Centrifugal micro-fluidic chip integrated with functionalized porous membrane and detection method

Abstract

The invention provides a centrifugal micro-fluidic chip integrated with a functional porous membrane and a detection method, and belongs to the field of micro-fluidic electrochemical biosensing. The chip is integrated with an electrochemical detection module and comprises a sample treatment cavity, a first incubation cavity, a second incubation cavity, a functionalized porous membrane fixer, a first electrode, a second electrode, a waste liquid cavity and a plurality of siphon micro-channels. A specific probe is fixed in a pore channel of the functionalized porous membrane, and the concentration of a target object can be converted into an electric signal. During detection, directional transfer of an electrolyte, a cleaning solution and a sample is realized through synergy of the siphon micro-channel and centrifugal force; the electrochemical detection module is driven to rotate by 180 degrees, so that a sample flows across a membrane in an incubation cavity in a reciprocating manner, and the probe-target binding efficiency is remarkably enhanced. The problems that an existing electrochemical biosensor based on a porous membrane is low in mass transfer efficiency and tedious in operation, and integrated detection is difficult to achieve are solved, and the electrochemical biosensor can be adapted to detection of multiple targets such as nucleic acid, protein and metabolite.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic electrochemical biosensing, specifically relating to a centrifugal microfluidic chip with integrated functionalized porous membrane and a detection method. Background Technology

[0002] Electrochemical biosensors, with their advantages of high sensitivity, rapid response, ease of operation, and miniaturization, have been widely used in clinical diagnostics, food safety testing, and environmental monitoring. Among these, electrochemical sensing platforms based on functionalized porous membranes (such as nuclear pore membranes and track-etched membranes) achieve label-free quantitative detection of various targets, including nucleic acid, proteins, and small molecule metabolites, by immobilizing nucleic acid probes, antibodies, or functional nanomaterials on the inner walls of the membrane pores. This is achieved by utilizing the specific binding of the probes to target molecules, which alters the ion transport properties within the pores. In existing technologies, porous membrane-based electrochemical biosensors generally employ static diffusion to bind the sample to the probe within the membrane pores. Target molecules primarily rely on passive diffusion to enter the pores, resulting in a slow mass transfer rate. Furthermore, the sample can only cross the membrane once, with unbound target matter being expelled from the pores, severely limiting the collision frequency and binding probability between the probe and the target. For the detection of low-abundance biomarkers, incubation times of tens of minutes or even hours are often required, and the detection sensitivity is insufficient to meet application requirements. In addition, traditional testing methods generally suffer from drawbacks such as complex equipment, cumbersome operation, and high sample consumption. They are highly dependent on professional laboratory environments and operators, resulting in a testing cycle that can take several hours or even days, making it difficult to meet the timely diagnostic needs of emergency or primary healthcare.

[0003] Microfluidic chip technology enables precise manipulation of fluids through a network of microchannels, integrating the entire process of sample processing, separation, reaction, and detection. It exhibits significant advantages in biochemical analysis and clinical diagnostics, including miniaturization, automation, low consumption, and high throughput. Centrifugal microfluidic chips, driven by centrifugal force generated by rotation, can achieve directional fluid delivery, valve-controlled switching, and volumetric measurement simply by adjusting the rotation speed, eliminating the need for complex external pump and valve systems. This has made it a crucial platform for achieving fully integrated "sample-in, answer-out" analysis. However, current technologies do not disclose a solution for integrating functionalized porous membrane electrochemical detection into centrifugal microfluidic chips and actively controlling the mass transfer process within the membrane pores using centrifugal rotation characteristics and chamber repositioning.

[0004] Therefore, developing a centrifugal microfluidic chip for integrated functionalized porous membrane electrochemical detection, and a detection method that utilizes centrifugal rotation and chamber position control to achieve efficient transmembrane incubation, are technical problems that urgently need to be solved in this field. Summary of the Invention

[0005] To overcome the problems of low mass transfer efficiency, single sample transmembrane crossing, and limited probe-target binding probability in existing porous membrane-based electrochemical biosensors, this invention proposes a centrifugal microfluidic chip and detection method integrating a functionalized porous membrane to solve the above-mentioned technical pain points.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A centrifugal microfluidic chip with integrated functionalized porous membrane includes at least one electrochemical detection module, which is integrated on a rotatable chip body and comprises:

[0008] The sample processing chamber is connected to the sample injection port through the sample inlet channel. The sample processing chamber is used for pre-processing the sample to be tested.

