A portable microfluidic chip, a detection method and a detection device

Abstract

The application discloses a portable micro-fluidic chip, a detection method and a detection device, and belongs to the field of micro-fluidic chips, and 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 paper strips; the reaction chamber is pre-buried with a reactant; one end of the micro-channel is communicated with the bottom end of the quantitative chamber, the other end 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 paper 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. The application can reduce the difficulty and complexity of fluid control and improve the portability of the micro-fluidic chip.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microfluidic chip, more particularly, relates to a portable microfluidic chip, a detection method and a detection device. BACKGROUND

[0002] Lateral flow antigen tests are fast and convenient, filling an important gap in the medical system, but they are generally less sensitive and can miss potential infections. Compared with antigen tests, nucleic acid-based tests are more easily adapted to changing targets and are more sensitive. 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, well-trained specialists, and reliable infrastructure, and sample transportation and result reporting times 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 for these infections. Therefore, a fast, 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) has become a powerful point-of-care tool for pathogen molecular diagnostics. In short, Cas proteins (such as Cas12a, Cas12b, Cas13a, and Cas14a) are activated by CRISPR RNA (crRNA) paired with complementary DNA, then release a powerful trans -cleavage activity that non-specifically cleaves a designed single-stranded DNA / RNA reporter probe, generating a target-specific detection signal. Due to the sequence specificity between crRNA and target, detection specificity is guaranteed, but the sensitivity of CRISPR-based nucleic acid direct detection is limited to the picomolar range. To further improve the detection sensitivity, CRISPR-driven detection strategies are usually combined with isothermal amplification methods such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP), developing sensitive and specific CRISPR-based molecular diagnostic techniques 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 in CRISPR-based molecular detection because it is highly sensitive, requires only one temperature (37-42°C), is easily implemented 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 device, and multiplexing capability. To perform nucleic acid testing (NAT) at home, the patient-collected sample must be accepted and made available for subsequent testing through simple processing. However, current magnetic bead-based nucleic acid extraction involves multiple reagent washing and elution, increasing the complexity of the detection, which is not convenient for home users. Although the proteinase K-based nucleic acid extraction method simplifies the process, it still requires multiple temperatures 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 the extraction and detection process, which can increase the risk of carrying contamination and is not ideal for point-of-care applications. Recent studies have shown that various "one-pot" CRISPR-Dx is a better solution. However, a major obstacle is that isothermal amplification and CRISPR systems are not compatible in one-step detection, because once the Cas protein is activated, its powerful 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 suboptimal protospacer adjacent motifs (PAM) (NTV and TTNT), or developing light-controlled short photosensitive nucleotides. Although progress has been made, 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, and 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 uses pumps and valves to complete the two-step reaction. MiCaR integrates CRISPR-Cas12a and multiplex RPA with a star-shaped microchip (SS-Chip) to 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 two-step reactions. These studies demonstrate the practicality of microfluidic technology, but they mostly use active pump strategies to control fluid flow, which is 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 the fluorescence detection signal, which increases the cost of detection and is not suitable for home self-testing.

[0005] The progress of microfluidic technology makes the driving and control of microfluids more flexible, accurate and convenient, and promotes the development of instant diagnosis, biochemical analysis, cell culture and other related fields. The traditional fluid driving mode relies on the support of expensive devices, and needs traditional fluid pumps such as peristaltic pumps and injection pumps. Although the traditional fluid driving mode has the advantages of accurate flow and flow rate, it is not conducive to the miniaturization of microfluidic devices and the application in the field of instant diagnosis.

