Portable micro-fluidic chip, detection method and detection device

Abstract

The invention discloses a portable micro-fluidic chip, a detection method and a detection device, and belongs to the field of micro-fluidic chips, the portable micro-fluidic chip comprises a chip body and M detection modules on the chip body, the detection module comprises a quantitative chamber, a micro-channel, a reaction chamber, N first connecting channels and N test strips; reactants are pre-buried in the reaction chamber; one end of the micro-channel is communicated with the bottom end of the quantitative chamber, the other end of the micro-channel is communicated with the reaction chamber, and the connecting point of the micro-channel and the reaction chamber is lower than the bottom end of the quantitative chamber; the bottom ends of the N first connecting channels are all communicated with the top end of the reaction chamber, and the top ends of the N first connecting channels are respectively communicated with the top ends of the N test strips; the flow resistance of the first connecting channel is smaller than that of the micro-channel; m and N are positive integers greater than or equal to 1. According to the invention, the difficulty and complexity of fluid control can be reduced, and the portability of the micro-fluidic chip is improved.

Description

Technical Field

[0001] The present invention belongs to the field of microfluidic chips, and more specifically, relates to a portable microfluidic chip, a detection method and a detection device. Background Art

[0002] Lateral flow antigen tests are rapid, convenient, and fill an important gap in the healthcare system, but their sensitivity is modest and they may miss potentially infected people. Compared with antigen tests, nucleic acid-based tests are more easily adapted to changing targets and have higher sensitivity. However, the gold standard for nucleic acid testing, quantitative polymerase chain reaction (qPCR), is not suitable for field use, especially in remote areas with limited resources, because it requires expensive benchtop instruments, trained experts, and reliable infrastructure, and sample transportation and result reporting time also greatly exceed the turnaround time of centralized testing. In addition, testing for sexually transmitted viruses (STVs) such as human papillomavirus (HPV) in a doctor's office can be inconvenient, and personal fears and social taboos often hinder effective screening and treatment of these infections. Therefore, a rapid, effective, and affordable home nucleic acid diagnostic method is essential for disease prevention.

[0003] As an alternative to PCR, CRISPR / Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) have emerged as a powerful point-of-care tool for molecular diagnosis of pathogens. In short, Cas proteins (such as Cas12a, Cas12b, Cas13a, and Cas14a) are activated by CRISPR RNA (crRNA) paired with complementary DNA, and then release strong trans-cleavage activity to non-specifically cleave the designed single-stranded DNA / RNA reporter probe to generate target-specific detection signals. Due to the sequence specificity between crRNA and the target, the detection specificity is guaranteed, but the sensitivity of direct nucleic acid detection based on CRISPR is limited to the picomolar range. To further improve the detection sensitivity, CRISPR-driven detection strategies are often combined with isothermal amplification methods such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) to develop sensitive and specific CRISPR-based molecular diagnostic technologies, such as SHERLOCK (Specific High Sensitivity Enzymatic Reporter Unlocking) and DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter). RPA is one of the most popular amplification techniques for CRISPR-based molecular assays because it is highly sensitive, requires only one temperature (37-42°C), can be easily achieved with a simple heater, and is compatible with the optimal temperature of the CRISPR-Cas system.

[0004] Unfortunately, these platform technologies still face three key bottlenecks in home testing applications: nucleic acid extraction, lack of integration, detection equipment, and multiplex detection capabilities. To perform nucleic acid testing (NAT) at home, samples collected from patients must be accepted and made available for subsequent testing through simple processing. However, current magnetic bead-based nucleic acid extraction involves washing and elution with multiple reagents, which increases the complexity of the test and is not convenient for home users. Although proteinase K-based nucleic acid extraction methods simplify the process, multiple temperatures are still required to control the activity of proteinase K to prevent it from interfering with subsequent reactions. In addition, most CRISPR-Dx detection methods require multiple manual transfers of samples or amplification products during extraction and detection, which may increase the risk of carryover contamination and is not ideal for point-of-care applications. Recent studies have shown that various "one-pot" CRISPR-Dx are better solutions. However, a major obstacle is that isothermal amplification and CRISPR systems are incompatible in one-step detection, because once the Cas protein is activated, its strong cleavage will inevitably destroy the nucleic acid amplification template, resulting in reduced amplification efficiency and detection sensitivity. To solve this problem, some researchers have made some attempts, such as spatial isolation of reaction reagents, screening of suboptimal protospacer adjacent motifs (PAM) (NTV and TTNT), or development of light-controlled short photosensitive nucleotides. Despite the progress, the process of screening the best PAM site requires a lot of preliminary work, and short nucleotides require additional chemical modifications to achieve the effect of blocking crRNA. Microfluidic technology has the advantages of automation, miniaturization and integration. By integrating CRISPR-Dx strategies, it has been used for nucleic acid detection of various pathogens. DISCoVER integrates LAMP and CRISPR / Cas13 on a microfluidic chip and completes the two-step reaction with pumps and valves. MiCaR integrates the star microchip (SS-Chip) with CRISPR-Cas12a and multiplex RPA, which can effectively detect nine HPV subtypes. mutaSCAN integrates RT-LAMP and CRISPR / Cas12a into a microfluidic chip with variable throughput, solving the problem of temperature incompatibility in the two-step reaction. These studies have demonstrated the practicality of microfluidics, but most of them use active pump strategies to control fluid flow, which are highly dependent on external power sources / fields or actuators (such as pneumatic actuators, centrifugal pumps, syringe pumps, and magnetic pumps). In addition, fluorescence-based methods require dedicated instruments to read fluorescence detection signals, which increases the cost of detection and is not suitable for home self-testing.

[0005] The progress of microfluidic technology has made the driving and control of microfluidics more flexible, accurate and convenient, promoting the development of instant diagnosis, biochemical analysis, cell culture and other related fields. Traditional fluid driving methods rely on the support of expensive devices and require traditional fluid pumps such as peristaltic pumps and syringe pumps. Although they have the advantages of precise adjustment of flow rate and flow rate, they are not conducive to the miniaturization of microfluidic devices and their application in the field of instant diagnosis.