[0009] The first incubation chamber and the second incubation chamber are arranged at intervals in the centrifugal radial direction;

[0010] A functionalized porous membrane immobilizer is disposed between the first incubation chamber and the second incubation chamber to isolate the two chambers and ensure that fluid exchange between the two chambers must pass through the functionalized porous membrane in the immobilizer; the functionalized porous membrane has probes that can specifically bind to the target substance fixed in its pores to convert the concentration signal of the target substance into a detectable electrical signal, thereby realizing the detection of the target substance in the test solution;

[0011] The first electrode and the second electrode are respectively disposed on the cavity walls of the first incubation cavity and the second incubation cavity, and are used to detect the current signal flowing across both sides of the functionalized porous membrane;

[0012] The waste liquid chamber is located on a side further away from the centrifugation center relative to the second incubation chamber;

[0013] The system includes multiple siphon microchannels for controlling the directional transfer of fluids, comprising: a first siphon microchannel, whose inlet end is connected to the sample processing chamber and whose outlet end is connected to the first incubation chamber, for controlling the sample to be tested to enter the first incubation chamber from the sample chamber; a second siphon microchannel, whose inlet end is connected to the electrolyte distribution network and whose outlet end is connected to the first incubation chamber, for controlling the electrolyte to enter the first incubation chamber; a third siphon microchannel, whose inlet end is connected to the cleaning solution distribution network and whose outlet end is connected to the first incubation chamber, for controlling the cleaning solution to enter the first incubation chamber; and a fourth siphon microchannel, whose inlet end is connected to the second incubation chamber and whose outlet end is connected to the waste liquid chamber, for controlling each liquid to be discharged from the second incubation chamber to the waste liquid chamber.

[0014] Furthermore, the electrochemical detection module is configured to be mounted as an independent unit on a chip body, and when the chip body remains stationary, it can rotate 180° relative to the chip body around its own geometric center to realize the interchange of the inner and outer positions of the first incubation cavity and the second incubation cavity in the centrifugal radial direction.

[0015] Furthermore, the first siphon microchannel, the second siphon microchannel, the third siphon microchannel, and the fourth siphon microchannel all achieve valve control through the combined action of capillary action and centrifugal force.

[0016] Furthermore, the functionalized porous membrane is a polyethylene terephthalate (PET) membrane, a polycarbonate (PC) membrane, or a polyimide (PI) membrane, with a pore size of 0.01~12μm and a pore density of 1×10⁻⁶. 3 ~1×10 15 / cm 2 It can ensure sufficient specific surface area while taking into account the hydrodynamic characteristics of liquid flow.

[0017] Furthermore, the inner wall of the functionalized porous membrane is activated to generate functional groups, and the probe is bound to the functional groups through covalent bonds.

[0018] Furthermore, it also includes, integrated on the chip body: an electrolyte distribution network having at least one electrolyte injection port and an electrolyte channel connecting to the inlet end of a second siphon microchannel of each of the electrochemical detection modules; and a cleaning fluid distribution network having at least one cleaning fluid injection port and a cleaning fluid channel connecting to the inlet end of a third siphon microchannel of each of the electrochemical detection modules.

[0019] This invention also provides a detection method for a centrifugal microfluidic chip with integrated functionalized porous membrane, comprising the following steps:

[0020] S1. Initial signal measurement: Electrolyte is injected into the electrolyte injection port, allowing the electrolyte to enter the first incubation chamber through the electrolyte distribution network and the second siphon microchannel, and then permeate through the functionalized porous membrane to the second incubation chamber; the initial current signal I0 is obtained by measuring the first electrode and the second electrode.

[0021] S2. Drain the electrolyte: Start the centrifuge and run it at the first speed so that the electrolyte is discharged into the waste liquid chamber through the fourth siphon microchannel under the action of centrifugal force.

[0022] S3. Initial cleaning: Inject cleaning solution into the cleaning solution inlet, allowing the cleaning solution to enter the first incubation chamber through the cleaning solution distribution network and the third siphon microchannel, and then permeate through the functionalized porous membrane to the second incubation chamber for cleaning; then start centrifugation at the first speed to discharge the cleaning solution into the waste liquid chamber, and repeat this step 1 to 2 times.

[0023] S4. Loading the sample: Inject the sample to be tested into the sample injection port so that it enters the sample processing chamber for sample pretreatment;

[0024] S5. Initial incubation: Start centrifugation at the second speed to allow the sample to enter the first incubation chamber through the first siphon microchannel and permeate through the functionalized porous membrane into the second incubation chamber, so that the target analyte can specifically bind to the probe fixed in the membrane pores.

