Microfluidic detection test paper and microfluidic detection method

Abstract

The application discloses a microfluidic detection test paper, comprising: a sample adding port for adding a sample to be detected; a flow channel connected with the sample adding port; an air bag connected with the flow channel and having a first intersection point with the flow channel, the air bag having a compressed state and an expanded state, and the switching between the two states being controllable; and the flow channel being formed with a detection zone located downstream of the first intersection point. The application also discloses a microfluidic detection method. The application can carry out reaction and detection without introducing additional liquid or gas, has simple overall structure, and consumes less energy; the channel is directly arranged at a detection terminal point, and a fourth control layer is correspondingly arranged, so that waste liquid can be directly absorbed by the fourth control layer through micropores after reaction or detection is completed, and the risk of backflow is completely avoided; two or more air bags work in cooperation, and the distance through which liquid flows in the channel of the test paper is highly controllable.

Description

Technical Field

[0001] This invention belongs to the field of test strip technology, and in particular relates to a microfluidic test strip and a microfluidic detection method. Background Technology

[0002] Microfluidic chips are widely researched and applied due to their ability to manipulate small volumes of reaction liquids, enable automated control, and achieve high experimental throughput. A microfluidic chip consists of a substrate and microchannels within the substrate. Through the coordinated action of valves and pumps, samples and reagents are precisely delivered to target locations, enabling sample detection.

[0003] Currently available microfluidic chips cannot achieve closed-loop reaction and detection without introducing additional liquids or gases besides the sample to be tested, nor can they collect waste liquid after the reaction without the risk of backflow. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention provides a microfluidic test paper and a microfluidic detection method that are simple in structure, can avoid the risk of backflow, and have high controllability.

[0005] The technical solution adopted by this invention to solve its technical problem is: a microfluidic test paper, comprising:

[0006] The sample dispensing port is used to add the sample to be tested;

[0007] The flow channel is connected to the sample dispensing port;

[0008] An airbag is connected to the circulation channel and has a first intersection point with the circulation channel. The airbag has a compressed state and an inflated state, and the switching between the two states is controllable.

[0009] The flow channel forms a detection zone, which is located downstream of the first intersection point.

[0010] Furthermore, a sensing area is provided between the first intersection point and the detection area to form a quantitative zone between the first intersection point and the sensing area, which is used to determine the amount of sample to be tested delivered to the detection area.

[0011] Furthermore, the first control layer serves as a cover layer; the second control layer is made of an elastic material; and the third control layer serves as a sensing layer. The first, second, and third control layers are stacked together to form the sample inlet, flow channel, and airbag.

[0012] Furthermore, the airbag includes at least a first airbag and a second airbag, and the detection area is located in the flow channel between the first airbag and the second airbag, or the detection area is located in the flow channel downstream of the second airbag.

[0013] Furthermore, the flow channel between the first airbag and the second airbag is bent, and the detection area is located within this bent area.

[0014] Furthermore, the sample has a first end, a second end, a third end, and a fourth end with the first intersection point as the interruption point. The sample with the first end as the starting point and the second end as the ending point is located in the quantitative zone, while the sample with the fourth end as the starting point and the third end as the ending point can flow upstream to the sample dispensing port.

[0015] Furthermore, the flow channel is bent to form an M-shape with the opening facing to the side.

[0016] Furthermore, the third control layer is equipped with electrodes, and the current generated by the reaction is transmitted to the detection instrument through the electrodes and electrode contacts.

[0017] Furthermore, it also includes a fourth control layer with a waste liquid area, which is connected to the flow channel through the first channel. This fourth control layer is stacked together with the first control layer, the second control layer, and the third control layer.

[0018] Furthermore, the first control layer has a gas channel, and the airbag is located upstream of the gas channel.

[0019] Furthermore, the downstream of the flow channel has multiple branch channels, and each branch channel has an opening for a gas passage on its sidewall.

[0020] This invention also discloses a microfluidic detection method, utilizing the aforementioned microfluidic test paper, comprising the following steps:

[0021] The airbag is in a compressed state;

[0022] Place the sample into the sample dispensing port;

[0023] The compressed air bladder is released to adsorb the sample flow to the quantification zone, which is used to determine the sample volume required for the reaction;

[0024] The airbag continues to release pressure, and a fixed amount of sample continues to flow to the detection area;

[0025] The sample reacts with a pre-placed reagent in the detection area;

[0026] The instrument detects the reaction results;

[0027] or,

[0028] The airbag is inflated;

[0029] Place the sample in the sample inlet, and the sample will flow under the siphon effect;

[0030] Pressure is applied to the inflated air bladder to allow the sample to flow and to determine the sample volume required for the reaction;

[0031] The airbag continues to apply pressure, and the quantified sample continues to flow into the detection area;

[0032] The sample reacts with a pre-placed reagent in the detection area;

[0033] The instrument detects the reaction results.

[0034] Furthermore, this includes the following steps:

[0035] The first airbag is inflated, and the second airbag is compressed.

[0036] Place the sample into the sample dispensing port;

[0037] The compressed second air bladder is released to adsorb the sample flowing to the quantification zone, which is used to determine the sample volume required for the reaction;

[0038] The second airbag stops releasing pressure and applies pressure to the first airbag, driving the quantitative sample to continue flowing to the detection area;

[0039] The sample reacts with a pre-placed reagent in the detection area;

[0040] The instrument detects the reaction results.

[0041] Furthermore, the process includes a thorough mixing step, where the compressed second air bladder is released to adsorb the sample flow to the quantitative zone, the second air bladder is pressurized, and the sample flows back to the dispensing port. The above actions are repeated until the mixture is thoroughly mixed.

[0042] Furthermore, it also includes a full reaction step.

[0043] Depressurize the second airbag and apply pressure to the first airbag;

[0044] The detection area has a first sensing position and a second sensing position. The sample flows downstream from the first end until the second end of the sample flows past the second sensing position.

[0045] Apply pressure to the second airbag and release pressure to the first airbag;

[0046] The sample flows upstream from the second end until the first end of the sample flows past the first sensing position.

[0047] Repeat the above steps to allow the sample and pre-prepared reagents to react fully.

[0048] Furthermore, it also includes a waste discharge step, in which pressure is applied to both the first and second airbags, with the pressure applied to the second airbag being less than the pressure applied to the first airbag, until the sample enters the waste liquid area.

[0049] Furthermore, it also includes a catalytic reaction step, in which a potential is applied to the electrode in the detection region to induce a catalytic reaction.

[0050] Furthermore, the instrument is an electrochemical instrument, and the current generated by the reaction is transmitted to the electrochemical instrument through electrodes and electrode contacts.

[0051] Furthermore, the instrument is a photochemical instrument, which emits detection light into the detection area, which is reflected back to the photochemical instrument after being reflected by the detection area.

[0052] Furthermore, the instrument is equipped with a temperature control module.

[0053] The beneficial effects of this invention are: 1) It can perform reactions and detections without introducing additional liquids or gases, has a simple overall structure, and consumes little energy; 2) It directly sets a channel at the detection endpoint and correspondingly sets a fourth control layer, so that the waste liquid after the reaction or detection is completed can be directly absorbed by the fourth control layer through the micropores, completely avoiding the risk of backflow; 3) Two or more airbags work together, and the distance the liquid flows in the channel of the test strip is highly controllable; 4) The setting of the bending section can reduce the chip size and production cost, and provide a buffer for the liquid flow, assisting the airbags to more effectively control the flow of liquid in the channel; 5) The setting of the sensing area can accurately determine the position of the liquid in the channel, provide more accurate start or stop signals to the compressed or released airbags, and apply potential to promote catalytic reactions; 6) It can be applied to the detection of various indicators, including electrochemical detection, photochemical detection, small molecule detection, macromolecule detection, antigen-antibody detection, and nucleic acid molecule detection. Attached Figure Description

[0054] Figure 1 This is a schematic diagram of the test strip in Embodiment 1 of the present invention.

[0055] Figure 2 This is a schematic diagram of the detection process in Embodiment 1 of the present invention.

[0056] Figure 3 This is a schematic diagram of the full reaction step in Embodiment 1 of the present invention.

[0057] Figure 4 This is a schematic diagram of the test strip in Embodiment 2 of the present invention.

[0058] Figure 5 This is a schematic diagram of the detection process in Embodiment 2 of the present invention.