[0006] Compared with traditional fluid pumps, micropumps can better realize the miniaturization of microfluidic devices. Micropumps include active and passive (active, passive). Active micropumps are driven by small external power supplies or actuator platforms, such as small peristaltic pumps, electrowetting, electroosmotic pumps, centrifugal pumps and magnetic pumps. Active micropumps are smaller than traditional fluid pumps and can be more integrated, but their dependence on external power supplies and actuator platforms greatly limits their use in environments with limited conditions. Passive micropumps do not require external power supplies. With the help of some physical principles such as capillary action, siphon action, osmotic pressure, gravity, etc., they can be driven spontaneously, or combined with manpower and specific mechanical structures, such as manually driven syringe pumps, manual centrifugal pumps, and finger-pressed pumps, to achieve liquid driving. Because they are free from dependence on external power supplies and actuator platforms, passive micropumps are widely used in the field of instant diagnosis. However, microfluidic chips based on passive microvalves require complex valve control design and processing, and involve multi-step manual pressing operations. Since different people may use different pressures, and the effect of each compression may vary, fluid manipulation may not be precise enough. Summary of the invention

[0007] In view of the defects of the prior art and the need for improvement, the present invention provides a portable microfluidic chip, a detection method and a detection device, the purpose of which is to reduce the difficulty and complexity of fluid manipulation and improve the portability of the microfluidic chip by improving the flow channel structure in the microfluidic chip.

[0008] To achieve the above object, according to one aspect of the present invention, a portable microfluidic chip is provided, comprising: a chip body, and M detection modules on the chip body;

[0009] The detection module includes: a quantitative chamber, a microchannel, a reaction chamber, N first connecting channels and N test strips;

[0010] The reaction chamber is pre-buried with reactants;

[0011] One end of the microchannel is connected to the bottom end of the quantitative chamber, and the other end is connected to the reaction chamber, and the connection point between the microchannel and the reaction chamber is lower than the bottom end of the quantitative chamber; the bottom ends of the N first connecting channels are all connected to the top end of the reaction chamber, and the top ends of the N first connecting channels are respectively connected to the top ends of the N test strips;

[0012] The flow resistance of the first connecting channel is smaller than the flow resistance of the microchannel;

[0013] Wherein, M and N are both positive integers greater than or equal to 1.

[0014] In some optional embodiments, N>1.

[0015] Further, the detection module further comprises: an enhancement reagent chamber and N second connection channels;

[0016] The enhancement reagent chamber is provided with an enhancement reagent;

[0017] The bottom ends of the N second connecting channels are all connected to the top of the enhancement reagent chamber, and the top ends of the N second connecting channels are respectively connected to the top ends of the N test strips;

[0018] The flow resistance of the second connecting channel is greater than the flow resistance of the first connecting channel.

[0019] Furthermore, the detection module further comprises: N detection chambers, each for pre-embedding N test strips;

[0020] Water absorbing materials are respectively arranged at both ends of the detection chamber.

[0021] In some optional embodiments, the connection point between the microchannel and the reaction chamber is higher than the bottom end of the reaction chamber;

[0022] The chip body includes a plurality of chip layers, and the microchannels and the reaction chambers are located in different chip layers.

[0023] In some optional embodiments, the connection point between the microchannel and the reaction chamber is located at the bottom end of the reaction chamber;

[0024] The chip body comprises a plurality of chip layers, and the microchannel and the reaction chamber are located in the same chip layer.

[0025] In some optional embodiments, the quantitative chamber is in a "V" shape, and the apex of the "V" shape is the bottom end of the quantitative chamber.

[0026] According to another aspect of the present invention, a microfluidic detection method for non-medical diagnosis purposes based on the above-mentioned portable microfluidic chip is provided, comprising the following steps:

[0027] S1: Place the portable microfluidic chip upright, add the sample to be tested into the quantitative chamber of each detection module, and complete the sample quantification;

[0028] S2: applying a positive force to the portable microfluidic chip, so that the samples in each quantitative chamber break through the microchannel and transfer to the reaction chamber, react with the reactants in the reaction chamber, and obtain reaction products;

[0029] S3: After the sample in the chamber to be quantified is completely transferred to the reaction chamber and the reaction in the reaction chamber is completed, the portable microfluidic chip is placed upside down to allow the reaction product to flow to the test strip through the first connecting channel;

[0030] S4: After a preset time, read the test result of the test strip.

[0031] Furthermore, if each detection module in the portable microfluidic chip further includes an enhanced reagent chamber, then in step S4, after a preset time, before reading the detection result of the test strip, the following steps are further included:

[0032] A reverse force is applied to the portable microfluidics to make the enhancement reagent in the enhancement reagent chamber break through the second connecting channel and enter the test strip.

[0033] According to another aspect of the present invention, there is provided a microfluidic detection device, comprising: a base and K portable microfluidic chips;

[0034] K slots are arranged on the bottom bracket, and K portable microfluidic chips are respectively fixed in the K slots;

[0035] Among them, the portable microfluidic chip is the above-mentioned portable microfluidic chip provided by the present invention; K is a positive integer greater than or equal to 1.

[0036] In general, the above technical solutions conceived by the present invention can achieve the following beneficial effects:

[0037] (1) The portable microfluidic chip provided by the present invention integrates one or more detection modules, and the relative positions and flow resistances of different flow channels in each detection module are specially designed. Specifically, the quantitative chamber is connected to the reaction chamber through the microfluidic channel, and the connection point between the microfluidic channel and the reaction chamber is lower than the bottom of the quantitative chamber; the top of the reaction chamber is connected to the bottom of the first connecting channel, the top of the first connecting channel is connected to the top of the test strip, and the flow resistance of the first connecting channel is smaller than the flow resistance of the microfluidic channel; based on such a flow channel design, after the sample is quantified by the quantitative chamber, the sample can be transferred to the reaction chamber for reaction by applying a positive force, and then the reaction chamber is inverted, so that the reaction product in the reaction chamber can enter the first connecting channel without entering the microfluidic channel, and the reaction product entering the first connecting channel can be further transferred to the test strip through the first connecting channel, which is convenient for reading the detection result; that is, based on the design of the flow channel structure, the present invention can realize complex fluid control in the detection process by simply controlling the placement direction of the chip, which greatly reduces the difficulty and complexity of fluid manipulation and improves the portability of the microfluidic chip.

[0038] (2) In a preferred embodiment of the present invention, a plurality of test strips are provided in the same detection module, and a plurality of first connection channels are provided accordingly, so that different tests can be completed in parallel for the same sample, thereby improving the detection efficiency.