[0025] S6, Repeated Incubation:

[0026] S6.1 Stop centrifugation and drive the electrochemical detection module to rotate 180° relative to the chip body, so that the inner and outer positions of the first incubation chamber and the second incubation chamber are interchanged in the centrifugation radial direction;

[0027] S6.2 Start centrifugation at the third speed to transfer the sample from the second incubation chamber on the radial inner side to the first incubation chamber on the radial outer side through the functionalized porous membrane;

[0028] S6.3 Stop centrifugation and drive the electrochemical detection module to rotate 180° in the opposite direction to reset;

[0029] S6.4 Start centrifugation at the third speed to transfer the sample from the first incubation chamber on the radial inner side to the second incubation chamber on the radial outer side through the functionalized porous membrane;

[0030] S6.5 Repeat steps S6.1 to S6.4, performing 2 to 4 reciprocating transmembrane transfers to promote the binding of the target analyte to the probe;

[0031] S7. Empty the sample: Start the centrifugation at the first speed and drain the incubated sample into the waste liquid chamber through the fourth siphon microchannel.

[0032] S8. Second cleaning: Repeat step S3;

[0033] S9. Detection signal measurement: Repeat step S1 to measure and obtain the current signal I, and calculate the current change rate (I0 - I) / I0. Quantify the target substance concentration based on the current change rate.

[0034] S10, Final evacuation: Start centrifugation at the first speed to discharge the electrolyte into the waste liquid chamber through the fourth siphon microchannel.

[0035] Furthermore, the first rotational speed, the second rotational speed, and the third rotational speed satisfy: first rotational speed > second rotational speed ≥ third rotational speed; wherein, the first rotational speed is configured to overcome the capillary force of the fourth siphon microchannel to drain the liquid; the second rotational speed is configured to overcome the capillary force of the first siphon microchannel to drive the sample transfer and pass through the functionalized porous membrane for the first time, while ensuring that it remains in the second incubation chamber and is not drained by the fourth siphon microchannel; the third rotational speed is configured to drive the sample to reciprocate across the membrane between the first incubation chamber and the second incubation chamber after the position is interchanged, while ensuring that it is not discharged from the first to the fourth siphon microchannels.

[0036] Technical principle of the invention:

[0037] This invention achieves reciprocating transmembrane flow of samples across a functionalized porous membrane through the synergistic effect of centrifugal force and chamber position interchange, significantly improving the mass transfer efficiency and binding probability between the probe and the target, thereby efficiently converting the target concentration into a quantitatively detectable electrical signal.

[0038] Specifically, the chip integrates a first incubation chamber and a second incubation chamber separated by a functionalized porous membrane. Probes (such as nucleic acids and antibodies) specifically recognizing targets are immobilized on the inner walls of the membrane pores. During detection, by precisely controlling the centrifugation speed and direction, combined with the capillary valve control characteristics of the siphon microfluidic channel, the electrolyte, washing solution, and sample are sequentially loaded and directionally transferred. In the core "reciprocating incubation" stage, the electrochemical detection module is rotated 180° relative to the chip body, exchanging the inner and outer positions of the first and second incubation chambers in the centrifugal radial direction. Centrifugation is then initiated, driving the sample to repeatedly pass through the functionalized porous membrane under the influence of gravity and centrifugal force, transferring it from one chamber to the other. This process is repeated multiple times, allowing the sample to flow through the membrane pores repeatedly, significantly increasing the collision frequency and binding opportunity between target molecules and immobilized probes, thereby achieving efficient capture of low-abundance targets. By measuring the rate of change of transmembrane ion current before and after binding ((I0-I) / I0) using the electrochemical detection module, precise quantitative analysis of the target concentration can be achieved.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] 1. Overcoming mass transfer limitations and significantly improving binding efficiency: Addressing the issue of slow mass transfer rates in existing technologies where target molecules rely solely on passive diffusion or single-pass transmembrane crossings, this invention drives the electrochemical detection module to rotate 180°, causing the sample to flow back and forth across the membrane between the first and second incubation chambers. This design forces the sample to pass through the porous membrane channels multiple times, significantly increasing the collision frequency and binding probability between the target and the immobilized probe. This results in highly efficient capture in a shorter time, overcoming the technical bottlenecks of limited sensitivity and long incubation times for low-abundance targets.

[0041] 2. Achieve end-to-end automation and simplify operation: Addressing the shortcomings of traditional methods, such as complex equipment, cumbersome operation, and high dependence on specialized laboratory environments, this invention integrates functionalized porous membrane electrochemical detection into a centrifugal microfluidic chip. Utilizing the synergistic effect of siphon microchannels and centrifugal force, it achieves fully automated control of the entire process, including sample pretreatment, electrolyte / washing solution loading, transmembrane incubation, and waste liquid discharge. No complex external pump and valve system is required; fluid direction transfer can be achieved simply by adjusting the rotation speed, reducing operational difficulty and truly realizing integrated detection with "sample in, result out."

[0042] 3. Precise control of fluid behavior, improving detection reliability: This invention combines differentiated rotation speeds (first speed > second speed ≥ third speed) with the capillary valve control characteristics of the siphon microfluidic channel to ensure precise execution of each step, including incubation, cleaning, and emptying. This design overcomes the risk of liquid residue or unexpected leakage in microfluidic systems, guaranteeing the repeatability of the detection process and the reliability of the results.

