Microfluidic analysis device and method for electrochemiluminescence immunoassay detection

Abstract

The present invention relates to a microfluidic analysis device and method for electrochemiluminescence immunoassay detection. The device includes a chip body and a heater. The chip body includes functional areas, capillary channels, air connection channels, a detection chamber, an air pressure chamber, and a three-electrode sensor. A fluid flow unit with a height difference and a capillary structure is constructed on the chip body. Two adjacent functional areas are connected through corresponding air connection channels. A capillary main channel is connected to the detection chamber. The capillary main channel has multiple branch sections that are connected one-to-one with corresponding functional areas. A capillary secondary channel connects the detection chamber with the air pressure chamber. The three-electrode sensor is arranged in the detection chamber, and the heater is arranged on the back of the air pressure chamber. The present invention gets rid of the dependence on a syringe pump or an external mechanical fixture, realizes precise control and automated operation of the liquid in the microfluidic chip, and completes high-sensitivity biomolecule detection on the microfluidic chip.

Description

Technical Field

[0001] The present invention belongs to the field of microfluidics and bioanalysis, and in particular relates to a microfluidics analysis device and method for electrochemiluminescence immunoassay detection. Background Art

[0002] Electrochemiluminescence (ECL) detection is a highly sensitive and selective analytical technique widely used in fields such as biosensing, environmental monitoring, and clinical diagnostics. However, traditional ECL detection systems often rely on large instrumentation and complex operating procedures, resulting in high system detection time and cost, which in turn limits the widespread application of ECL in rapid on-site testing. Precise control of reagent and sample dosage is crucial in bioanalysis, especially in ECL immunoassays, where precise requirements are even more stringent. Therefore, achieving precise management of reagent and sample dosage is a key step in improving analytical accuracy and reliability. Microfluidics, a technology for precisely controlling and manipulating microscale fluids, offers the potential for integrated bioanalytical 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 often rely on manual operation or external precision syringe pumps to drive the fluid, which not only increases system complexity but also limits their ease of use in on-site applications. 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 purpose of the present invention is achieved through the following technical solutions:

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

[0006] The chip body includes at least two functional areas, a capillary channel, several 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 includes a main capillary channel and a secondary capillary channel. The main capillary channel is connected to the detection chamber. The main capillary channel has multiple branch sections that are connected one-to-one with the corresponding functional areas. The secondary capillary channel connects the detection chamber with the air pressure chamber. All functional areas, capillary channels, all air connection channels, detection chamber, and air pressure chamber together constitute a fluid flow unit with a height difference and a capillary structure. The three-electrode sensor is disposed in the detection chamber and is communicatively connected to an external control circuit.

[0007] a heater, 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. At the end of the capillary sub-channel, it is blocked by the capillary structure and terminates 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 fluid in all functional areas flows 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. 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 connecting channels include a first air connecting channel and a second air connecting 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. The incubation chamber can be connected to the outside world 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 serving as a substrate, a second chip layer attached to a front surface of the first chip layer, and a third chip layer attached to a front surface 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 some 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-detectable 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 injected into the incubation chamber, buffer chamber, and capillary main channel microfluidic regions through the chip body in sequence, wherein the incubation chamber and buffer chamber belong to the functional area, and the reagents will automatically fill the corresponding microfluidic region along the corresponding flow path. The reagent injection is sequential, and the injection of the next level of reagents must wait until the current level of reagents completely fills its corresponding microfluidic region before it can begin. Moreover, after the reagents fill the corresponding functional area, the PBS buffer reagent injected from the inlet 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 terminated 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 gas pressure acts on the PBS solution in the capillary side channel through the gas-liquid contact surface at the interface between the air pressure chamber and the capillary side channel. Due to the nearly incompressible nature of liquids, the negative gas pressure pulls the liquid toward the air pressure chamber. The PBS solution stored in the capillary main channel also flows toward the air pressure chamber. According to the Bernoulli principle of fluid, when a fluid accelerates in a pipe or flow channel, its increased velocity causes a corresponding decrease in the hydrostatic pressure of the fluid. At this time, the negative gas pressure in the air pressure chamber is transmitted in the form of liquid negative pressure along the capillary main channel to each microchannel area along the way.