[0039] (3) In a preferred embodiment of the present invention, an enhancement reagent chamber is further provided in the detection module, wherein an enhancement reagent is provided, and the enhancement reagent chamber is connected to the top of the test strip through a second connecting channel, and the flow resistance of the second connecting channel is greater than the flow resistance of the first connecting channel. Based on the design of the channel flow resistance, when the chip is placed upside down to transfer the reaction product in the reaction chamber to the test strip, the enhancement reagent in the enhancement reagent chamber cannot break through the second connecting channel, that is, the enhancement reagent will not interfere with the process of transferring the reaction product to the test strip; after the reaction product is completely transferred to the test strip, the enhancement reagent can be transferred to the test strip by applying a reverse force to the chip, thereby enhancing the detection result on the test strip and improving the detection sensitivity.

[0040] (4) In a preferred embodiment of the present invention, a detection chamber for placing a test strip is specifically provided in the detection module, and water-absorbing materials are provided at both ends of the detection chamber, thereby improving the efficiency of transferring the reaction product in the reaction chamber or the enhancement reagent in the enhancement reagent chamber to the test strip.

[0041] (5) In some optional embodiments of the present invention, the connection point between the microchannel and the reaction chamber is higher than the bottom of the reaction chamber, which facilitates the transfer of samples in the quantitative chamber to the reaction chamber; In some optional embodiments of the present invention, the connection point between the microchannel and the reaction chamber is higher than the bottom of the reaction chamber, so that the microchannel and the reaction chamber can be set on the same chip layer, simplifying the layered structure design of the chip.

[0042] (6) In a preferred embodiment of the present invention, the quantitative chamber is in a "V" shape, and the top of the "V" shape is the bottom of the quantitative chamber, which facilitates the quantification of the sample and the transfer of the sample in the quantitative chamber to the reaction chamber. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 A schematic diagram of the structure of a portable microfluidic chip provided in Example 1 of the present invention;

[0044] Figure 2 A schematic diagram of the layered structure of the portable microfluidic chip provided in Example 1 of the present invention;

[0045] Figure 3 Schematic diagrams of other portable microfluidic chip structures of the present invention; wherein (a) is a schematic diagram of a portable microfluidic chip comprising three detection modules, and (b) is a schematic diagram of a portable microfluidic chip comprising only one detection module;

[0046] Figure 4 A schematic diagram of the flow chart of the microfluidic detection method provided in Example 2 of the present invention;

[0047] Figure 5 This is a schematic diagram of the structure of a portable microfluidic chip provided in Example 3 of the present invention; wherein (a) is a schematic diagram of a detection module in the chip, and (b) is a schematic diagram of a layered structure of the chip;

[0048] Figure 6 A schematic diagram of the flow chart of the microfluidic detection method provided in Example 4 of the present invention;

[0049] Figure 7 A schematic diagram of the structure of a portable microfluidic chip provided in Example 5 of the present invention; wherein (a) is a schematic diagram of a detection module in the chip, and (b) is a schematic diagram of a layered structure of the chip;

[0050] Figure 8 A schematic diagram of the flow chart of the microfluidic detection method provided in Example 6 of the present invention;

[0051] Fig. 9 A schematic diagram of the structure of a portable microfluidic chip provided in Example 7 of the present invention; wherein (a) is a schematic diagram of a detection module in the chip, and (b) is a schematic diagram of a layered structure of the chip;

[0052] Fig.10 A schematic diagram of the flow chart of the microfluidic detection method provided in Example 8 of the present invention;

[0053] Fig.11 A schematic diagram of a microfluidic detection device provided in Example 9 of the present invention;

[0054] Fig.12 The experimental results of temperature control provided by the present invention; wherein (a) is the temperature change of two chemical heat release devices, a heating pack and a warming patch; (b) the RPA and CRISPR results corresponding to the release of DNA by chemical lysing agents in combination with different heat sources, the gray value ratio of the test line to the quality control line in the test strip is (T / C); (c) the difference between the warming patch and the water bath in performing RPA and CRISPR, the gray value ratio of the test line to the quality control line in the test strip is (T / C);

[0055] Fig.13 Experimental results related to primer validation provided by the present invention; wherein (a) is the detection principle of RPA, CRISPR and test strips, (b) is the single-plex RPA validation result of primers for four HPV subtypes (HPV 6, HPV 11, HPV 16 and HPV 18), and (c) is the multiplex RPA validation result of primers for four HPV subtypes (HPV 6, HPV 11, HPV 16 and HPV 18);

[0056] Fig.14 The specificity and sensitivity test results of four HPV subtypes provided by the present invention; wherein, (a) is a schematic diagram of specificity detection, (b) is a specificity test result based on a test strip, the test strip result on the left, and a heat map of the ratio of the test line and the quality control line on the right, (c) is the sensitivity of the unamplified plasmid, from left to right are HPV 6, HPV 11, HPV 16, HPV 18, (d) is the sensitivity of the amplified plasmid, from left to right are HPV 6, HPV 11, HPV 16, HPV 18;

[0057] Fig.15 The interpretation results of 140 HPV clinical samples provided by the present invention; wherein, (a) is the detection process, (b) is the images of some typical clinical samples, (c) is the heat map of the on-chip detection results of 4 subtypes of 140 HPV clinical samples, (d) is the ROC analysis result of 4 HPVs, (e) is the grouping scatter plot of 4 HPVs, the critical value is set to 0.54 (≤0.54 is positive; >0.54 is negative), and (f) is a four-grid table of the on-chip and clinical detection results of 4 HPV subtypes;

[0058] Fig.16 The interpretation results of 70 spiked respiratory virus samples provided by the present invention; wherein, (a) is the detection process, (b) is the image of some spiked samples, (c) is the on-chip detection result of the chip for three respiratory viruses in 70 spiked samples, (d) ROC analysis of three respiratory viruses on the chip, (e) grouped scatter plot of three respiratory viruses, the critical value is set to 0.65 (≤0.65 is positive; >0.65 is negative), and (f) is a four-grid table of the on-chip and spiked detection results of three respiratory viruses;

[0059] In all drawings, the same reference numerals are used to denote the same elements or structures, wherein:

[0060] 1-Chip body; 2-Detection module;