[0059] Figure 6 This is a schematic diagram of the test strip in Embodiment 3 of the present invention.

[0060] Figure 7 This is a schematic diagram of the test strip in Embodiment 4 of the present invention.

[0061] Figure 8This is a schematic diagram of the reaction steps for detecting antigens using the competitive method in Embodiment 4 of the present invention.

[0062] Figure 9 This is a schematic diagram of the test strip in Embodiment 5 of the present invention.

[0063] Figure 10 This is a schematic diagram of the test strip in Embodiment Six of the present invention.

[0064] Figure 11 This is a schematic diagram of the detection process in Embodiment Six of the present invention.

[0065] Figure 12 This is a schematic diagram of the test strip in Embodiment 9 of the present invention.

[0066] Figure 13 This is a schematic diagram of the first control layer in Embodiment 9 of the present invention.

[0067] Figure 14 This is a schematic diagram of the second control layer in Embodiment 9 of the present invention.

[0068] Figure 15 This is a schematic diagram of the third control layer in Embodiment 9 of the present invention.

[0069] Figure 16 This is a schematic diagram of the fourth control layer in Embodiment 9 of the present invention.

[0070] Figure 17 This is a schematic diagram of the test strip in Embodiment 10 of the present invention.

[0071] Figure 18 This is a schematic diagram of the first control layer in Embodiment 10 of the present invention.

[0072] Figure 19 This is a schematic diagram of the second control layer in Embodiment 10 of the present invention.

[0073] Figure 20 This is a schematic diagram of the third control layer in Embodiment 10 of the present invention.

[0074] Figure 21 This is a schematic diagram of the fourth control layer in Embodiment 10 of the present invention.

[0075] Among them, 1-first control layer, 11-gas channel, 2-second control layer, 21-sample dispensing port, 22-flow channel, 221-branch channel, 222-tilted guide section, 23-airbag, 231-first airbag, 2311-first intersection point, 232-second airbag, 24, 26-detection area, 3-third control layer, 31-first channel, 32-electrode, 33-sensing carbon block, 4-fourth control layer, 41-waste liquid collection area, 51-fourth end, 52-third end, 53-second end, 54-first end. Detailed Implementation

[0076] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0077] A microfluidic test strip includes a sample inlet 21 for adding a sample to be tested, a flow channel 22 connected to the sample inlet 21, and an air bladder 23 connected to the flow channel 22. The air bladder 23 has a compressed state and an inflated state, and the switching between the two states is controllable. The air bladder 23 and the flow channel 22 have a first intersection point, and the flow channel 22 forms a detection area 24 located downstream of the first intersection point.

[0078] A sensing area is provided between the first intersection point and the detection area 24, thereby forming a quantitative zone between the first intersection point and the sensing area, which is used to determine the amount of sample to be tested delivered to the detection area 24. The sample has a first end, a second end, a third end and a fourth end with the first intersection point as the interruption point. The sample with the first end as the starting point and the second end as the ending point is located in the quantitative zone, and the sample with the fourth end as the starting point and the third end as the ending point can flow upstream to the sample dispensing port 21.

[0079] The aforementioned sample inlet 21, flow channel 22, and airbag 23 can be formed by stacking a first control layer 1, a second control layer 2, and a third control layer 3 into a single unit. The first control layer 1 serves as a cover layer, the second control layer 2 is made of an elastic material and has a certain thickness, and the third control layer 3 serves as a sensing layer. Of course, in other embodiments, the aforementioned sample inlet 21, flow channel 22, and airbag 23 can also be a single unit, and there is no specific limitation.

[0080] It may also include a fourth control layer 4 with a waste liquid area, which is connected to the flow channel 22 through the first channel, and the fourth control layer 4 is stacked as a whole with the first control layer 1, the second control layer 2 and the third control layer 3.

[0081] The first control layer 1 may have a gas channel 11, with an airbag 23 located upstream of the gas channel 11. The number of airbags 23 may be one, or it may include at least a first airbag 231 and a second airbag 232. A detection area 24 is located in the flow channel 22 between the first airbag 231 and the second airbag 232. Specifically, the flow channel 22 between the first airbag 231 and the second airbag 232 is bent, and the detection area 24 is located within this bent area. Specifically, the flow channel 22 may be bent to form an M-shape with the opening facing to the side.

[0082] The third control layer 3 is equipped with electrodes, and the current generated by the reaction is transmitted to the detection instrument through the electrodes and electrode contacts.

[0083] The instrument is a photochemical instrument that emits detection light into the detection area. After being reflected by the detection area, the light returns to the photochemical instrument, which absorbs the reflected light and calculates the detection result according to the preset algorithm in the photochemical instrument. If the reaction product to be detected is a liquid, it is discharged to the waste liquid collection area after the detection is completed. If the reaction product to be detected is not a liquid, the waste liquid is discharged to the waste liquid collection area before the detection.

[0084] Example 1

[0085] like Figures 1-3 As shown, a microfluidic test strip includes a first control layer 1, a second control layer 2, and a third control layer 3. The first control layer 1 is hydrophilically treated to improve sample injection smoothness. This test strip can be used to detect blood glucose, blood ketones, uric acid, cholesterol, lactic acid, etc., contained in samples. Of course, the test strip can be used for any analyte in body fluids, as well as for the detection of antibodies and antigens, and the use of nucleic acid aptamers.

[0086] In its initial state, the sample application port 21 of the test strip, composed of the first control layer 1, the second control layer 2, and the third control layer 3, is pre-loaded with sample processing reagents. These reagents may include, but are not limited to, buffer systems, sample lysing agents, red blood cell filtration structures, stabilizers, enhancers, enzyme reaction substrates, immobilized enzyme-linked antibodies, and immobilized enzyme-linked antigens. (The immobilized enzyme-linked antibody (or antigen) can flow with the sample to the next region; the enzyme linked to the antibody may include glucose oxidase, glucose dehydrogenase, uricase oxidase, lactate oxidase, β-hydroxybutyrate dehydrogenase, cholesterol oxidase, etc., and the antibody and enzyme are linked by a covalent bond.) However, the above sample processing reagents are not applicable to electrochemical detection of blood glucose, blood ketones, uric acid, etc., i.e., no sample processing reagents are present in these areas.

[0087] The flow channel 22 between sensing regions 33' and 33" is the detection zone 24', which can be loaded with the enzyme and mediator of the analyte, or with an enzyme capable of independently performing substrate oxidation and electron transfer. In both cases, sample processing reagents need to be loaded simultaneously in the 24' region. Alternatively, the detection zone 24' can be an immobilized secondary antibody and an immobilized mediator. The substances loaded here must not flow with the sample into the next region. The mediator may include potassium ferricyanide, ferrocene and its derivatives, phenazines, flavins, organic dyes, quinones and their derivatives, tetrathiofulvalene, or fullerene derivatives.

[0088] Sensing area 34 is located upstream of sensing area 33', and the flow channel 22 between it and the first intersection point 2311 of the first airbag 231 and the flow channel 22 is a quantitative zone. Sensing area 35 is located downstream of sensing area 33', and sensing area 36 is located at the inlet of waste liquid collection area 41.

[0089] The testing instrument has a sealing cap at the top 211 of the sample application port 21. The instrument is equipped with a central signal processor (MCU). When the test strip is inserted into the instrument, the sensing area on the test strip contacts the contacts in the instrument, and the signal collected by the test strip is synchronously fed back to the MCU. The instrument also has a motor that applies or releases pressure to the airbag. The motor is electrically connected to the MCU, and the MCU issues commands to the motor to apply or release pressure based on the signals collected and fed back by the test strip.

[0090] When in use, the first step is initial sample quantification, which involves using test tubes, pipettes, or other materials to quantify the sample. This step is optional; it can be skipped for detecting substances such as blood glucose, but is mandatory for detecting antibodies or antigens. It should be noted that this step is applicable to antibody and antigen detection and nucleic acid aptamer reactions, but not to electrochemical methods such as blood glucose, blood ketones, and uric acid.