[0023] Due to the negative pressure environment formed by the air pressure chamber, the fluids in all functional areas flow into the detection chamber and the air pressure chamber in sequence through the capillary channel. During this process, the procalcitonin sandwich complex solution enters the detection chamber through the capillary main channel and is 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 enters 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. A pulse signal is applied to the working electrode in the detection chamber through an external circuit, causing the detection chamber to generate a luminescence signal reflecting the presence and content of the molecule to be tested and transmitting 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 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 microfluidic area of ​​the next level (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 injection pump or other complex mechanical components, and gets rid of the dependence on the injection 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 and reduces the cost and volume of the system, but also improves the stability and reliability of the entire device, and is particularly suitable for on-site rapid 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, and is particularly suitable for on-site rapid detection and emergency response. By optimizing the chip design and 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 following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

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

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

[0031] Figure 3 This is a structural diagram of the second chip layer provided by an embodiment of the present invention.

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

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

[0037] Figure 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, as 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. 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 to the detection chamber 27. The capillary main channel 26 has multiple branch sections that are connected one-to-one with the corresponding functional areas. The capillary sub-channel connects the detection chamber 27 with the air pressure chamber 21. 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 the 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 world 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 each have an inlet and an outlet at both ends along their length direction. In addition, the incubation chamber 23 is connected to 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 outlet of the incubation chamber 23 and the buffer chamber 25. The incubation chamber 23 is connected to 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 to 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 base, a second chip layer 2 attached to the front of the first chip layer 1, and a third chip layer 3 for top packaging and attached to the front of the second chip layer 2. Figure 3 and Figure 4 The third chip layer 3 is provided with an incubation chamber 23, a buffer chamber 25, all air connection channels, a capillary channel, a detection chamber 27, and an air pressure chamber 21. The third chip layer 3 is connected to 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 first chip layer 1 is made of glass, and the second chip layer 2 and the third chip layer 3 are both made of 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 7As shown in the figure, a first injection port 31, a second injection port 32, a third injection port 33, a first air hole 34 and a second air hole 35 are provided on the third chip layer 3 in a vertical direction. The first injection port 31, the second injection port 32, the third 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 incubation chamber 23, the buffer chamber 25, the inlet end of the main section of 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 with an external control circuit, such as Figure 8 As shown, the three-electrode sensor 4 consists 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 consists 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 Detection chamber 27 includes a circular reaction chamber in which a three-electrode sensor 4 attached to first chip layer 1 is disposed. The three-electrode sensor 4 can collect and detect reaction signals from detection chamber 27. In some embodiments, the three-electrode sensor 4 is an electrochemiluminescent signal sensor or an optical sensor.

[0046] The heater 5 is arranged on the back of the air pressure chamber in the chip body and is connected to an external switch circuit, such as Figure 9 As shown, the heater 5 consists of a heater mask 51, a heating wire 52 and a heater base 53, wherein the heating wire 52 is designed as 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 stop valve is installed at the first air hole of the air pressure chamber 21 to control the flow of gas. When the valve is open, the gas in the air pressure chamber 21 can escape to the outside; when the valve is closed, the gas in the air pressure chamber 21 can only flow within the flow path 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 a 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 the labeled antibody and the capture antibody, and the mixture is incubated at room temperature for 9 minutes to form a specific immune sandwich complex sample of a labeled antibody-detectable marker-capture antibody.

[0051] The procalcitonin sandwich complex solution, tri-n-propylamine (TNA) buffer solution, and PBS buffer solution are injected into the microfluidic regions of 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, respectively. 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 along the corresponding flow path. The reagent injection is sequential (the areas for reagent injection are the incubation chamber 23, the buffer chamber 25, and the capillary main channel 26 in sequence). The injection of the next level of reagent must wait until the current level of reagent completely fills its corresponding microchannel area before it can begin. Moreover, after the reagent fills the corresponding functional area, the PBS buffer reagent injected from the inlet 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. It will be blocked by the capillary structure and terminated at the junction of the capillary side channel and the air pressure chamber. In addition, the function 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 has demonstrated the cut-off valve structure cross-section design between the incubation chamber 23 and the first air connecting passage 22.Specifically, the first cut-off valve interface 231 in the middle of the cut-off valve structure is a shallow flow channel, and the incubation chamber 23 side is relatively deep, and the first air connecting passage 22 side is then deeper.According to the capillary action principle, reagent first enters the first cut-off valve interface 231 from the incubation chamber 23. When reagent attempts to further flow into the first air connecting passage 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 can prevent the further flow of reagent at the microfluidic channel junction.In addition, the filling incubation chamber 23 is also regarded as " liquid plug " between described first air connecting passage 22, the second air connecting passage 24 and the capillary main channel 26, plays an important role in 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 successively transform into a liquid / liquid interface, but the pressure on both sides of the stop valve remains 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 (in one embodiment, the heater 5 is a micro heater) is started to heat the gas in the 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, closing the first air hole 34, so that the pressure chamber is closed from the outside and a negative pressure environment (approximately 0.65 to 0.78 standard atmospheres) is formed inside; this negative pressure then triggers the start of the liquid chain reaction.