[0061] 21-quantitative chamber; 22-microchannel, 221-first connecting hole; 23-reaction chamber; 24-first connecting channel, 241-second connecting hole; 25-test strip, 251-detection line, 252-quality control line; 26-detection chamber, 261-water-absorbing material; 27-enhanced reagent chamber; 28-second connecting channel. DETAILED DESCRIPTION

[0062] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0063] It should be understood by those skilled in the art that these embodiments are only used to explain the technical principles of the present invention, and are not intended to limit the scope of protection of the present invention. It should be noted that in the description of the present invention, the terms "upper", "lower", "left", "right", "inner", "outer" and the like indicating directions or positional relationships are based on the directions or positional relationships shown in the drawings, which are only for the convenience of description, and do not indicate or imply that the device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation of the present invention. In addition, it should be noted that in the description of the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected" and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected, or indirectly connected through an intermediate medium, or it can be the internal connection of two elements. For those skilled in the art, the specific meanings of the above terms in the present invention can be understood according to the specific circumstances. The terms "first", "second" and the like (if any) in the present invention and the drawings are used to distinguish similar objects, and do not have to be used to describe a specific order or sequence.

[0064] In order to reduce the difficulty and complexity of fluid manipulation and improve the portability of microfluidic chips, the present invention provides a portable microfluidic chip, a detection method and a detection device, and improves the flow channel structure in the microfluidic chip, specifically including a special design of the relative positions between the flow channels and chambers and the relative relationship of the flow resistance between the flow channels, so that the fluid can be manipulated during the detection process by simply manipulating the placement direction of the chip.

[0065] It is easy to understand that in the present invention, the descriptions of "top", "bottom", "high", "low", etc. are all relative to the chip in an upright state, and when the chip is in an upright state, the highest point of each structure is the top of the corresponding structure, and the lowest point of each structure is the bottom of the corresponding structure. "Forward force" and "reverse force" are relative to the top and bottom of the chip. The force from the top of the chip to the bottom of the chip is the forward force, and conversely, the force from the bottom of the chip to the top of the chip is the reverse force.

[0066] The following are examples.

[0067] Embodiment 1:

[0068] A portable microfluidic chip, such as Figure 1-Figure 2 As shown, it includes: a chip body 1, and five detection modules 2 on the chip body;

[0069] The detection module 2 includes: a quantitative chamber 21, a microchannel 22, a reaction chamber 23, a first connecting channel 24 and a test strip 25;

[0070] The reaction chamber 23 is pre-buried with reactants;

[0071] One end of the microchannel 22 is connected to the bottom end of the quantitative chamber 21, and the other end is connected to the reaction chamber 23, and the connection point between the microchannel 22 and the reaction chamber 23 is lower than the bottom end of the quantitative chamber 21; the bottom end of the first connecting channel 24 is connected to the top end of the reaction chamber 23, and the top end of the first connecting channel 24 is connected to the top end of the test strip 25;

[0072] The flow resistance of the first connecting channel 24 is smaller than the flow resistance of the microchannel 22 .

[0073] This embodiment uses the above-mentioned flow channel structure design to achieve complex fluid control during the detection process by simply controlling the placement direction of the chip, which greatly reduces the difficulty and complexity of fluid manipulation and improves the portability of the microfluidic chip. Specifically, after the sample is quantified through the quantitative chamber, the sample can be transferred to the reaction chamber for reaction by applying a positive force through centrifugation or other means, and then the reaction chamber is inverted, so that the reaction product in the reaction chamber can enter the first connecting channel and will not enter the microchannel. The reaction product entering the first connecting channel will be further transferred to the test strip through the first connecting channel, which is convenient for reading the test results.

[0074] The portable microfluidic chip provided in this embodiment can be used for multi-virus nucleic acid detection, HPV subtype detection, respiratory virus detection, etc.

[0075] It is easy to understand that the reaction product in the reaction chamber can be set according to the specific detection target. Similarly, the test strip (LFA) can be selected according to the specific detection target, and the reading of the test result is realized by comprehensively interpreting the display results of the test line 251 (T) and the quality control line 252 (C) on the test strip. Optionally, in this embodiment, the test strip used is a negative signal test strip. If the test line 251 and the quality control line 252 show the test results at the same time, it indicates that the test result is negative; if only the quality control line 252 shows the test result, it indicates that the test result is positive; in other cases, it indicates that the test result is invalid.

[0076] In practical applications, the control of flow resistance of the flow channel can be achieved by designing the flow channel dimensions, by modifying the inner wall of the flow channel, or by other methods.

[0077] Further Figure 1 As shown, in this embodiment, the connection point between the microchannel 22 and the reaction chamber 24 is higher than the bottom end of the reaction chamber 23, and the bottom end of the microchannel 22 is specifically connected to the reaction chamber 23 through the first connecting hole 221. This structural design can facilitate the transfer of samples in the quantitative chamber to the reaction chamber.

[0078] Optionally, the top of the first connecting channel 24 is connected to the top of the test strip 25 through the second connecting hole 241 .

[0079] Further Figure 1 As shown, in this embodiment, the detection module 2 further includes: a detection chamber 26 for pre-embedding the test strip 25;

[0080] Water absorbing materials 261 are respectively disposed at both ends of the detection chamber 26 .

[0081] By pre-embedding the test strip in the detection chamber and arranging water-absorbing materials at both ends of the detection chamber, the efficiency of the reaction product in the reaction chamber being transferred to the test strip through the first connecting channel can be improved under the suction effect of the water-absorbing materials. In practical applications, the water-absorbing materials can be arranged as water-absorbing paper, water-absorbing sponge, etc.

[0082] As a preferred embodiment, the quantitative chamber in this embodiment is in a "V" shape, and the top of the "V" shape is the bottom of the quantitative chamber. This structure facilitates the quantification of samples and the transfer of samples in the quantitative chamber to the reaction chamber. In addition, optionally, as Figure 1 As shown, in this embodiment, the quantitative chambers of the five detection modules integrated on the same chip body are interconnected, so that simultaneous quantification of the five quantitative chambers can be achieved by adding samples only through the leftmost quantitative chamber or the quantitative chamber.

[0083] It is easy to understand that in order to facilitate the transfer of fluid between different chambers or channels, in this embodiment, air holes are also provided for each chamber to connect the chamber with the atmosphere.