[0043] 4. Expanding the scope of detection applications and enhancing application value: The functionalized porous membrane in this invention can immobilize various probes such as nucleic acids and antibodies, and is suitable for the detection of various targets such as nucleic acids, proteins, and metabolites. The chip supports the parallel integration of multiple detection modules, and combined with a shared electrolyte and cleaning solution distribution network, it can realize the simultaneous analysis of multiple samples or different targets, which has broad application prospects in clinical diagnosis, point-of-care testing, and other fields. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of a single electrochemical detection module.

[0045] Figure 2 This is a schematic diagram of the overall structure of a centrifugal microfluidic chip with an integrated functional porous membrane.

[0046] Figure 3 The image shows the IV curves of the response of different concentrations of tau217 protein.

[0047] Figure 4 The graph shows the ΔIC quantification curves of the rate of change of current in response to different concentrations of tau217 protein.

[0048] Figure 5 IV curves showing the response of different concentrations of Mycoplasma pneumoniae.

[0049] Figure 6 The ΔIC quantification curves represent the rate of change of current in response to different concentrations of Mycoplasma pneumoniae.

[0050] Figure label:

[0051] 100. Electrochemical detection module; 110. Sample processing chamber; 111. Sample injection port; 112. Sample inlet channel; 120. First incubation chamber; 121. First electrode; 130. Second incubation chamber; 131. Second electrode; 140. Functionalized porous membrane fixator; 141. Functionalized porous membrane; 150. Waste liquid chamber; 161. First siphon microchannel; 162. Second siphon microchannel; 163. Third siphon microchannel; 164. Fourth siphon microchannel; 200. Chip body; 210. Electrolyte distribution network; 211. Electrolyte injection port; 220. Cleaning solution distribution network; 221. Cleaning solution injection port. Detailed Implementation

[0052] The present invention will be further described below with reference to specific embodiments.

[0053] Example 1

[0054] This embodiment specifically provides a centrifugal microfluidic chip with integrated functionalized porous membrane, the structure of which is as follows: Figure 1 and Figure 2 As shown.

[0055] Reference Figure 1 A single electrochemical detection module 100 is integrated on a rotatable chip body 200, and mainly includes: a sample processing chamber 110, a first incubation chamber 120, a second incubation chamber 130, a functionalized porous membrane fixator 140, a waste liquid chamber 150, and multiple siphon microchannels.

[0056] The sample processing chamber 110 is a fan-shaped cavity located radially inner to the centrifuge. It is connected to the sample injection port 111 via the sample inlet channel 112. The sample injection port 111 is located at the edge of the chip for easy external sample addition. The sample processing chamber 110 is used for pretreatment of the test sample, including but not limited to dilution, filtration, or isothermal amplification. The corresponding pretreatment system can be matched according to the type of test sample. The first incubation chamber 120 and the second incubation chamber 130 are arranged at intervals in the centrifuge radial direction, and both have the same volume. The first incubation chamber 120 is located radially inner to the centrifuge, and the second incubation chamber 130 is located radially outer to the centrifuge, and the two are connected by a functionalized porous membrane holder 140. The functionalized porous membrane 141 is clamped between the functionalized porous membrane holders 140 and is sealed to the open ends of the first incubation chamber 120 and the second incubation chamber 130 to ensure that fluid exchange between the two chambers must be through the micropores on the functionalized porous membrane 141. The inner wall of the membrane pores was activated by the following steps: The PET membrane was placed in a mixed solution prepared with 0.1 M MES buffer (pH 6.0), containing 10 mg / mL EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and 1 mg / mL NHS (N-hydroxysuccinimide), and reacted at room temperature for 1 hour to activate the carboxyl functional groups on the inner wall of the porous membrane pores. Subsequently, the activated membrane was immersed in an aminated capture probe solution for immobilization for 45 minutes. After the immobilization reaction was completed, it was washed with MES buffer solution to remove unbound capture probes and other impurities. Then, the remaining active sites on the membrane were blocked with bovine serum albumin (BSA) solution to finally obtain the functionalized porous membrane 141. The first electrode 121 and the second electrode 131 were respectively disposed on the cavity walls of the first incubation cavity 120 and the second incubation cavity 130 for detecting transmembrane current signals. The electrode material can be an inert conductive material such as gold, platinum, or carbon, suitable for electrochemical detection. The waste liquid chamber 150 is located outside the centrifugation chamber 130 and is used to collect waste liquid generated in each step to avoid cross-contamination between samples.

[0057] To achieve directional fluid transfer, the chip integrates multiple siphon microchannels, each with a rectangular cross-section, utilizing the synergistic effect of capillary action and centrifugal force to achieve valve-controlled liquid transfer.