[0091] The second step is sample addition. The test strip is placed into the instrument. The motor in the instrument acts on the air bladders of the test strip, causing the first air bladder 231 to inflate and the second air bladder 232 to compress. The sample to be tested is added to the sample application port 21 through the top 211. For samples testing blood glucose, blood ketones, etc., sample processing reagents are not necessary; if the sample is testing for antigens or antibodies, it must be processed by the sample processing reagent in the sample application port 21. The corresponding sealing cap on the instrument seals the sample application port. The instrument's MCU receives a signal that the test strip has arrived and commands the motor to release the pressure on the second air bladder 232.

[0092] The third step is to thoroughly mix the sample with the sample processing reagent. This step is optional and depends on the detection process of the analyte. The motor receives and executes the command from the MCU, and the second airbag 232 gradually releases, applying suction to the sample, assisting it to reach position 37 and generating an induced current, which is synchronously fed back to the instrument's MCU. Then, according to preset logic, the MCU commands the motor to gradually pressurize the second airbag 232. At this time, the sample will flow back to the sample dispensing port 21. The first airbag 231 can also be gradually pressurized as needed to assist in the sample backflow. This step can be performed 1-100 times. After reaching position 37, the sample returns to the sample dispensing port 21. This repeated process ensures thorough mixing of the sample and sample processing reagent.

[0093] If the antigen or antibody is being detected, the reaction formula here is: (1) 'Substrate + Enzyme (Active State) = Substrate Product + Enzyme (Inactive State). The substrate is glucose, uric acid, lactic acid, β-hydroxybutyric acid, cholesterol, etc., and the substrate should match the enzyme immobilized in the detection zone 24. This reaction occurs after the sample and enzyme-linked antibody bind, but because there is no mediator, the reaction stops when all the enzyme is converted to the inactive state. It should be noted that this step is applicable to the detection of antibodies and antigens and nucleic acid aptamer reactions, but not to electrochemical reactions such as blood glucose, blood ketones, and uric acid.

[0094] The fourth step is sample quantification. The motor receives and executes the command from the MCU, and the second airbag 232 gradually releases, applying suction to the sample and drawing it to the quantification area. At this time, the sensing area 34 collects the current and synchronously feeds it back to the instrument's MCU. According to the preset logic, the MCU commands the motor to stop releasing pressure on the second airbag 232 and begin applying pressure to the first airbag 231. The motor receives and executes the command from the MCU, the second airbag 232 stops releasing pressure, the sample flow stops at the front, and the first airbag 231 begins applying pressure. The sample, with the first intersection point 2311 of the first airbag and the flow channel 22 as the interruption point, has a first end 54, a second end 53, a third end 52, and a fourth end 51. According to Pascal's Law: when an incompressible, stationary fluid experiences a pressure increase at any point due to an external force, this pressure increase is instantaneously transmitted to all points in the stationary fluid. Therefore, the pressure applied by the first airbag 231 at the first intersection point 2311 will be evenly distributed to all parts of the sample solution, giving the sample solution a force far from the first intersection point 2311. The sample originating from the fourth end 51 and ending at the third end 52 flows upstream to the sample inlet 21 and eventually remains there. The sample originating from the first end 54 and ending at the second end 53 flows downstream, thus completing the sample quantification. During this process, due to the strong hydrophilicity of the channel wall, the surface tension of the solution itself is not disrupted, so air pressure does not form bubbles inside the solution, and the solution inside the pipe provides airtightness for the detection process.

[0095] The fifth step is the reaction. According to preset logic, the MCU commands the motor to continue releasing pressure on the second airbag 232. The motor receives and executes the MCU's command, and the second airbag 232 continues to release pressure, accelerating the sample's downstream flow to the detection area 24'. At this point, both sensing areas 33' and 33" collect current. The lengths of sensing areas 33' and 33" are less than or equal to the sample length, and this is synchronously fed back to the instrument's MCU. According to preset logic, the MCU commands the motor to stop applying pressure to the first airbag 231 and releasing pressure to the second airbag 232 for a certain period. This time can be calculated and determined during application based on the dimensions of the pipes and airbags, as well as the reaction time required for detecting the substrate. The sample reacts with the pre-loaded substance. If detecting substances such as blood glucose or ketones, these substances will be oxidized by the corresponding enzymes and transfer electrons to the electrodes via a medium, flowing with the sample to the next area. The reaction formulas are: (1) analyte + enzyme (active state) = analyte product + enzyme (inactive state), (2) enzyme (inactive state) + mediator (active state) = mediator (inactive state) + enzyme (inactive state). A potential is applied to 64' and 64'' of the detection zone 24', and the catalytic reaction occurs. The reaction formula is: (3) mediator (inactive state) = mediator (active state) + electron. This reaction only occurs after a potential is applied to the detection zone. This reaction can convert the inactive mediator into an active mediator in a timely manner, thereby making the inactive enzyme in reaction formula (1) convert into an active enzyme in a timely manner. This makes reaction formula (1-3) form a sustainable reaction. Since the enzyme and mediator concentrations at 24' are fixed, the current collected by the MCU through the electrode is proportional to the concentration of the analyte in the sample, thereby quantitatively analyzing the content of the analyte in the sample and obtaining the final result.

[0096] For electrochemical reactions involving blood glucose, blood ketones, and uric acid, this concludes the process. In other words, the reaction steps are steps two, four, and five. In some cases, to improve accuracy, steps seven and eight can be added, and steps two, four, and five can be repeated, averaging the two results. In other cases, steps two, four, five, seven, and eight can be repeated more than once before repeating steps two, four, and five, averaging all results.

[0097] If the antigen being detected is an antigen, the reaction formula here is: (2) 'Enzyme (inactive state) + Mediator (active state) = Enzyme (active state) + Mediator (inactive state). This reaction occurs after the enzyme-linked primary antibody binds to the secondary antibody through the antigen and comes into contact with the loaded mediator. The reaction stops when all the mediator is converted to an inactive state. Because the mediator cannot spontaneously and promptly convert from an inactive state to an active state, the reaction progress is also limited.

[0098] The sixth step is to ensure a complete reaction. This step may be optional, but its presence ensures a thorough and complete reaction. Before this step, the sample from the fourth end 51 and the third end 52 can be pressurized by an airbag to flow towards point 21. This step prevents the sample from the third end 52 from entering the channel after the first intersection point 2311 during the complete reaction, thus preventing changes in sample volume. To achieve this function, a sensing region 37 is established between the sample inlet 21 and the first intersection point 2311, with electrodes 613 and 614 forming the sensing function. The flow channel distance between the first intersection point 2311 and the sensing region 37 should be much greater than the flow channel distance between the first intersection point 2311 and the sensing region 34. Alternatively, the flow channel distance between the first intersection point 2311 and the sensing region 37 should be much greater than the length of the sample in the flow channel, i.e., greater than the distance from the second end 53 to the first end 54.

[0099] 6-1: According to the preset logic, the MCU commands the motor to release pressure on the second airbag 232 and apply pressure to the first airbag 231. The motor receives the command from the MCU and executes it. The sample flows downstream from the first end 54 until the second end 53 of the sample flows through the position corresponding to the sensing area 33”. The sensing area 33” can no longer collect current signals.

[0100] 6-2: According to preset logic, the MCU commands the motor to switch the mode of action on the first airbag 231 and the second airbag 232, applying pressure to the second airbag 232 and releasing pressure on the first airbag 231. The motor receives and executes the command from the MCU, and the sample flows upstream from the second end 53 until it flows through the sensing area 33' at the first end 54, where the sensing area 33' can no longer collect a current signal. According to preset logic, the MCU commands the motor to switch the mode of action on the first airbag 231 and the second airbag 232.

[0101] Repeat steps 6-2 to 6-1 at least once, mixing the pre-loaded enzyme-linked antibody with the sample repeatedly, ensuring that the antigen to be tested and the enzyme-linked antibody in the sample react fully.

[0102] The seventh step is waste removal. According to preset logic, the MCU commands the motor to begin applying pressure to the second airbag 232 and increases the pressure applied to the first airbag 231. The pressure applied to the second airbag 232 is less than the pressure applied to the first airbag 231, allowing the sample to flow downstream without entering the second airbag 232. The motor is then commanded to simultaneously apply pressure to both the first and second airbags until the second end 53 of the sample flows through the sensing area 36. The sensing area 36 can no longer collect a current signal, at which point waste removal ends, and the MCU commands the motor to stop applying pressure to both the first and second airbags 231 and 232.