[0084] In accordance with the above structural design, as an optional implementation, in this embodiment, the layered design of the chip is as follows: Figure 2 As shown, the chip layers are, from bottom to top, bottom plate layer, test strip layer, water absorption layer, chamber layer, connection layer, quantitative layer, microchannel layer and top cover layer; the test strip is pre-buried in the test strip layer, the water absorption material is arranged in the water absorption layer, the reaction chamber and the connection channel are arranged in the chamber layer, the first quantitative chamber is arranged in the quantitative layer, and the microchannel is arranged in the microchannel layer; corresponding connecting holes are arranged in the connection layer. Optionally, in this embodiment, each chip layer is composed of PMMA.

[0085] It should be noted that, in this embodiment, the number of detection modules integrated in the chip body is not limited to 5 in this embodiment, and can be set according to actual detection requirements. For example, in some other embodiments of the present invention, it can be set to 3, such as Figure 3 As shown in (a) in FIG. 1 , in some other embodiments of the present invention, it can also be set to 1, such as Figure 3 More examples will not be listed here one by one.

[0086] In addition, in this embodiment, the specific chip layering design scheme is only an optional implementation method. In other embodiments of the present invention, on the basis of ensuring the relationship between each structure and the realization of corresponding functions, the number of chip layers and the distribution of each structure in each chip layer can be flexibly designed.

[0087] As an optional implementation, according to actual detection needs, other reactants such as amplification reagents may be pre-embedded in the quantitative chamber so that the corresponding reaction is completed before the sample is transferred to the reaction chamber.

[0088] Embodiment 2:

[0089] A microfluidic detection method of a portable microfluidic chip provided based on the above-mentioned embodiment 1, the present embodiment is used to realize multi-virus nucleic acid detection. As a preferred embodiment, in the present embodiment, the first detection module on the chip is selected as a negative control group, and accordingly, the reaction chamber in the detection module does not contain crRNA, and only contains Cas12a and the CRISPR detection mixture of the reporter gene; the remaining detection modules are used as normal detection groups, and accordingly, the pre-buried reactants in the reaction chambers in each detection module are Cas12a, crRNA and the CRISPR detection mixture of the reporter gene. Optionally, the reporter gene used in the present embodiment is an oligonucleotide sequence simultaneously labeled with biotin and FAM, i.e., TBA11 (GGTTGGTGTGG), which has a higher sensitivity than normal ssDNA reporter genes. In addition, an amplification reagent is pre-buried in the quantitative chamber in each detection module.

[0090] In practical applications, the number of detection modules on the chip and the type of crRNA can be changed according to the needs of target detection, thereby achieving personalized customization of detection needs.

[0091] like Figure 4 As shown, this embodiment includes the following steps:

[0092] S1: Place the portable microfluidic chip upright, add the sample to be tested into the quantitative chamber, divide it into 5 parts by the "V"-shaped quantitative chamber, achieve quantification, and perform multiple recombinase polymerase amplification (RPA); during multiple RPA, the chip can be heated; at this time, due to the large flow resistance of the microchannel, the sample in the quantitative chamber will not enter the reaction chamber;

[0093] S2: applying a positive force to the portable microfluidic chip, so that the sample after multiple RPA breaks through the microchannel and enters the reaction chamber, reacts with the reactants in the reaction chamber, and generates reaction products;

[0094] S3: After the sample in the chamber to be quantified is completely transferred to the reaction chamber and the reaction in the reaction chamber is completed, the portable microfluidic chip is placed upside down so that the reaction product flows to the test strip through the first connecting channel; since the flow resistance of the microchannel is greater than the flow resistance of the first connecting channel, the reactants in the reaction chamber will preferentially enter the first connecting channel and will not flow back to the quantification chamber through the microchannel;

[0095] S4: After a preset time, read the test result of the test strip; in this embodiment, the preset time is specifically 5 minutes. When the target DNA matches the crRNA, the corresponding nucleic acid test strip will cause the signal intensity of the test line to disappear or weaken due to the cutting off of the reporter gene.

[0096] Based on the portable microfluidic chip provided in the above-mentioned Example 1, the microfluidic detection method provided in this example has the following advantages: (1) There is no need to extract and purify nucleic acids, which simplifies the detection process and the entire reaction (including sample preparation) can be completed within 1 hour; (2) The combined use of chemical lysis reagents and chemical exothermic devices enables the chip to stably perform rapid detection in an environment lacking professional heating instruments such as water baths and incubators; (3) Through the sophisticated layout of the chip microchannels, no external instruments are required, and the direction of the chip can be simply manipulated in combination with the application of external force through the sophisticated layout of the chip microchannels; (4) The combined use of RPA and CRISPR significantly improves the detection sensitivity and specificity. Experiments show that the detection limits of unamplified and amplified samples based on LFA readings are 0.05nM and 10 –18 M, with a diagnostic accuracy of 98.57%; (5) It has strong universality and can be used for various nucleic acid tests such as COVID-19, influenza A, and influenza B.

[0097] In general, the proposed portable microfluidic chip can overcome the challenges of traditional PCR in terms of portability, cost, and analytical performance, and can realize microfluidic detection quickly, at low cost, and conveniently.

[0098] Embodiment 3:

[0099] A portable microfluidic chip, such as Figure 5This embodiment is similar to the above-mentioned embodiment 1, except that in order to further improve the enhanced color development effect of the test strip and improve the detection sensitivity, Figure 5 As shown in (a), in this embodiment, each detection module 2 further includes an enhancement reagent chamber 27 and a second connecting channel 28;

[0100] The enhancement reagent chamber 27 is provided with an enhancement reagent;

[0101] The bottom end of the second connecting channel 28 is connected to the top end of the enhanced reagent chamber 27, and the top end of the second connecting channel 28 is connected to the top end of the test strip 25;

[0102] The flow resistance of the second connecting channel 28 is greater than the flow resistance of the first connecting channel 24 .

[0103] In this embodiment, an enhancement reagent chamber is provided, which is connected to the top of the test strip through a second connecting channel, and the flow resistance of the second connecting channel is greater than the flow resistance of the first connecting channel. Based on the design of the channel flow resistance, when the chip is placed upside down to transfer the reaction product in the reaction chamber to the test strip, the enhancement reagent in the enhancement reagent chamber cannot break through the second connecting channel, that is, the enhancement reagent will not interfere with the process of transferring the reaction product to the test strip; after the reaction product is completely transferred to the test strip, the enhancement reagent can be transferred to the test strip through the second connecting channel by applying a reverse force to the chip, thereby enhancing the detection result on the test strip and improving the detection sensitivity.