[0054] The negative gas pressure acts on the PBS solution in the capillary secondary channel 28 through the gas-liquid interface at the interface between the air pressure chamber 21 and the capillary secondary channel 28. Due to the nearly incompressible nature of liquids, the negative gas pressure pulls the liquid toward the air pressure chamber 21. The PBS solution stored in the capillary main channel 26 also flows toward the air pressure chamber 21. According to the Bernoulli principle of fluids, when a fluid accelerates in a pipe or flow channel, its increased velocity causes a corresponding decrease in the hydrostatic pressure of the fluid. At this time, the negative gas pressure in the air 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 negative pressure of the liquid reaches the first fluid interface 232 along the main capillary channel 26, the procalcitonin sandwich mixture solution, acting as a "liquid plug" in the incubation chamber 23, under the combined effects of the negative pressure and the atmospheric pressure applied at the first shut-off valve interface 231, breaks through the pressure threshold of the shut-off valve at the connection to the first air connection channel 22, and the incubation chamber 23 begins to release. The procalcitonin sandwich mixture liquid, acting as a "liquid plug," is drawn into the main capillary channel 26 and toward the air pressure chamber 21. When the incubation chamber 23 is emptied, the "liquid plug" disappears, and the air link connecting to the outside world extends through the second shut-off valve interface 233 to the second air connection channel 24. Atmospheric pressure acts directly on the third shut-off valve interface 251. Simultaneously, the negative pressure of the liquid reaches the second fluid interface 252 along the main capillary channel 26. Furthermore, atmospheric pressure does not break through the first fluid interface 231 and enter the main capillary channel 26. As can be seen from the cross-sectional structure of the incubation chamber 23 shown in the first vertical section 201, the opening of the first fluid interface 231 is shallower and narrower. Due to the principle of capillary action, the first fluid interface 231 has a smaller cross-sectional area and higher flow resistance. This smaller capillary resistance and shape, compared to the stop valve interface, 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 liquid negative pressure reaches the second fluid interface 252, the tri-n-propylamine buffer solution in the buffer chamber 25, which acts as a "liquid plug", will break through the pressure threshold of the shut-off valve at the connection of the second air connection channel 24 under the combined effect of the liquid negative pressure and the atmospheric pressure at the third shut-off valve interface 251, and the buffer chamber 25 begins to release. The tri-n-propylamine buffer solution, which acts as a "liquid plug", is pulled into the capillary main channel 26 and pulled toward 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 solution 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 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 sequence 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 27 through the capillary main channel 26 and is 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 enters the detection chamber 27 from the capillary main channel 26, completing the flushing of the detection chamber 27 and providing a buffer solution 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, causing the detection chamber 27 to generate a luminescent signal reflecting the presence and content of the molecule to be detected, and the signal is transmitted 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 connecting channel 22. The air-liquid interface between the first air connecting channel 22 and the incubation chamber 23 is affected by the transmitted negative air pressure, causing the relative hydraulic pressure of the solution in the incubation chamber 23 to rise, first breaking through the hydraulic constraints of the PBS solution in the capillary main channel 26, and allowing the sandwich complex sample solution to enter 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 connecting channel 24 is opened, and the negative air pressure continues to be transmitted to the second air connecting channel 24, transmitting the negative pressure to the buffer chamber 25. The process of the negative air pressure acting on the tri-n-propylamine buffer in the buffer chamber 25 is similar to the process of the negative pressure acting on the incubation chamber. The tri-n-propylamine buffer then breaks through the hydraulic constraints of the PBS solution and enters the capillary main channel 26 until it is completely emptied.