[0058] The first siphon microchannel 161 has an inlet end connected to the bottom of the sample processing chamber 110 and an outlet end connected to the top of the first incubation chamber 120. The channel is shaped like an inverted "U". When the chip is stationary, capillary action retains the liquid at the inlet end; when the centrifugal force exceeds the capillary force, the liquid passes over the apex of the channel, realizing the directional transfer of the sample.

[0059] The second siphon microchannel 162 has an inlet end connected to the electrolyte distribution network 210 and an outlet end connected to the first incubation chamber 120, and is used to introduce electrolyte into the incubation chamber.

[0060] The third siphon microchannel 163 has an inlet end connected to the cleaning fluid distribution network 220 and an outlet end connected to the first incubation chamber 120, and is used to introduce cleaning fluid into the incubation chamber.

[0061] The fourth siphon microchannel 164 has an inlet end connected to the bottom of the second incubation chamber 130 and an outlet end connected to the waste liquid chamber 150. Its flow channel parameters are designed to ensure that the liquid can only overcome capillary action and flow into the waste liquid chamber when the centrifugal speed reaches the first speed (1500~2000 r / min), while the liquid is stably retained in the incubation chamber at low speeds.

[0062] Referring to Figure 2, multiple electrochemical detection modules 100 can be integrated onto the same chip body 200 to form a multi-channel parallel detection system. The chip body 200 also integrates:

[0063] Electrolyte distribution network 210: includes one electrolyte injection port 211 and multiple electrolyte channels, which are respectively connected to the inlet end of the second siphon microchannel 162 of each detection module;

[0064] Cleaning fluid distribution network 220: includes one cleaning fluid injection port 221 and multiple cleaning fluid channels, which are respectively connected to the inlet end of the third siphon microchannel 163 of each detection module.

[0065] Electrolyte and cleaning solution can be loaded at once through the injection port and supplied to each detection module synchronously via the distribution network, greatly simplifying the operation process of multi-channel detection.

[0066] Specifically, the electrochemical detection module 100 is constructed as an independent structural unit that can rotate 180° relative to the chip body 200. When the chip body 200 remains stationary, the detection module can be rotated around its geometric center by an external driving mechanism, thereby enabling the interchange of the inner and outer positions of the first incubation chamber 120 and the second incubation chamber 130 in the centrifugal radial direction. This design is the core structural basis for realizing reciprocating transmembrane incubation.

[0067] Example 2

[0068] This embodiment uses the detection of tau217 protein (an Alzheimer's disease-related biomarker) as an example to illustrate the non-amplified target detection process of a centrifugal microfluidic chip based on the integrated functionalized porous membrane. The functionalized porous membrane 141 has a pore size of 0.2 μm and a pore density of 5 × 10⁻⁶. 8The track etching process is performed on a polyethylene terephthalate (PET) film at a depth of / cm². Corresponding capture probes are fixed inside the pores.

[0069] The testing process specifically includes the following steps:

[0070] 1. Initial Signal Measurement: PBS electrolyte is injected through electrolyte injection port 211. The electrolyte enters the first incubation chamber 120 via electrolyte distribution network 210 and the second siphon microchannel 162, and naturally permeates through the functionalized porous membrane 141 into the second incubation chamber 130 under gravity and capillary action. After the liquid level stabilizes (approximately 30 seconds), a scanning voltage of -1 V to 1 V is applied through the first electrode 121 and the second electrode 131 to measure the transmembrane ion current, which is recorded as the initial current signal I0.

[0071] 2. Drain the electrolyte: Start the centrifuge and run it at the first speed (1500-2000 rpm) for 30 seconds to allow the electrolyte to be completely drained into the waste liquid chamber 150 through the fourth siphon microchannel 164 under the action of centrifugal force.

[0072] 3. Initial cleaning: Inject cleaning solution through cleaning solution inlet 221. The cleaning solution enters the first incubation chamber 120 via the cleaning solution distribution network 220 and the third siphon microchannel 163, and then permeates through the functionalized porous membrane 141 into the second incubation chamber 130 to clean the membrane pores and chambers. The cleaning solution is then drained by centrifugation at the first speed for 30 seconds. Repeat this step once.

[0073] 4. Sample loading: Take the sample to be tested and inject it into the sample processing chamber 110 through the sample injection port 111. The sample processing chamber contains pre-lyophilized sample diluent. After the sample is injected, it is reconstituted and mixed after standing for 2 minutes to complete the sample pretreatment.

[0074] 5. Initial Incubation: Start centrifugation at the second rotation speed (600-800 rpm) for 1 minute, allowing the pretreated sample to enter the first incubation chamber 120 through the first siphon microchannel 161 and slowly permeate through the functionalized porous membrane 141 into the second incubation chamber 130. During this process, the tau217 protein in the sample undergoes initial specific binding with the probe immobilized within the membrane pores.