[0104] Alternatively, if Figure 5 As shown in (b), this embodiment adopts the same chip layering design as the above-mentioned embodiment 1. In this embodiment, the enhanced actual chamber, the second connecting channel, the reaction chamber and the first connecting channel are arranged in the same chip layer, that is, the chamber layer.

[0105] The specific implementation of the remaining structures in this embodiment is the same as that of the above-mentioned embodiment 1, and the details can be referred to the description of the above-mentioned embodiment 1.

[0106] Embodiment 4:

[0107] A microfluidic detection method based on the portable microfluidic chip provided in the above embodiment 3. This embodiment is similar to the above embodiment 2, except that Figure 6 As shown, in this embodiment, in step S4, after a preset time, before reading the test result of the test strip, the following steps are also included:

[0108] A reverse force is applied to the portable microfluidics to make the enhancement reagent in the enhancement reagent chamber break through the second connecting channel and enter the test strip.

[0109] Under the enhancing effect of the enhancing reagent, the color development effect of the test strip can be enhanced and the detection sensitivity can be improved.

[0110] Embodiment 5:

[0111] A portable microfluidic chip, such as Figure 7 This embodiment is similar to the above-mentioned embodiment 1, except that Figure 7 As shown in (a), in this embodiment, the connection point between the microchannel 22 and the reaction chamber 23 is located at the bottom end of the reaction chamber 23. Based on this structural design, in this embodiment, the microchannel 22 and the reaction chamber 23 can be arranged on the same chip layer.

[0112] Optionally, in this embodiment, the layered structure of the chip is as follows: Figure 7 As shown in (b), compared with the above-mentioned embodiment 1, this embodiment can reduce the number of chip layers and simplify chip design.

[0113] Embodiment 6:

[0114] A microfluidic detection method based on the portable microfluidic chip provided in Example 5 above. Figure 8 As shown, the detection process steps of this embodiment are the same as those of the above-mentioned embodiment 2, and the specific implementation of each step can refer to the description in the above-mentioned embodiment 2.

[0115] Embodiment 7:

[0116] A portable microfluidic chip, such as Fig. 9 This embodiment is similar to the above-mentioned embodiment 1, except that Fig. 9 As shown in (a), in this embodiment, the number of the test strips 25 is 3, and accordingly, the number of the first connecting channels 24 and the number of the detection chambers 26 are both 3;

[0117] Three test strips 25 are pre-buried in three detection chambers 26 respectively, and water absorbing materials 262 are provided at both ends of the three detection chambers 26;

[0118] The bottom ends of the three first connecting channels 24 are all connected to the top end of the reaction chamber 23 , and the top ends of the three first connecting channels 24 are respectively connected to the top ends of the three test strips 25 .

[0119] Based on this structural design, after the sample is transferred from the quantitative chamber to the reaction chamber and the reaction is completed, the portable microfluidic chip is inverted, and the reaction products in the reaction chamber will be evenly distributed to the three first channels to achieve secondary quantitative distribution, and then transferred to the three test strips through the three first connecting channels to complete the reading of the test results.

[0120] By increasing the number of test strips and setting different test strips, this embodiment can complete multiple tests on the same sample at one time.

[0121] In this embodiment, an amplification reagent chamber can also be set in the detection module. In this case, the number of second connecting channels is 3, and the bottom ends of the three second connecting channels are connected to the top of the enhancement reagent chamber, and the top ends of the three second connecting channels are respectively connected to the top ends of the three test strips.

[0122] In this embodiment, the same layered design scheme as in the above-mentioned embodiment 1 is adopted, such as Fig. 9 As shown in (b) in .

[0123] Embodiment 8:

[0124] A microfluidic detection method based on the portable microfluidic chip provided in Example 7. Fig.10 As shown, the detection process steps of this embodiment are the same as those of the above-mentioned embodiment 2, and the specific implementation of each step can refer to the description in the above-mentioned embodiment 2.

[0125] Embodiment 9:

[0126] A microfluidic detection device, such as Fig.11 As shown, it includes: a base and 5 portable microfluidic chips;

[0127] There are 5 slots on the bottom bracket, and 5 portable microfluidic chips are fixed in the 5 slots respectively;

[0128] Wherein, the portable microfluidic chip is the portable microfluidic chip provided in the above-mentioned embodiment 1.

[0129] This embodiment integrates multiple portable microfluidic chips into the same microfluidic detection device, so as to realize multiple detections at the same time and improve the detection throughput.

[0130] In some other embodiments of the present invention, the number of portable microfluidic chips in the device can be flexibly adjusted to other numbers according to detection needs; the portable microfluidic chips can also select other chips provided in the above embodiments, or other chips implemented based on the above concepts.

[0131] The following is a further analysis and explanation of the technical effects that can be achieved by the present invention in combination with specific experimental results.

[0132] Fig.12These are the experimental results related to temperature control, where (a) is the temperature change of two chemical heat release devices, a heating pack and a warming patch; (b) is the RPA and CRISPR results corresponding to the release of DNA by chemical lysing agents combined with different heat sources. The grayscale value ratio of the test line to the quality control line in the test strip is (T / C); (c) compares the differences between the warming patch and the water bath in RPA and CRISPR. The grayscale value ratio of the test line to the quality control line in the test strip is (T / C).

[0133] The present invention uses clinical samples infected with HPV 16 as a proof of concept to verify the feasibility of the chemical heat release device. First, their temperature changes are recorded. The temperature of the heating pack can be maintained at 90°C for about 10 minutes, which meets the temperature conditions required for cell lysis, such as Fig.12 Similarly, the temperature of the warming patch can be maintained at 45°C for about 60 minutes, which is sufficient to complete RPA amplification and CRISPR detection, as shown in the left figure of (a). Fig.12 As shown in the right figure of (a) in the figure. The cervical swab was added to a sample processing tube containing a chemical lysis reagent, mixed and divided into three parts, and then heated using room temperature, a heating pack, and a water bath to release HPV DNA. The electrophoresis results showed that there was no obvious target band in the positive sample when it was left at room temperature, while the target was successfully amplified using a chemical lysis reagent combined with a heating pack or a water bath. At the same time, the CRISPR detection results based on the test strip showed that there was no difference between negative and positive detection using a chemical lysis reagent alone, while the other two methods would significantly reduce the signal strength of the detection line, such as Fig.12 As shown in (b) in the figure. Therefore, the chemical lysis reagent combined with the chemical exothermic device can replace the physical heating device driven by the power supply. Finally, the present invention uses a heating patch and a water bath as heating devices to compare the effects of RPA and CRISPR. The target band content and intensity produced by the two heating methods are comparable, and there is no significant difference between negative and positive, as shown in Figure 2. Fig.12 The above data show that the chemical exothermic device can enable HID-MACRO to achieve home self-testing without power supply.