[0103] Normally, the detection process ends here. However, when the analyte is an antigen or antibody, steps two through seven, i.e., step eight, can be repeated in some cases to reintroduce a sample. This process can be repeated 1-20 times to ensure that there are no unbound enzyme-linked antibodies (primary antibodies) in the detection area. This step can maximize the detection signal.

[0104] The ninth step is a catalytic reaction, which occurs when the analyte is an antigen or antibody. According to preset logic, the MCU applies a potential to the sensing regions 33' and 33" of the detection zone 24', triggering the catalytic reaction. The reaction formula is: Mediator (inactive state) = Mediator (active state) + Electron. This reaction occurs only after a potential is applied to the detection zone. This reaction can promptly convert the inactive mediator from the fifth step into an active mediator, thereby enabling the inactive enzyme from the third step to be converted into an active enzyme. This allows the reactions in the third, fifth, and ninth steps to form a closed catalytic cycle, amplifying the signal. Due to the design of the previous reaction steps, the substrate is in large excess; therefore, the collected current is directly proportional to the amount of enzyme, allowing for quantitative analysis of the content of the analyte antigen in the sample, yielding the final result.

[0105] Example 2

[0106] like Figure 4 , Figure 5 As shown, this test strip can be used to detect the antigen content contained in a sample.

[0107] The parts of Example 2 that are the same as those in Example 1 will not be repeated. The difference is that in this example, the flow channel 22 between sensing regions 33' and 33" is a detection zone 24, pre-loaded with immobilized enzyme-linked antibody (primary antibody), which can flow with the sample into the next zone. The enzyme linked to the antibody may include glucose oxidase, glucose dehydrogenase, uricase oxidase, lactate oxidase, β-hydroxybutyrate dehydrogenase, cholesterol oxidase, etc., and the antibody and enzyme are linked by a covalent bond. The flow channel 22 between sensing regions 33'" and 33"" is a detection zone 26, loaded with immobilized secondary antibody and immobilized media, which cannot flow with the sample into the next zone.

[0108] In use, the first step is sample addition. The test strip is placed into the instrument. The motor in the instrument acts on the air bladders of the test strip, causing the first air bladder 231 to inflate and the second air bladder 232 to compress. The sample to be tested is added to the sample dispensing port 21 through the top 211. The antigen in the sample is treated by the sample processing reagent in the sample dispensing port 21. The corresponding sealing cap on the instrument seals the sample dispensing port. The instrument's MCU receives a signal that the test strip has been placed and commands the motor to release the pressure on the second air bladder 232.

[0109] The second step is sample quantification. The motor receives and executes the command from the MCU, and the second airbag 232 gradually releases, applying suction to the sample and drawing it to the quantification area. At this time, the sensing area 34 collects the current and synchronously feeds it back to the instrument's MCU. According to preset logic, the MCU commands the motor to stop releasing pressure on the second airbag 232 and begin applying pressure to the first airbag 231. The motor receives and executes the command from the MCU, the second airbag 232 stops releasing pressure, the sample flow stops at the front, and the first airbag 231 begins applying pressure. The sample, with the first intersection point 2311 of the first airbag 231 and the flow channel 22 as the interruption point, has a first end 54, a second end 53, a third end 52, and a fourth end 51. According to Pascal's Law: when an incompressible, stationary fluid experiences a pressure increase at any point due to an external force, this pressure increase is instantaneously transmitted to all points in the stationary fluid. Therefore, the pressure applied by the first airbag 231 at the first intersection point 2311 will be evenly distributed to all parts of the sample solution, giving the sample solution a force far from the first intersection point 2311. The sample starting from the fourth end 51 and ending at the third end 52 flows upstream to the sample inlet 21 and eventually remains at the sample inlet, while the sample starting from the first end 54 and ending at the second end 53 flows downstream, thus completing the sample quantification.

[0110] The third step is the reaction. According to preset logic, the MCU commands the motor to continue releasing pressure on the second airbag 232. The motor receives and executes the MCU's command, and the second airbag 232 continues to release pressure, accelerating the sample's downstream flow and reaching the detection area 24. At this time, both sensing areas 33' and 33" collect current, which is synchronously fed back to the instrument's MCU. According to preset logic, the MCU commands the motor to stop applying pressure to the first airbag 231 and releasing pressure to the second airbag 232 for a certain period. This time is related to the time required for each type of reaction and the size of the airbag. In practice... In this application, accurate timing can be obtained through repeated experiments and designed into the corresponding product. The sample reacts with pre-loaded immobilized enzyme-linked antibody and flows to the next region with the sample. The reaction formula is: substrate + enzyme (active state) = substrate product + enzyme (inactive state), where the substrate can be glucose, uric acid, lactic acid, β-hydroxybutyrate, cholesterol, etc., and the substrate should match the enzyme immobilized in detection zone 24. This reaction occurs after the sample and enzyme-linked antibody bind, but because there is no mediator, the reaction stops when all the enzyme is converted to the inactive state.

[0111] The fourth step is to ensure a complete reaction. This step may be omitted, but its presence ensures a complete and thorough reaction. It is the same as the sixth step, the complete reaction, in Example 1, and will not be described again.

[0112] The fifth step is the reaction. According to preset logic, the MCU commands the motor to stop releasing pressure on the second airbag 232. The sample flows downstream from the first end 54 until it reaches the sensing area 35. The sensing area 35 collects the current signal and synchronously feeds it back to the instrument's MCU. According to preset logic, the MCU commands the motor to start applying pressure to the second airbag 232 and increases the pressure applied to the first airbag 231. The pressure applied to the second airbag 232 is less than the pressure applied to the first airbag 231, ensuring the sample flows downstream without entering the second airbag 232. When the second end 53 of the sample flows past the sensing area 35, the sensing area 35 can no longer collect the current signal, and the MCU commands the motor to stop applying pressure to the second airbag 232. The sample flows downstream and reaches the detection area 26. At this point, both sensing areas 33'" and 33"" collect current and synchronously feed it back to the instrument's MCU. According to preset logic, the MCU commands the motor to stop applying pressure to the first airbag 231, and the sample reacts with the immobilized secondary antibody and immobilized media. The reaction formula is: enzyme (inactive state) + mediator (active state) = enzyme (active state) + mediator (inactive state). This reaction occurs after the enzyme-linked primary antibody binds to the secondary antibody through the antigen and comes into contact with the loaded mediator. The reaction stops when all the mediator is converted to an inactive state. Intervention is required to convert the mediator from an inactive state to an active state so that it can continue to participate in the reaction.

[0113] The sixth step is to ensure a full reaction. The same method as in the fourth step can be used to make the reaction complete. However, in this step, at the same time, the MCU commands the motor to act on the first airbag 231 and the second airbag 232 in the same way. At the same time, pressure is applied to make the second end 53 of the sample flow through the sensing area 33””, and pressure is released to make the first end 54 of the sample flow through the sensing area 33’”.

[0114] The seventh step is waste removal. According to the preset logic, the MCU commands the motor to apply pressure to the first airbag 231 and the second airbag 232 simultaneously until the second end 53 of the sample flows through the sensing area 36. The sensing area 36 can no longer collect current signals, the waste removal ends, and the MCU commands the motor to stop applying pressure to the first airbag 231 and the second airbag 232.

[0115] Normally, the detection process ends here. However, in some cases, steps one through seven, i.e. step eight, can be repeated to reintroduce a sample. This process can be repeated 1-20 times to ensure that there are no unbound enzyme-linked antibodies (primary antibodies) in the detection area.

[0116] The ninth step is a catalytic reaction, which is the same as the ninth step of the catalytic reaction in Example 1, and will not be described again.

[0117] Example 3

[0118] like Figure 6 As shown, unlike Example 2, this test strip can be used to detect the antibody content in a sample. The parts of Example 3 that are the same as those in Example 2 will not be repeated here; the differences are as follows:

[0119] The flow channel 22 between sensing regions 33' and 33" is the detection zone 24, which contains pre-loaded immobilized enzyme-linked antigen (antigen protein). This antigen can flow with the sample into the next zone. The enzymes linked to the antigen may include glucose oxidase, glucose dehydrogenase, uricase oxidase, lactate oxidase, β-hydroxybutyrate dehydrogenase, cholesterol oxidase, etc., and are covalently linked. The flow channel 22 between sensing regions 33'" and 33"" is the detection zone 26, which contains immobilized secondary antibody and immobilized media. This zone cannot flow with the sample into the next zone.