[0075] 6. Repeated incubation:

[0076] 6.1 Stop centrifugation and drive the electrochemical detection module 100 to rotate 180° relative to the chip body 200, so that the inner and outer positions of the first incubation chamber 120 and the second incubation chamber 130 are interchanged in the centrifugation radial direction.

[0077] 6.2 Start centrifugation at the third speed (400-500 rpm) and run for 1 minute to transfer the sample from the second incubation chamber 130 on the radially inner side to the first incubation chamber 120 on the radially outer side through the functionalized porous membrane 141.

[0078] 6.3 Stop centrifugation and drive the electrochemical detection module 100 to rotate 180° in the opposite direction to reset.

[0079] 6.4 Start centrifugation at the third speed (400-500 rpm) and run for 1 minute to transfer the sample from the first incubation chamber 120 on the radially inner side to the second incubation chamber 130 on the radially outer side through the functionalized porous membrane 141.

[0080] 6.5 Repeat steps 6.1 to 6.4 a total of 3 times (i.e., complete 4 transmembrane transfers) to significantly increase the collision frequency and binding probability of tau217 protein and probe.

[0081] 7. Empty the sample: Centrifuge at the first speed (1500-2000 rpm) for 30 seconds, and discharge the incubated sample into the waste liquid chamber 150 through the fourth siphon microchannel 164.

[0082] 8. Second cleaning: Repeat the cleaning process in step 3 (inject cleaning solution, centrifuge and drain, repeat once).

[0083] 9. Detection signal measurement: Repeat step 1, inject electrolyte and measure the transmembrane ion current, denoted as I. Calculate the rate of change of current (I0-I) / I0.

[0084] 10. Final evacuation: Centrifuge at the first speed (1500-2000 rpm) for 30 seconds, and discharge the electrolyte into the waste liquid chamber 150 through the fourth siphon microchannel 164 to complete the detection.

[0085] Following steps 1-10 of Example 2, seven different concentrations of tau217 protein standards (0.1 pg / mL, 1 pg / mL, 5 pg / mL, 10 pg / mL, 20 pg / mL, 50 pg / mL, and 100 pg / mL) were tested. The initial current signal I0 (step 1) and the post-binding current signal I (step 9) were recorded for each test, and current-voltage (IV) curves at different concentrations were plotted, as shown below. Figure 3 As shown. Based on step 9, calculate the current change rate (I0 - I) / I0 corresponding to each concentration. Plot a quantification curve with the target concentration on the x-axis and the current change rate on the y-axis, as shown. Figure 4 As shown. In actual sample testing, simply measure the rate of change of current in the unknown sample using the same procedure, and then substitute it into the standard curve to calculate the concentration of tau217 protein in the sample.

[0086] Example 3 This embodiment uses the detection of Mycoplasma pneumoniae nucleic acid as an example to illustrate the isothermal amplification detection process based on the chip. In this embodiment, the functionalized porous membrane 141 is selected with a pore size of 0.2 μm and a pore density of 5 × 10⁻⁶. 8 Track-etched polyethylene terephthalate (PET) membranes with a diameter of / cm². Mycoplasma pneumoniae-specific nucleic acid capture probes are immobilized within the pores.

[0087] The testing process specifically includes the following steps: 1. Initial signal measurement: Same as step 1 in Example 2, inject PBS electrolyte and measure the initial current signal I0.

[0088] 2. Drain the electrolyte: Same as step 2 in Example 2, centrifuge at the first speed (1500-2000 rpm) to drain the electrolyte.

[0089] 3. Initial cleaning: Same as step 3 in Example 2, inject cleaning solution to clean and drain, repeat once.

[0090] 4. Sample loading and isothermal amplification: The Mycoplasma pneumoniae DNA sample to be tested is directly injected into the sample processing chamber 110 through the sample injection port 111. The sample processing chamber contains pre-lyophilized LAMP amplification reaction reagents, which are incubated at 63°C for 30 minutes to allow the target nucleic acid to undergo exponential amplification within the sample processing chamber 110.

[0091] 5. Initial Incubation: After amplification, centrifuge at the second rotation speed (600-800 rpm) for 1 minute, allowing the reaction solution containing the amplification product to enter the first incubation chamber 120 through the first siphon microchannel 161 and slowly permeate through the functionalized porous membrane 141 into the second incubation chamber 130. During this process, the amplification product undergoes initial specific binding with the capture probe within the membrane pores.

[0092] 6. Reciprocating incubation: Same as step 6 in Example 2, by reciprocating rotation-centrifugation 3 times (each rotation 180°, the third rotation speed 400-500 rpm, run for 1 minute), the reaction solution containing the amplification product is driven to repeatedly flow across the membrane between the two chambers, promoting the full binding of the amplification product and the probe.