[0134] Fig.13 These are the experimental results related to primer validation, where (a) is the detection principle of RPA, CRISPR and test strips, (b) is the single-plex RPA validation result of primers for four HPV subtypes (HPV 6, HPV 11, HPV 16 and HPV 18), and (c) is the multiplex RPA validation result of primers for four HPV subtypes (HPV 6, HPV 11, HPV 16 and HPV 18).

[0135] Fig.13(a) in the figure shows the principle of CRISPR / Cas12 detection based on RPA and test strips. First, the sample is exponentially amplified by RPA, which can significantly improve the detection sensitivity. Subsequently, when crRNA recognizes the HPV target in the RPA product, the trans-cutting ability of Cas12a is activated, and then the TBA11-FB reporter is cut into smaller elements, resulting in a decrease in the signal of the test strip due to the reduction of TBA11-FB-SA-AuNPs complexes captured by the test line. The performance of these primers was determined using single-plex RPA, and agarose gel electrophoresis showed that the four subtype primers worked well, showing clear and dense bands, and there was no nonspecific amplification of the negative control, as shown in Figure 2. Fig.13 The RPA product was then used for strip-based CRISPR detection, and the results showed that all four HPV subtypes were clearly distinguished from the negative control, indicating that all targets were successfully amplified, as shown in Figure 2. Fig.13 In addition, multiplexed RPA analysis within the chip can save test time and reduce reagent consumption, so the optimal primer structure and concentration are crucial to avoid primer aggregation and obtain sufficient amplicons for each target. The present invention uses multiplexed RPA to verify four primers, such as Fig.13 As shown in (c), all targets were successfully amplified without obvious primer dimer formation. Therefore, this primer structure was used in subsequent detection.

[0136] Fig.14 Shown are the specificity and sensitivity test results of four HPV subtypes, where (a) is a schematic diagram of specificity detection, (b) is the specificity detection result based on the test strip, the test strip result on the left, and the heat map of the ratio of the test strip detection line and the quality control line on the right, (c) is the sensitivity of the unamplified plasmid, from left to right are HPV 6, HPV 11, HPV 16, HPV 18, (d) is the sensitivity of the amplified plasmid, from left to right are HPV 6, HPV 11, HPV 16, HPV 18.

[0137] Since there is a high degree of homology between sequences of different HPV subtypes, crRNA designed for a specific subtype can recognize other subtypes. Therefore, a 4×4 matrix test (4 crRNAs × 4 HPV RPA amplified fragments) was designed to screen for cross-reactions of crRNA. In addition, a control group without any target gene was set up. Only when the target gene matches the crRNA will Cas12a be activated and cleave the reporter probe, resulting in a weakened signal on the test strip test line, such as Fig.14 The actual results of CRISPR based on the test strip are the same as the theoretical analysis. When the crRNA corresponds to the target, the signal intensity of the test line is significantly weakened or disappears, as shown in (a). Fig.14As shown in the left figure of (b), no extensive cross-reactivity was observed. Fig.14 As shown in the right figure of (b) in FIG. These highly specific crRNAs are then used for multiple nucleic acid detection.

[0138] Sensitivity determines the detection rate of samples, so the present invention evaluates the detection sensitivity of direct CRISPR and RPA combined with CRISPR. First, the present invention uses plasmid samples of 4 HPV subtypes (without pre-amplification) to verify the sensitivity of direct nucleic acid detection based on CRISPR. Fig.14 As shown in (c), with the increase of plasmid concentration, the signal intensity of the detection line gradually decreased, and the sensitivity of HPV 6, HPV 11, HPV 16 and HPV 18 were 0.05nM, 0.1nM, 0.05nM, and 0.05nM, respectively. In order to further improve the detection sensitivity, the present invention introduced RPA to amplify the plasmid sample before the lysis detection. The CRISPR detection based on the test strip showed that the sensitivity of HPV 6, HPV 11, HPV 16 and HPV 18 was 1×10 -16 M, 1×10 -16 M, 1×10 -18 M, 1×10 -18 M, such as Fig.14 As shown in (d), the sensitivity of the HID-MACRO system was improved by a million times compared with the non-amplified target (0.05 nM). The difference in detection performance between HPV 6 and HPV11 may be due to the different efficiencies in the RPA amplification and CRISPR detection processes. In summary, these results show that RPA can significantly improve the detection sensitivity, and the HID-MACRO system has a good sensitivity for both non-amplified (0.05 nM) and RPA amplified (1×10 -18 M) HPV plasmid has a high detection sensitivity.

[0139] Fig.15 The following table shows the results of the interpretation of 140 HPV clinical samples, where (a) is the detection process, (b) is the images of some typical clinical samples, (c) is the heat map of the on-chip detection results of 140 HPV clinical samples for 4 subtypes analysis, (d) is the ROC analysis result of 4 HPVs, (e) is the grouped scatter plot of 4 HPVs, and the critical value is set at 0.54 (≤0.54 is positive; >0.54 is negative), and (f) is a four-grid table of the on-chip and clinical detection results of 4 HPV subtypes.