[0120] When in use, the first step is to add the sample, the second step is to quantify the sample, the third step is to react, the fourth step is to allow the reaction to proceed to completion, and the sixth step is to allow the reaction to proceed to completion. These steps are the same as in Example 2 and will not be repeated here.

[0121] The fifth step is the reaction, which occurs when the secondary antibody captures the test antibody that has bound the enzyme-linked antigen and comes into contact with the loaded mediator. The reaction stops when all the mediator is converted to an inactive state. This is because the mediator cannot spontaneously and promptly convert from an inactive to an active state; it requires intervention. Once the mediator has converted from an inactive to an active state, the reaction continues. The rest is the same as in Example 2 and will not be repeated.

[0122] The seventh step is waste removal, which is the same as in Example 2 and will not be repeated here. It ultimately ensures that there are no unbound enzyme-linked antigens in the detection area.

[0123] The ninth step is a catalytic reaction, which is the same as the ninth step in Example 1 and will not be described again. Finally, the content of the antibody to be tested in the sample is quantitatively analyzed to obtain the final result.

[0124] Example 4

[0125] like Figure 6 As shown, this test strip can be used to detect the antigen content contained in a sample.

[0126] The parts of Example 4 that are the same as those in Example 2 will not be repeated. The difference is that an enzyme-linked antigen is loaded in detection zone 24 to compete with the antigen in the sample for limited antibody binding sites. The signal intensity in detection zone 26 is inversely proportional to the antigen content: if the antigen concentration in the analyte is high, the proportion of enzyme-linked antigen after mixing with detection zone 24 is reduced, and the number of enzyme-linked antigens bound in the detection zone is less, thus resulting in a low signal intensity; if the antigen concentration in the analyte is low, the proportion of enzyme-linked antigen increases, the number of enzyme-linked antigens bound in the detection zone increases, and the detection signal intensity is enhanced.

[0127] In the initial state, a fixed amount of immobilized enzyme-linked antigen (antigen protein, with the same binding site on the antibody as the analyte antigen) is pre-loaded. The enzyme linked to the antigen may include glucose oxidase, glucose dehydrogenase, uricase oxidase, lactate oxidase, β-hydroxybutyrate dehydrogenase, cholesterol oxidase, etc., and the antigen and enzyme are linked by a covalent bond. The flow channel 22 between sensing regions 33'” and 33”” is the detection region 26, which is loaded with immobilized antibody and immobilized mediator. The mediator may include potassium ferricyanide, ferrocene and its derivatives, phenazines, flavins, organic dyes, quinones and their derivatives, tetrathiofulvalene, or fullerene derivatives. The enzyme-linked antigen loaded in the detection region 24 has the same binding site as the antigen in the analyte, and both can specifically bind to the antibody immobilized in the detection region 26. They compete for the limited antibody binding sites, forming a competitive relationship.

[0128] The third step is a reaction, which differs from Example 2 in that... Figure 8 As shown, the sample is mixed with pre-loaded immobilized enzyme-linked antigen. The reaction formula is: substrate + enzyme (active state) = substrate product + enzyme (inactive state), where the substrate can be glucose, uric acid, lactic acid, β-hydroxybutyrate, cholesterol, etc., and the substrate should match the enzyme immobilized in detection zone 24. This reaction occurs after the sample and enzyme-linked antigen bind, but because there is no mediator present, the reaction stops when all the enzyme is converted to the inactive state.

[0129] The fourth step is to ensure a complete reaction. This step may be omitted, but if it is included, it ensures that the two antigens are thoroughly mixed and homogeneous. It is the same as the sixth step of the complete reaction in Example 1, and will not be described again.

[0130] The fifth step is the reaction. The sample is mixed with the immobilized antibody and the immobilized medium. Both antigens in the sample, when mixed evenly, will be captured by the immobilized antibody with equal probability. The enzyme-related reaction formula is: Enzyme (inactive state) + Medium (active state) = Enzyme (active state) + Medium (inactive state). This reaction occurs after the enzyme-linked antigen binds to the immobilized antibody and comes into contact with the immobilized medium. The reaction stops when all the medium is converted to an inactive state. Because the medium cannot spontaneously and promptly convert from an inactive state to an active state, it can only be achieved through the influence of an intervention factor. Once the medium converts from an inactive state to an active state, the reaction continues. The other steps in this process are the same as in Example 2 and will not be repeated.

[0131] The sixth step is to allow the reaction to proceed fully, and the seventh step is to discharge waste. These steps are the same as in Example 2 and will not be repeated here.

[0132] Normally, the detection process ends here. However, in some cases, steps one through seven, or step eight, can be repeated to reintroduce a sample. This process can be repeated 1-20 times to ensure that there are no unbound enzyme-linked antigens in the detection area.

[0133] The ninth step is catalysis. According to preset logic, the MCU applies a potential to the sensing regions 33'" and 33"" of the detection area 26, initiating a catalytic reaction. The reaction formula is: Mediator (inactive state) = Mediator (active state) + Electron. This reaction occurs only after a potential is applied to the detection area. This reaction can promptly convert the inactive mediator from the fifth step into an active mediator, thereby enabling the inactive enzyme from the third step to be converted into an active enzyme. This allows the reactions in the third, fifth, and ninth steps to form a closed catalytic cycle, amplifying the signal. Due to the design of the previous reaction steps, the substrate is in large excess; therefore, the collected current is directly proportional to the amount of enzyme, but inversely proportional to the concentration of the analyte antigen (the two compete for limited antibody binding sites, creating a competitive relationship). This allows for quantitative analysis of the analyte antigen content in the sample, yielding the final result.

[0134] Example 5

[0135] like Figure 9 As shown, this embodiment adds a constant temperature module based on embodiment two.

[0136] The instrument has an internal temperature control module that communicates with the MCU to monitor and control the reaction temperature in real time. This module includes a heating element and temperature sensors. When the test strip is inserted into the instrument, the temperature control module is positioned above (or below) the test strip. Multiple sensors are distributed within the temperature control module to monitor the temperature in real time, with a focus on the temperatures of detection zones 24 and 26. The heating element is activated when the average temperature of the channel area is below 36˚C and stops when the temperature reaches 36˚C. If the temperature sensor temperature subsequently drops below 35˚C, the heating element is activated again. This process repeats to maintain a relatively constant temperature in the area. The current setting is 35-37°C, but the temperature range can be adjusted according to actual conditions.

[0137] Subsequent detection steps are the same as in Example 2. With the support of the constant temperature module, the reaction and detection time will be shortened, and the constant temperature can also ensure the consistency of the reaction and improve the detection accuracy.

[0138] Example 6

[0139] This embodiment, based on Example 2, replaces the enzyme used with one that can independently complete the oxidation of the substrate and the transfer of electrons. This type of enzyme does not require a mediator to participate in the reaction, achieving the effect of electron transfer. It can be used to detect the antigen content contained in a sample.

[0140] The parts of Example 6 that are the same as those in Example 2 will not be repeated. The difference is that the pre-loaded immobilized third-generation enzyme-linked antibody (primary antibody) can flow with the sample into the next region. The enzyme linked to the antibody may include glucose oxidase, glucose dehydrogenase, uricase oxidase, lactate oxidase, β-hydroxybutyrate dehydrogenase, cholesterol oxidase, etc., and the antibody and enzyme are linked by a covalent bond. The flow channel 22 between the sensing regions 33'" and 33"" is the detection region 26, where the immobilized secondary antibody cannot flow with the sample into the next region.

[0141] The first step is to add the sample, and the second step is to quantify the sample, which is the same as in Example 2 and will not be repeated here.

[0142] The third step is the reaction, which differs from Example 2 in that the sample reacts with a pre-loaded, immobilized third-generation enzyme-linked antibody and flows with the sample to the next region. The reaction formula is: substrate + enzyme (active state) = substrate product + enzyme (active state) + electrons, where the substrate can be glucose, uric acid, lactic acid, β-hydroxybutyrate, cholesterol, etc., and the substrate should match the enzyme immobilized in detection region 24. This reaction occurs after the sample and enzyme-linked antibody bind and continues to the next step without the need for a mediator.