[0093] 7. Empty the sample: Same as step 7 in Example 2, centrifuge at the first speed (1500~2000 rpm) for 30 seconds, and discharge the incubated reaction solution into the waste liquid chamber 150 through the fourth siphon microchannel 164.

[0094] 8. Second cleaning: Same as step 8 in Example 2, inject cleaning solution to clean and drain, repeat once.

[0095] 9. Detection signal measurement: Same as step 9 in Example 2, inject electrolyte and measure the transmembrane ion current, denoted as I. Calculate the current change rate (I0-I) / I0.

[0096] 10. Final drainage: Same as step 10 in Example 2, drain the electrolyte by centrifugation at the first speed (1500-2000 rpm) to complete the detection.

[0097] Similarly, taking the Mycoplasma pneumoniae nucleic acid detection in Example 3 as an example, a series of concentration gradient experiments were performed. Following steps 1-10 of Example 3, seven different concentrations of Mycoplasma pneumoniae genomic DNA standards (10... 2 copies / μL, 10 3 copies / μL, 10 4 copies / μL, 10 5 copies / μL, 10 6 copies / μL, 10 7 copies / μL, 10 8 The samples were detected at concentrations of (copies / μL). The initial current signal I0 (step 1) and the post-binding current signal I (step 9) were recorded for each detection. Current-voltage (IV) curves were plotted at different concentrations, as shown below. Figure 5 As shown. Based on step 9, calculate the current change rate (I0-I) / I0 corresponding to each concentration. Plot a quantification curve with the target concentration on the x-axis and the current change rate on the y-axis, as shown. Figure 6 As shown. In actual testing, simply measure the rate of change of current in the unknown sample using the same procedure, and then substitute it into the standard curve to calculate the concentration of Mycoplasma pneumoniae nucleic acid in the sample.

Claims

1. A centrifugal microfluidic chip integrating a functionalized porous membrane, characterized in that, Includes at least one electrochemical detection module (100), said electrochemical detection module (100) being integrated on a rotatable chip body (200) and comprising: The sample processing chamber (110) is connected to the sample injection port (111) through the sample inlet channel (112), and the sample processing chamber (110) is used to preprocess the sample to be tested; The first incubation chamber (120) and the second incubation chamber (130) are arranged at intervals in the centrifugal radial direction; A functionalized porous membrane fixator (140) is disposed between the first incubation chamber (120) and the second incubation chamber (130) to isolate the two chambers and ensure that fluid exchange between the two chambers must pass through the functionalized porous membrane (141); the functionalized porous membrane (141) has probes that can specifically bind to the target substance fixed in its pores to convert the concentration signal of the target substance into a detectable electrical signal, thereby realizing the detection of the target substance in the test solution; The first electrode (121) and the second electrode (131) are respectively disposed on the cavity walls of the first incubation cavity (120) and the second incubation cavity (130) for detecting the current signal flowing across both sides of the functionalized porous membrane (141); The waste liquid chamber (150) is located on the side further away from the centrifugation center relative to the second incubation chamber (130); And a plurality of siphon microchannels for controlling the directional transfer of fluid, including: a first siphon microchannel (161), the inlet end of which is connected to the sample processing chamber (110) and the outlet end of which is connected to the first incubation chamber (120); a second siphon microchannel (162), the inlet end of which is connected to the electrolyte distribution network (210) and the outlet end of which is connected to the first incubation chamber (120); a third siphon microchannel (163), the inlet end of which is connected to the cleaning fluid distribution network (220) and the outlet end of which is connected to the first incubation chamber (120); and a fourth siphon microchannel (164), the inlet end of which is connected to the second incubation chamber (130) and the outlet end of which is connected to the waste liquid chamber (150).

2. The centrifugal microfluidic chip with integrated functionalized porous membrane according to claim 1, characterized in that, The electrochemical detection module (100) is configured to be installed on the chip body (200) as an independent unit, and to rotate 180° relative to the chip body (200) around its own geometric center when the chip body (200) is stationary, so as to realize the interchange of the inner and outer positions of the first incubation cavity (120) and the second incubation cavity (130) in the centrifugal radial direction.

3. The centrifugal microfluidic chip with integrated functionalized porous membrane according to claim 1, characterized in that, The first siphon microchannel (161), the second siphon microchannel (162), the third siphon microchannel (163) and the fourth siphon microchannel (164) all achieve valve control through the combined action of capillary action and centrifugal force.

4. The centrifugal microfluidic chip with integrated functionalized porous membrane according to claim 1, characterized in that, The functionalized porous membrane (141) is a polyethylene terephthalate (PET) membrane, a polycarbonate (PC) membrane, or a polyimide (PI) membrane, with a pore size of 0.01~12μm and a pore density of 1×10⁻⁶. 3 ~1×10 15 / cm 2 .