[0140] The entire on-chip assay time is approximately 60 minutes, including sample preparation, RPA, CRISPR assay, and readout. Fig.15 As shown in (a) in . Fig.15(b) in the figure shows some typical results on the chip. It can be seen from the figure that the signal intensity of the detection lines on the four subtype detection strips corresponding to the negative control is significantly weaker than the negative control, indicating that the samples are positive for these subtypes. Fig.15 (c) in the figure shows the on-chip results of 140 samples for 4 HPV subtypes. Compared with the previous clinical results, no samples showed false positives, which shows that HID-MACRO has high specificity. Unfortunately, 8 samples showed false negatives (indicated by green boxes), which may be due to DNA degradation caused by long storage time of samples or low viral load. In order to systematically analyze the performance of the HID-MACRO detection platform, a receiver operating characteristic (ROC) analysis was performed by comparing the chip and clinical test results. Fig.15 As shown in (d) in Figure 3, in a total of 560 tests (4 HPV subtypes × 140 clinical samples), the area under the curve (AUC) was 0.9636, indicating that the results of the two methods were highly consistent. When the critical value was set to 0.54 (≤0.54, positive; >0.54, negative), HID-MACRO was able to significantly distinguish between negative and positive, as shown in Figure 3. Fig.15 Overall, HID-MACRO showed good performance in detecting patient samples, with a sensitivity and specificity of 92.73% and 100%, respectively. Fig.15 These results indicate that HID-MACRO based on naked eye reading can be used to accurately identify HPV in clinical samples, providing a low-cost, portable, and reliable point-of-care diagnostic tool for cervical cancer screening.

[0141] Fig.16 The results of 70 spiked samples of respiratory viruses, where (a) is the detection process, (b) is the image of some spiked samples, (c) is the on-chip detection result of the chip for three respiratory viruses in 70 spiked samples, (d) ROC analysis of three respiratory viruses on the chip, (e) grouped scatter plot of the three respiratory viruses, with the critical value set at 0.65 (≤0.65 is positive; >0.65 is negative), and (f) is a four-grid table of the on-chip and spiked detection results of the three respiratory viruses.

[0142] The present invention tested 70 spiked samples of three respiratory viruses. Fig.16 (a) shows the brief process of on-chip detection, which first uses multiplex RPA to amplify the spiked sample, followed by a strip-based CRISPR detection. Fig.16 (b) in the figure shows the typical on-chip detection results of some spiked samples. When the sample contains a certain viral plasmid, the signal intensity of the detection line on the test paper is significantly reduced. Fig.16(c) in Figure 3 shows the on-chip detection results of 70 spiked samples. No false positives or false negatives were found in any of the samples, indicating that HID-MACRO has high specificity and sensitivity. Fig.16 As shown in (d) in Figure 2, a total of 210 tests were performed (3 respiratory viruses × 70 spiked samples), and the area under the curve for the three respiratory viruses was 1.0. When the critical value of the three respiratory viruses was set to 0.65 (≤0.65, positive; >0.65, negative), HID-MACRO was able to significantly distinguish between negative and positive, as shown in Figure 2. Fig.16 The sensitivity and specificity of the three respiratory viruses were 100% (n=98) and 100% (n=112), respectively. Fig.16 The above data show that HID-MACRO can be used as a general strategy for nucleic acid detection.

[0143] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A portable microfluidic chip, characterized in that: include: A chip body, and M detection modules on the chip body; The detection module includes: a quantitative chamber, a microchannel, a reaction chamber, N first connecting channels and N test strips; The reaction chamber is pre-buried with reactants; One end of the microchannel is connected to the bottom end of the quantitative chamber, and the other end is connected to the reaction chamber, and the connection point between the microchannel and the reaction chamber is lower than the bottom end of the quantitative chamber; the bottom ends of the N first connecting channels are all connected to the top end of the reaction chamber, and the top ends of the N first connecting channels are respectively connected to the top ends of the N test strips; The flow resistance of the first connecting channel is smaller than the flow resistance of the microchannel; Wherein, M and N are both positive integers greater than or equal to 1.

2. The portable microfluidic chip according to claim 1, characterized in that: N>1。 3. The portable microfluidic chip according to claim 1, characterized in that: The detection module further includes: an enhancement reagent chamber and N second connection channels; The enhancement reagent chamber is provided with an enhancement reagent; The bottom ends of the N second connecting channels are all connected to the top of the enhancement reagent chamber, and the top ends of the N second connecting channels are respectively connected to the top ends of the N test strips; The flow resistance of the second connecting channel is greater than the flow resistance of the first connecting channel.

4. The portable microfluidic chip according to any one of claims 1 to 3, characterized in that: The detection module further includes: N detection chambers, each used to pre-embed the N test strips; Water absorbing materials are respectively arranged at two ends of the detection chamber.

5. The portable microfluidic chip according to any one of claims 1 to 3, characterized in that: The connection point between the microchannel and the reaction chamber is higher than the bottom end of the reaction chamber; The chip body includes a plurality of chip layers, and the microchannel and the reaction chamber are located in different chip layers.

6. The portable microfluidic chip according to any one of claims 1 to 3, characterized in that: The connection point between the microchannel and the reaction chamber is located at the bottom end of the reaction chamber; The chip body includes a plurality of chip layers, and the microchannel and the reaction chamber are located in the same chip layer.

7. The portable microfluidic chip according to any one of claims 1 to 3, characterized in that: The quantitative chamber is in a "V" shape, and the top of the "V" shape is the bottom of the quantitative chamber.

8. A microfluidic detection method for non-medical diagnosis purposes based on the portable microfluidic chip according to any one of claims 1 to 7, characterized in that: The steps include: S1: placing the portable microfluidic chip upright, adding the sample to be detected into the quantitative chamber of each detection module, and completing the sample quantification; S2: applying a positive force to the portable microfluidic chip, so that the sample in each quantitative chamber breaks through the microchannel and transfers to the reaction chamber, reacts with the reactant in the reaction chamber, and obtains a reaction product; S3: After the sample in the quantitative chamber is completely transferred to the reaction chamber and the reaction in the reaction chamber is completed, the portable microfluidic chip is placed upside down to allow the reaction product to flow to the test strip through the first connecting channel; S4: After a preset time, read the test result of the test strip.

9. The microfluidic detection method for non-medical diagnosis purposes according to claim 8, characterized in that: If each detection module in the portable microfluidic chip further includes an enhanced reagent chamber, then in step S4, after a preset time, before reading the detection result of the test strip, the following steps are further included: A reverse force is applied to the portable microfluidic system to make the enhancement reagent in the enhancement reagent chamber break through the second connecting channel and enter the test strip.

10. A microfluidic detection device, characterized in that: include: Base and K portable microfluidic chips; The base is provided with K slots, and the K portable microfluidic chips are respectively fixed in the K slots; Wherein, the portable microfluidic chip is the portable microfluidic chip described in any one of claims 1 to 7; K is a positive integer greater than or equal to 1.

Images