[0143] The fourth step is to allow the reaction to proceed fully, which is the same as in Example 2 and will not be described again.

[0144] The fifth step is the reaction, which differs from Example 2 in that the reaction formula is: substrate + enzyme (active state) = substrate product + enzyme (active state) + electron. This reaction has already started at the detection area 24 and is still ongoing.

[0145] The sixth step is to allow the reaction to proceed fully, and the seventh step is to discharge waste. These steps are the same as in Example 2 and will not be repeated here.

[0146] The ninth step is the catalytic reaction, with the following formula: Substrate + Enzyme (Active State) = Substrate Product + Enzyme (Active State) + Electrons. After a potential is applied to the detection region, electrons rapidly oxidize on the electrode surface. However, this reaction has already occurred in previous steps, generating a large number of electrons, leading to a strong background current. Due to the design of the previous reaction steps, the substrate is in significant excess; therefore, the collected current is directly proportional to the amount of enzyme, allowing for quantitative analysis of the content of the analyte antigen in the sample, thus obtaining the final result.

[0147] Example 7

[0148] This embodiment differs from Embodiment 2 in that the detection area 24 may not be provided, and both the reaction and detection occur in the detection area 26. The detection area 24 is located downstream of the second airbag 232.

[0149] The flow channel 22 between sensing regions 33''' and 33'''' is the detection zone 26, which can be loaded with the enzyme and mediator of the analyte, or an enzyme capable of independently completing substrate oxidation and electron transfer. In both cases, the detection zone 26 needs to be loaded with sample processing reagents simultaneously. Alternatively, the detection zone 26 can be an immobilized secondary antibody and an immobilized mediator. The substances loaded here must not flow with the sample to the next zone. The mediator may include potassium ferricyanide, ferrocene and its derivatives, phenazines, flavins, organic dyes, quinones and their derivatives, tetrathiofulvalene, or fullerene derivatives. Sensing region 34 is located upstream of sensing region 33''', and the flow channel 22 between it and the first intersection point 2311 of the first air bladder 231 and the flow channel 22 is the quantification zone. Sensing region 35 is located downstream of sensing region 33''''.

[0150] The third step is the reaction. According to the preset logic, the MCU commands the motor to continue releasing pressure on the second airbag 232. The motor receives the command from the MCU and executes it. The second airbag 232 continues to release. When the sample at the first end 54 flows to the sensing area 35, the sensing area 35 collects the current signal and synchronously feeds it back to the instrument's MCU. According to the preset logic, the MCU commands the motor to start applying pressure to the second airbag 232 and increase the pressure applied to the first airbag 231. The pressure applied to the second airbag 232 is less than the pressure applied to the first airbag 231, so that the sample flows downstream without entering the second airbag 232. When the sample flows through the second end 53 of the sensing area 35, the sensing area 35 can no longer collect current signals. The MCU commands the motor to increase the pressure applied to the second airbag 232, accelerating the sample to flow downstream and reach the detection area 26. At this time, the sensing areas 33''' and 33'''' collect current and synchronously feed it back to the instrument's MCU. According to the preset logic, the MCU commands the motor to stop applying pressure to the first airbag 231 and release pressure to the second airbag 232 for a certain period of time, and the sample reacts with the preloaded substance. For the detection of substances such as blood glucose and blood ketones, they will be oxidized by the corresponding enzymes here, and electrons will be transferred to the electrodes through the medium and flow with the sample. The reaction formula is: (1) analyte + enzyme (active state) = analyte product + enzyme (inactive state), (2) enzyme (inactive state) + medium (active state) = medium (inactive state) + enzyme (inactive state). When a potential is applied to 33''' and 33'''' in the detection area 26, the catalytic reaction occurs. The reaction formula is: (3) Mediator (inactive state) = Mediator (active state) + electron. This reaction only occurs after a potential is applied to the detection area. This reaction can convert the inactive mediator into an active mediator in a timely manner, thereby making the inactive enzyme in reaction formula (1) convert into an active enzyme in a timely manner. This makes the reaction formula (1-3) form a sustainable reaction. Since the enzyme and mediator concentrations are fixed at 24', the current collected by the MCU through the electrode is proportional to the concentration of the analyte in the sample, thereby quantitatively analyzing the content of the analyte in the sample and obtaining the final result.

[0151] Example 8

[0152] In this embodiment, the number of airbags is one, that is, the first airbag 231 is not provided, and the second airbag 232 is retained.

[0153] The flow channel 22 between sensing regions 33' and 33'' is the detection zone 24', which can be loaded with the enzyme and mediator of the analyte, or with an enzyme capable of independently performing substrate oxidation and electron transfer. In both cases, sample processing reagents need to be loaded simultaneously in the 24' region. Alternatively, the detection zone 24' can contain immobilized secondary antibody and immobilized mediator. The substances loaded here must not flow with the sample into the next region. Mediators may include potassium ferricyanide, ferrocene and its derivatives, phenazines, flavins, organic dyes, quinones and their derivatives, tetrathiofulvalene, or fullerene derivatives.

[0154] The first step is sample addition. The test strip is placed into the instrument, and the motor in the instrument acts on the air bladder of the test strip, compressing the second air bladder 232. The sample to be tested is added to the sample addition port 21 from the top of the sample addition port 211. The added sample is a quantitative sample.

[0155] The second step is the reaction. According to preset logic, the MCU commands the motor to release pressure on the second airbag 232. The motor receives and executes the command from the MCU, and the second airbag 232 begins to release pressure. The sample flows downstream from the sample inlet 21 and reaches the detection area 24'. At this time, both sensing areas 33' and 33" collect current, which is synchronously fed back to the instrument's MCU. According to preset logic, the MCU commands the motor to stop releasing pressure on the second airbag 232 for a certain period, allowing the sample to react with the pre-loaded substance. Since the enzyme and mediator concentrations in 24' are fixed, the current collected by the MCU through the electrodes is directly proportional to the concentration of the analyte in the sample, thus enabling quantitative analysis of the analyte content in the sample and obtaining the final result.

[0156] Example 9

[0157] In this embodiment, the downstream of the flow channel 22 has multiple branch channels, and the sidewall of each branch channel is provided with an opening of the gas channel 22.

[0158] like Figures 12-16 As shown, in this embodiment, there is one airbag 23, located upstream of the gas channel 11. The gas channel 11 has been changed from an opening to a long, thin ventilation strip, and the downstream of the flow channel 22 has multiple branch channels 221, such as... Figure 14 As shown, the gas channel 11 extends along the width of the test paper and intersects with multiple branch channels 221, thereby allowing the sample to be adsorbed into each branch channel 221 by siphon effect through the gas channel 11.

[0159] like Figure 15As shown, the third control layer 3 has electrodes 32 in the same number and position as the branch channels 221, and first channels 31 in the same number and position as the branch channels 221. An inductive carbon block 33, which can be electrically connected to a detection instrument, is also disposed upstream of the first channel 31, and is located upstream of the electrodes 32.

[0160] The flow channel 22 has an inclined guide section 222, to which the airbag 23 is connected. The inclined guide section 222 provides a buffer for the liquid sample, maximizing the effect of the airbag 23 on the liquid. The airbag 23 allows the liquid to flow continuously towards the detection area 24. However, the elasticity of the airbag 23 is limited, and the inclined guide section 222 can overcome the insufficient force on the liquid caused by the limited elasticity of the airbag 23. Without the inclined guide section 222, the airbag 23 would need to be very large, or the airbag 23 might not be able to allow enough liquid to flow to the detection area 24.

[0161] A microfluidic detection method includes the following steps:

[0162] Insert the microfluidic test paper into the instrument;

[0163] When the sample is placed in the sample inlet 21, the sample flows into the gas channel 11 within the flow channel 22 under the siphon effect, because the first control layer 1 has a gas channel 11.

[0164] The instrument compresses the air bladder 23 downwards, putting it under negative pressure, and the sample flows to multiple branch channels 221;

[0165] The instrument stops compressing the airbag 23, and the liquid samples in each branch channel 221 remain in the reagent area 24 and react with the pre-placed reagents therein;

[0166] The reaction products flow into detection zone 26;

[0167] The instrument is a photochemical instrument that emits detection light into the detection area 26. After being reflected by the detection area 26, the light returns to the photochemical instrument. The photochemical instrument absorbs the reflected light and calculates the detection results according to the preset algorithm in the photochemical instrument. It detects the reaction results in each branch channel. If the reaction product to be detected is a liquid, it is discharged to the waste liquid layer after the detection is completed. If the reaction product to be detected is not a liquid, the waste liquid is discharged to the waste liquid layer before the detection.