5. A centrifugal microfluidic chip with integrated functionalized porous membrane according to claim 1, characterized in that, The functionalized porous membrane (141) has its pore inner walls activated to generate functional groups, and the probe binds to the functional groups via covalent bonds.

6. A centrifugal microfluidic chip with integrated functionalized porous membrane according to claim 1, characterized in that, It also includes an electrolyte distribution network (210) integrated on the chip body (200), having at least one electrolyte injection port (211) and an electrolyte channel connected to the inlet end of the second siphon microchannel (162) of each of the electrochemical detection modules (100); and a cleaning fluid distribution network (220), having at least one cleaning fluid injection port (221) and a cleaning fluid channel connected to the inlet end of the third siphon microchannel (163) of each of the electrochemical detection modules (100).

7. A detection method for a centrifugal microfluidic chip with an integrated functionalized porous membrane according to any one of claims 1-6, characterized in that, Includes the following steps: S1. Initial signal measurement: Electrolyte is injected into the electrolyte injection port (211), so that the electrolyte enters the first incubation chamber (120) through the electrolyte distribution network (210) and the second siphon microchannel (162), and permeates through the functionalized porous membrane (141) to the second incubation chamber (130); the initial current signal I0 is obtained by measuring the first electrode (121) and the second electrode (131); S2. Draining electrolyte: Start centrifugation and run at the first speed to drain the electrolyte into the waste liquid chamber (150) through the fourth siphon microchannel (164) under the action of centrifugal force. S3. Initial cleaning: Inject cleaning solution into the cleaning solution inlet (221), so that the cleaning solution enters the first incubation chamber (120) through the cleaning solution distribution network (220) and the third siphon microchannel (163), and permeates through the functionalized porous membrane (141) to the second incubation chamber (130) for cleaning; then start centrifugation at the first speed, so that the cleaning solution is discharged into the waste liquid chamber (150) through the fourth siphon microchannel (164) under the action of centrifugal force. Repeat this step 1 to 2 times. S4. Loading the sample: Inject the sample to be tested into the sample injection port (111) so that it enters the sample processing chamber (110) for sample pretreatment; S5. Initial incubation: Start centrifugation and run at the second speed to allow the sample to enter the first incubation chamber (120) through the first siphon microchannel (161) and permeate through the functionalized porous membrane (141) into the second incubation chamber (130), so that the target substance specifically binds to the probe fixed in the membrane pores; S6, Repeated Incubation: S6.1 Stop centrifugation, and rotate the electrochemical detection module (100) 180° relative to the chip body (200) to interchange the inner and outer positions of the first incubation chamber (120) and the second incubation chamber (130) in the centrifugation radial direction; S6.2 Start centrifugation at the third speed to transfer the sample from the second incubation chamber (130) on the current radial inner side to the first incubation chamber (120) on the radial outer side through the functionalized porous membrane (141). S6.3 Stop centrifugation and drive the electrochemical detection module (100) to rotate 180° in the opposite direction to reset; S6.4 Start centrifugation at the third speed to transfer the sample from the first incubation chamber (120) on the current radial inner side to the second incubation chamber (130) on the radial outer side through the functionalized porous membrane transfer (141). S6.5 Repeat steps S6.1 to S6.4, performing 2 to 4 reciprocating transmembrane transfers to promote the binding of the target analyte to the probe; S7. Empty the sample: Start the centrifugation at the first speed and drain the incubated sample into the waste liquid chamber (150) through the fourth siphon microchannel (164). S8. Second cleaning: Repeat step S3; S9. Detection signal measurement: Repeat step S1 to measure and obtain the current signal I, and calculate the current change rate (I0- I) / I0. Quantify the target substance concentration based on the current change rate. S10, Final emptying: Start centrifugation at the first speed and discharge the electrolyte into the waste liquid chamber (150) through the fourth siphon microchannel (164).

8. The detection method according to claim 7, characterized in that, The first rotational speed, the second rotational speed, and the third rotational speed satisfy the following condition: first rotational speed > second rotational speed ≥ third rotational speed; wherein, the first rotational speed is configured to overcome the capillary force of the fourth siphon microchannel (164) to drain the liquid; the second rotational speed is configured to overcome the capillary force of the first siphon microchannel (161) to drive the sample to transfer and pass through the functionalized porous membrane (141) for the first time, while ensuring that it remains in the second incubation chamber (130) and is not drained by the fourth siphon microchannel (164); the third rotational speed is configured to drive the sample to reciprocate across the membrane between the first incubation chamber (120) and the second incubation chamber (130) after the position is interchanged, while ensuring that it is not discharged from the first to the fourth siphon microchannels.

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