in that: The fluids input into the incubation chamber, the buffer chamber, and all the capillary main channels on the second chip layer are procalcitonin sandwich complex solution, tri-n-propylamine buffer, and PBS buffer, respectively.

6. The microfluidic analysis device for electrochemiluminescence immunoassay detection according to claim 1, characterized in that: The three-electrode sensor adopts an electrochemical luminescence signal sensor or an optical sensor.

7. The microfluidic analysis device for electrochemiluminescence immunoassay detection according to claim 3, characterized in that: The first chip layer is made of glass, and the second chip layer and the third chip layer are both made of methyl methacrylate.

8. The microfluidic analysis device for electrochemiluminescence immunoassay detection according to claim 2, characterized in that: The air pressure chamber has a waste liquid collection area.

9. A microfluidic analysis method for electrochemiluminescence immunoassay detection, characterized in that: A microfluidic analysis device suitable for electrochemiluminescence immunoassay detection according to any one of claims 1 to 8, comprising the following steps: First, a specific immune sandwich complex sample of labeled antibody-test marker-capture antibody, i.e., a procalcitonin sandwich complex solution, is constructed; the procalcitonin sandwich complex solution, tri-n-propylamine buffer, and PBS buffer are respectively injected into the incubation chamber, the buffer chamber, and the capillary main channel through the chip body, wherein the incubation chamber and the buffer chamber belong to the functional area, and the reagents will automatically fill the corresponding microfluidic area step by step along the corresponding flow path, wherein the reagent injection is in order, and the injection of the next level of reagents must wait until the current level of reagents completely fills its corresponding microfluidic area before it can start, and after the reagents fill the corresponding functional area, the PBS buffer reagent injected from the entrance of the capillary main channel will follow the path of the capillary main channel and flow into the detection chamber and the capillary side channel in sequence, and will be blocked by the capillary structure and terminate at the junction of the capillary side channel and the air pressure chamber; Then, the heater is started to heat the gas in the air pressure chamber, so that the air pressure chamber is closed from the outside and a negative pressure environment is formed inside; The negative pressure of the gas acts on the PBS solution in the capillary side channel through the gas-liquid contact surface at the interface between the gas pressure chamber and the capillary side channel. According to the almost incompressible property of the liquid, the negative pressure of the gas will pull the liquid toward the direction of the gas pressure chamber; the PBS solution stored in the capillary main channel will also flow toward the direction of the gas pressure chamber. According to the Bernoulli principle of fluid, when the fluid accelerates in the pipe or channel, the increase in its speed will cause the hydrostatic pressure of the fluid to decrease accordingly. At this time, the negative pressure of the gas in the gas pressure chamber is transmitted in the form of liquid negative pressure along the capillary main channel to each microchannel area along the way; The fluids in all functional areas flow into the detection chamber and the air pressure chamber in turn through the capillary channel due to the negative pressure environment formed by the air pressure chamber. During this process, the procalcitonin sandwich complex solution comes to the detection chamber through the capillary main channel, and will be specifically recognized and captured by the streptavidin modified in the coating layer attached to the working electrode in the detection chamber; then the tri-n-propylamine buffer will enter the detection chamber from the capillary main channel to complete the flushing of the detection chamber, and provide electrochemiluminescence co-reaction conditions for the terpyridine ruthenium labeled antibody, and apply a pulse signal to the working electrode in the detection chamber through an external circuit, so that the detection chamber generates a luminescence signal reflecting the presence and content of the molecule to be tested, and transmits the signal to the external circuit through a three-pole sensor to achieve electrochemiluminescence detection; Finally, after the fluid in one functional area is emptied due to negative pressure, the fluid in another adjacent functional area connected through the air connection channel begins to be discharged and is finally discharged into the air pressure chamber.

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