[0168] In this embodiment, there are four branch channels 221, and the enzymes and mediators loaded in the detection areas 24 of the four branch channels are: glucose dehydrogenase + potassium ferricyanide, uricase + potassium ferricyanide, lactate oxidase + ferrocene, and β-hydroxybutyrate dehydrogenase + tetrathione. The sample to be tested is blood.

[0169] The first step is sample addition. The test strip is placed into the instrument, and the motor in the instrument inflates the air bladder 23 of the test strip. The sample to be tested is added to the sample inlet 21. Under the action of the airflow channel 11, the sample flows downstream and reaches the corresponding position in the branch channel 221 of the airflow channel 11. The corresponding sealing cap on the instrument seals the sample inlet, and the instrument's MCU receives a signal that the test strip has reached its position. Preferably, while the corresponding sealing cap on the instrument seals the sample inlet, the instrument also uses an airflow channel sealant to seal the airflow channel. This sealant can be solid or gaseous, and pressure is applied to the airflow channel opening to prevent the sample to be tested from overflowing in subsequent processes.

[0170] The second step is quantification. The instrument's MCU commands the motor to apply pressure to the airbag 23. The sample to be tested has a first end 54, a second end 53, a third end 52, and a fourth end 51, with the intersection of the airbag 23 and the inclined guide section 222 as the interruption point. The sample starting from the first end 54 and ending at the second end 53 flows downstream, and the sample quantification is completed.

[0171] The third step is the reaction. The motor continues to apply pressure to the airbag 23. When the sample to be tested reaches the detection area 24, the sensing areas at both ends of the detection area 24 can collect current and synchronously feed it back to the instrument's MCU. According to the preset logic, the MCU commands the motor to stop applying pressure to the airbag 23 for a certain period of time, and the sample to be tested reacts with the preloaded substance. The reaction formula is: (1) analyte + enzyme (active state) = analyte product + enzyme (inactive state), (2) enzyme (inactive state) + mediator (active state) = mediator (inactive state) + enzyme (inactive state). When a potential is applied to the sensing areas at both ends of the detection area 24, the catalytic reaction occurs. The reaction formula is: (3) mediator (inactive state) = mediator (active state) + electron. This reaction only occurs after a potential is applied to the detection area. This reaction can convert the inactive mediator into an active mediator in time, thereby making the inactive enzyme in reaction formula (1) convert into an active enzyme in time. Thus, reaction formula (1-3) forms a sustainable reaction. Since the concentrations of enzyme and mediator loaded in the detection zone 24 are fixed, the current collected by the MCU through the sensing areas at both ends of the detection zone 24 is directly proportional to the concentration of the analyte in the sample, thereby enabling quantitative analysis of the content of the analyte in the sample and obtaining the final result. Because the reaction occurs in the detection zone 24, it can also be considered a reaction zone.

[0172] In other reactions, the final result is detected in the detection zone 26. After the sample to be tested reacts in the detection zone 24 for a certain period of time, the MCU commands the motor to continue applying pressure to the airbag 23 according to the preset logic, so that the reacted sample flows downstream to the detection zone 26. When the sensing areas at both ends of the detection zone 26 can collect current, the MCU commands the motor to stop applying pressure to the airbag 23 for a certain period of time, and calculates the final result through the current collected by the sensing areas at both ends of the detection zone 26.

[0173] The fourth step is waste discharge. According to the preset logic, the MCU commands the motor to start applying pressure to the airbag 23, so that the reacted sample flows downstream and enters the waste liquid collection area 41 of the fourth control layer through the channel 31. When the sensing area at the entrance of the channel 31 can no longer collect the current signal, the waste discharge ends and the MCU commands the motor to stop applying pressure to the airbag.

[0174] Example 10

[0175] A microfluidic test strip includes a first control layer 1 with a gas channel 11, a second control layer 2, a third control layer 3 with a first channel 31, and a fourth control layer 4 for absorbing waste liquid generated during a reaction. The first control layer 1, second control layer 2, third control layer 3, and fourth control layer 4 are stacked sequentially on top of each other. Figure 17 As shown.

[0176] like Figure 18 As shown, a gas channel 11 is provided on the first control layer 1, which is connected to the atmosphere so that the test strip is not absolutely sealed, and helps the liquid sample to be tested to enter the flow channel 22. In this embodiment, the gas channel 11 is an opening.

[0177] like Figure 19 As shown, the second control layer 2 is made of an elastic material and forms a sample inlet 21, a flow channel 22, and an air bladder 23. The flow channel 22 forms a reagent area 24, which is the area for reactions or adsorption processes. This reagent area 24 is located downstream of the air bladder 23 and can be pre-filled with reagents. The opening of the gas channel 11 on the first control layer 1 is located on the side wall of the flow channel 22. In other words, the gas channel 11 and the flow channel 22 are connected through the opening. The second control layer 2 is made of a soft rubber material, which gives the air bladder 23 the elasticity to be compressed and released.

[0178] A detection area 26 may also be provided downstream of the reagent area 24. The detection area 26 may or may not contain pre-placed reagents; there are no specific restrictions.

[0179] like Figure 20As shown, the third control layer 3 is used for detection. Specifically, an electrode 32 can be set in the third control layer 3, meaning the third control layer 3 is an electrode layer. The electrode 32 is used to obtain the result after the liquid reacts with the pre-placed reagent. In some cases, the electrode 32 is also used to monitor the specific location of the liquid during its flow. The induced current is transmitted to the detection instrument through the electrode 32 and the electrode contact 321. The third control layer 3 is also provided with an inductive carbon block 33 that can be electrically connected to the electrode 32 or the detection instrument, and it is located upstream of the electrode 32.

[0180] Specifically, before the reaction begins, during process monitoring, the liquid flowing past the two sensing carbon blocks 33 indicates that the liquid has reached the reagent area 24 corresponding to those two sensing carbon blocks 33. The instrument receives a signal indicating that the circuit is connected, which instructs the gasbag to stop functioning, allowing the liquid to react with the reagent or enabling the instrument to detect that area. The sensing carbon blocks 33 are connected to the electrode 32 or to the circuitry in the instrument to achieve this function.

[0181] like Figure 21 As shown, the fourth control layer 4 is connected to the flow channel 22 via the first channel 31. It is equipped with a waste liquid collection area 41 for absorbing waste liquid downstream of the flow channel 22. The waste liquid collection area 41 can be made of absorbent material. There is at least one first channel 31; increasing the number of channels helps waste liquid enter the waste liquid collection area 41 more quickly, reducing the compressible space required for the airbag. The fourth control layer 4 and the third control layer 3 are bonded together.

[0182] The gas channel 11 is located upstream of the airbag 23. There are at least two airbags 23. In this embodiment, there are two airbags 23, both of which are located upstream of the gas channel 11. A bend 25 is formed between adjacent airbags 23. Specifically, the first airbag 231 is located upstream of the bend 25, and the second airbag 232 is located downstream of the bend 25.

[0183] The aforementioned bend 25 provides a buffer for the liquid sample, maximizing the effect of the first airbag 231 on the liquid. The first airbag 231 allows the liquid to continuously flow towards the reagent area 24. However, the elasticity of the first airbag 231 is limited. The bend 25 overcomes the insufficient force on the liquid caused by the limited elasticity of the first airbag 231. Without the bend 25, the first airbag 231 would need to be very large, or it might not be able to allow enough liquid to flow into the reagent area 24.

[0184] A microfluidic detection method includes the following steps:

[0185] Insert the microfluidic test paper into the instrument;

[0186] The instrument compresses the second airbag 232 downwards, putting it into a negative pressure state;

[0187] When a liquid sample is placed in the sample inlet 21, the sample flows into the gas channel 11 within the flow channel 22 under the siphon effect, because the first control layer 1 has a gas channel 11.

[0188] The second airbag 232, which is in a compressed state, is slowly released under the control of the instrument. At this time, the second airbag 232 acts as the adsorption force, thereby adsorbing the sample and flowing upward to the bend section 25 between the adjacent airbags. The instrument stops releasing the second airbag 232, thereby determining the sample volume required for the reaction. That is, the liquid volume contained in the flow channel 22 between the first airbag 231 and the second airbag 232 is the volume required for the reaction.