[0061] After passing through detection chamber 27, the PBS solution, sandwich complex sample solution, and tri-n-propylamine buffer become waste liquid, entering pneumatic chamber 21 through capillary side channel 28. Due to the design characteristics of the microfluidic chip, pneumatic chamber 21 also serves as a waste liquid collector after the liquid chain reaction begins, ensuring a clean and efficient detection process. As waste liquid accumulates within pneumatic chamber 21 and the gas pressure within pneumatic chamber 21 approaches or returns to atmospheric pressure, the liquid chain reaction ceases.

[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 includes at least two functional areas, a capillary channel, several 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 includes a main capillary channel and a secondary capillary channel. The main capillary channel is connected to the detection chamber. The main capillary channel has multiple branch sections that are connected one-to-one with the corresponding functional areas. The secondary capillary channel connects the detection chamber with the air pressure chamber. All functional areas, capillary channels, all air connection channels, detection chamber, and air pressure chamber together constitute a fluid flow unit with a height difference and a capillary structure. The three-electrode sensor is disposed in the detection chamber and is communicatively connected to an external control circuit. a heater, 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. 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. At the end of the capillary sub-channel, it is blocked by the capillary structure and terminates 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 fluid in all functional areas flows 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. 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 the plurality of air connecting channels include a first air connecting channel and a second air connecting 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. The incubation chamber can be connected to the outside world 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 includes a first chip layer as a substrate, a second chip layer attached to the front surface of the first chip layer, and a third chip layer attached to the front surface of the second chip layer; 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 main channel in the second chip layer.

4. The microfluidic analysis device for electrochemiluminescence immunoassay 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 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, the capillary main channel, 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 according to claim 3, characterized in that: The fluids input into the incubation chamber, the buffer chamber, and the capillary main channel 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 according to claim 1, characterized in that: The three-electrode sensor adopts an electrochemiluminescence signal sensor.

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

8. The microfluidic analysis device for electrochemiluminescence immunoassay 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 claim 4, comprising the following steps: First, a specific immune sandwich complex sample of labeled antibody-detectable 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 injected into the incubation chamber, buffer chamber, and capillary main channel microfluidic regions through the chip body in sequence, wherein the incubation chamber and buffer chamber belong to the functional area, and the reagents will automatically fill the corresponding microfluidic region along the corresponding flow path. The reagent injection is sequential, and the injection of the next level of reagents must wait until the current level of reagents completely fills its corresponding microfluidic region before it can begin. Moreover, after the reagents fill the corresponding functional area, the PBS buffer reagent injected from the inlet 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 terminated at the junction of the capillary side channel and the air pressure chamber. Then, the heater is activated to heat the gas in the pressure chamber. The gas expands due to the heat and escapes to the outside through the first air hole. After a period of time, the heater completes the heating cycle and stops working, closing the first air hole. The pressure chamber is sealed off from the outside world and a negative pressure environment is formed inside. This negative pressure then triggers the start of the liquid chain reaction. The negative gas pressure acts on the PBS solution in the capillary side channel through the gas-liquid contact surface at the interface between the air pressure chamber and the capillary side channel. Due to the nearly incompressible nature of liquids, the negative gas pressure pulls the liquid toward the air pressure chamber. The PBS solution stored in the capillary main channel also flows toward the air pressure chamber. According to the Bernoulli principle of fluid, when a fluid accelerates in a pipe or flow channel, its increased velocity causes a corresponding decrease in the hydrostatic pressure of the fluid. At this time, the negative gas pressure in the air pressure chamber is transmitted in the form of liquid negative pressure along the capillary main channel to each microchannel area along the way. Due to the negative pressure environment formed by the air pressure chamber, the fluids in all functional areas flow into the detection chamber and the air pressure chamber in sequence through the capillary channel. During this process, the procalcitonin sandwich complex solution enters the detection chamber through the main capillary channel and is 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 enters the detection chamber from the main capillary channel to complete the flushing of the detection chamber and provide electrochemiluminescence co-reaction conditions for the terpyridine ruthenium-labeled antibody. A pulse signal is applied to the working electrode in the detection chamber through an external circuit, causing the detection chamber to generate a luminescence signal reflecting the presence and content of the molecule to be tested and transmitting 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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