[0189] The instrument slowly compresses the first airbag 231 downwards, making it under negative pressure. At this time, the first airbag 231 acts as a power source to blow air downstream, driving the sample to continue flowing to the reagent area 24. That is, the liquid between the first airbag 231 and the second airbag 232 moves upwards and reaches the reagent area 24.

[0190] The instrument stops compressing the first airbag 231, and the liquid sample remains in the reagent area 24 to react with the pre-placed reagents therein;

[0191] The instrument detects the reaction results. This instrument can be an electrochemical instrument. The current generated by the reaction is transmitted to the electrochemical instrument through electrode 32 and electrode contact 321. The instrument calculates the reaction results according to a preset algorithm.

[0192] The reacted sample enters the waste liquid collection area 41 of the fourth control layer 4 through the first channel 31 of the third control layer 3.

[0193] Of course, it is also possible that after the sample completes the reaction in the reagent area 24, the instrument continues to compress the first airbag 231, and the liquid after the reaction can continue to flow upward to the detection area 26 to react with other reagents preset in the detection area 26. The current generated by the reaction is transmitted to the instrument through the electrode 32 and the electrode contact 321. The instrument calculates the reaction result according to the preset algorithm, and the waste liquid after the reaction is discharged into the waste liquid collection area 41 of the fourth control layer 4 through the first channel 31.

[0194] Example 11

[0195] In this embodiment, the microfluidic test strip is used to detect subjects at risk of developing diabetes. The test sample is blood, and the test components include hemoglobin and glycated albumin. The pre-prepared reagents include sodium deoxycholate, sodium nitrite, buffer solution, sodium 2-hydroxy-3-m-toluidine propanesulfonate, glycated amino acid oxidase, bromocresol green reagent protease, 4-aminoantipyrine (4-AAP), and peroxidase.

[0196] Example 12

[0197] In this embodiment, the microfluidic test strip is used to detect subjects who may be at risk of atherosclerosis, coronary heart disease, hypertension, etc. The test sample is blood, the test components include total cholesterol, and the pre-prepared reagents include bovine serum albumin (BSA), carboxymethyl cellulose, methyl cellulose, cholesterol lipase, cholesterol oxidase, potassium ferricyanide, phosphate buffer, and Triton X-100.

[0198] Example 13

[0199] In this embodiment, the microfluidic test strip is used to detect specific proteins in biological fluids, such as antigens, antibodies, and enzymes. The test samples include blood, tissue fluid, urine, cerebrospinal fluid, tears, bronchoalveolar lavage fluid, saliva, or other biological fluids. The pre-contained reagents include bovine serum albumin (BSA), antigens, antibodies, labeled antigens, labeled antibodies, nucleic acid aptamers, phosphate buffer (pH=7.0; 0.1M), Triton X-100, and Proclin 300.

[0200] Example 14

[0201] In this embodiment, the microfluidic test strip is used to detect nucleic acids in biological fluids, where the specific nucleic acid can be DNA or RNA. The test samples include blood, tissue fluid, urine, cerebrospinal fluid, tears, bronchoalveolar lavage fluid, saliva, other biological fluids, or biological extracts such as hair, pharyngeal swabs, nasal swabs, and anal swabs. The pre-contained reagents include bovine serum albumin (BSA), Taq polymerase, phosphate buffer (pH=7.0; 0.1M), Triton X-100, and Proclin 300.

[0202] The above specific embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A microfluidic test strip, characterized in that, include: The sample dispensing port is used to add the sample to be tested; The flow channel is connected to the sample dispensing port; An airbag is connected to the circulation channel and has a first intersection point with the circulation channel. The airbag has a compressed state and an inflated state, and the switching between the two states is controllable. The flow channel has a detection zone located downstream of the first intersection point; A sensing area is provided between the first intersection point and the detection area to form a quantitative area between the first intersection point and the sensing area, which is used to determine the amount of sample to be tested delivered to the detection area; The airbag includes at least a first airbag and a second airbag, with the first airbag located upstream of the second airbag and the detection area located in the flow channel between the first airbag and the second airbag, or the detection area located in the flow channel downstream of the second airbag.

2. The microfluidic test paper according to claim 1, characterized in that, include: The first control layer serves as a cover plate layer; The second control layer is made of a flexible material; The third control layer serves as the sensing layer; The first control layer, the second control layer, and the third control layer are stacked together to form the sample inlet, the flow channel, and the airbag.

3. The microfluidic test paper according to claim 1 or 2, characterized in that: The flow channel between the first airbag and the second airbag is bent, and the detection area is located within the bent area.

4. The microfluidic test paper according to claim 1, characterized in that: The sample has a first end, a second end, a third end, and a fourth end, with the first intersection point as the interruption point. The sample with the first end as the starting point and the second end as the ending point is located in the quantitative zone, while the sample with the fourth end as the starting point and the third end as the ending point can flow upstream to the sample dispensing port.

5. The microfluidic test paper according to claim 1, characterized in that: The flow channel is bent to form an M-shape with the opening facing to the side.

6. The microfluidic test paper according to claim 2, characterized in that: The third control layer is equipped with electrodes, and the current generated by the reaction is transmitted to the detection instrument through the electrodes and electrode contacts.

7. The microfluidic test paper according to claim 2, characterized in that: It also includes a fourth control layer with a waste liquid area, which is connected to the flow channel through the first channel. The fourth control layer is stacked together with the first control layer, the second control layer and the third control layer.

8. The microfluidic test paper according to claim 2, 6, or 7, characterized in that: The first control layer has a gas channel, and the airbag is located upstream of the gas channel.

9. The microfluidic test paper according to claim 8, characterized in that: The downstream of the flow channel has multiple branch channels, and each branch channel has an opening for a gas passage on its side wall.

10. A microfluidic detection method, utilizing the microfluidic test paper as described in any one of claims 1-9, characterized in that, Includes the following steps: The first airbag is inflated, and the second airbag is compressed. Place the sample into the sample dispensing port; The compressed second air bladder is released to adsorb the sample flowing to the quantification zone, which is used to determine the sample volume required for the reaction; The second airbag stops releasing pressure and applies pressure to the first airbag, driving the quantitative sample to continue flowing to the detection area; The sample reacts with a pre-placed reagent in the detection area; The instrument detects the reaction results.

11. The microfluidic detection method according to claim 10, characterized in that: It also includes a thorough mixing step, where the compressed second air bladder is released to adsorb the sample flow to the quantitative zone, the second air bladder is pressurized, the sample flows back to the sample inlet, and the above actions are repeated until the mixture is thorough.

12. The microfluidic detection method according to claim 10, characterized in that: It also includes a full reaction step, Depressurize the second airbag and apply pressure to the first airbag; The detection area has a first sensing position and a second sensing position. The sample has a first end, a second end, a third end and a fourth end with the first intersection point as the interruption point. The sample with the first end as the starting point and the second end as the ending point is located in the quantitative area. The sample flows downstream from the first end until the second end of the sample flows through the second sensing position. Apply pressure to the second airbag and release pressure to the first airbag; The sample flows upstream from the second end until the first end of the sample flows past the first sensing position. Repeat the above steps to allow the sample and pre-prepared reagents to react fully.

13. The microfluidic detection method according to claim 10, characterized in that: It also includes a waste discharge step, in which pressure is applied to the first and second airbags, with the pressure applied to the second airbag being less than the pressure applied to the first airbag, until the sample enters the waste liquid area.

14. The microfluidic detection method according to claim 10, characterized in that: It also includes a catalytic reaction step, in which a potential is applied to the electrode in the detection area to induce a catalytic reaction.

15. The microfluidic detection method according to claim 10, characterized in that: The instrument is an electrochemical instrument, and the current generated by the reaction is transmitted to the electrochemical instrument through electrodes and electrode contacts.

16. The microfluidic detection method according to claim 10, characterized in that: The instrument is a photochemical instrument. The photochemical instrument emits detection light into the detection area, which is reflected back to the photochemical instrument after being reflected by the detection area.

17. The microfluidic detection method according to claim 10, characterized in that: The instrument is equipped with a constant temperature module.

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