[0006] Compared with the traditional fluid pump, the micro-pump can better realize the miniaturization of the microfluidic device. The micro-pump includes active and passive types (active and passive). The active micro-pump is driven by a small external power supply or an actuator platform, such as a small peristaltic pump, an electro-wetting pump, an electro-osmotic pump, a centrifugal pump and a magnetic force pump. The active micro-pump is smaller in size and higher in integration than the traditional fluid pump, but the dependence on the external power supply and the actuator platform greatly limits its use in limited conditions. The passive micro-pump does not need external power supply, and can be self-driven by means of some physical principles such as capillary action, siphon action, osmotic pressure and gravity, or can be driven by manual force combined with a specific mechanical structure, such as a manually driven injection pump, a manually driven centrifugal pump and a finger pressing pump, to realize the driving of the liquid. Because the dependence on the external power supply and the actuator platform is eliminated, the passive micro-pump has a wide application in the field of instant diagnosis. However, the microfluidic chip based on the passive micro-valve needs complex valve control design and processing, and involves multi-step manual pressing operation. Because the pressing force of different persons is quite different, and the effect of each pressing is different, the fluid operation may not be accurate enough. SUMMARY

[0007] In view of the defects of the prior art and the improvement needs, the present application provides a portable microfluidic chip, a detection method and a detection device, which aims to reduce the difficulty and complexity of fluid operation and improve the portability of the microfluidic chip by improving the flow channel structure in the microfluidic chip.

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

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

[0010] The reaction chamber is pre-embedded with a reactant;

[0011] One end of the microchannel is in communication with the bottom end of the quantitative chamber, the other end of the microchannel is in communication with the reaction chamber, and the connection point of 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 in communication with the top end of the reaction chamber, and the top ends of the N first connecting channels are in communication with the top ends of the N test strips, respectively;

[0012] The flow resistance of the first connecting channel is less than the flow resistance of the micro channel.

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

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

[0015] Further, the detection module further comprises an enhancing reagent chamber and N second connecting channels.

[0016] The enhancing reagent chamber is provided with an enhancing reagent.

[0017] The bottom ends of the N second connecting channels are in communication with the top end of the enhancing reagent chamber, and the top ends of the N second connecting channels are in communication with the top ends of the N test strips respectively.

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

[0019] Further, the detection module further comprises N detection chambers for embedding the N test strips respectively.

[0020] The detection chamber is provided with a water-absorbing material at both ends.

[0021] In some optional embodiments, the connecting point of the micro channel and the reaction chamber is higher than the bottom end of the reaction chamber.

[0022] The chip body comprises a plurality of chip layers, and the micro channel and the reaction chamber are located in different chip layers.

[0023] In some optional embodiments, the connecting point of the micro channel 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 micro channel and the reaction chamber are located in the same chip layer.

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

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

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

[0028] S2: Apply a positive force to the portable microfluidic chip, so that the sample in each quantitative chamber breaks through the microfluidic channel and is transferred to the reaction chamber, reacts with the reactant in the reaction chamber, and obtains the reaction product;

[0029] S3: after the sample in the quantification 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, so that the reaction product flows to the test strip through the first connecting channel;

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

[0031] Further, if each detection module in the portable microfluidic chip further comprises an enhancement reagent chamber, before the detection result of the test strip is read after the preset time in step S4, the following step is further included:

[0032] A reverse force is applied to the portable microfluidic chip, so that the enhancement reagent in the enhancement reagent chamber breaks through the second connecting channel and enters the test strip.

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

[0034] The base is provided with K slots, and the 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 application; K is a positive integer greater than or equal to 1.

[0036] Overall, the above technical solutions conceived by the present application can achieve the following beneficial effects:

[0037] (1) The portable microfluidic chip provided by the present application 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 quantification chamber is connected to the reaction chamber through a microflow channel, and the connection point of the microflow channel and the reaction chamber is lower than the bottom end of the quantification chamber; the top end of the reaction chamber is connected to the bottom end of the first connecting channel, the top end of the first connecting channel is connected to the top end of the test strip, and the flow resistance of the first connecting channel is smaller than that of the microflow channel; based on such flow channel design, after the sample is quantified by the quantification chamber, the sample can be transferred to the reaction chamber for reaction by applying a positive force, and then the reaction chamber is placed upside down, so that the reaction product in the reaction chamber enters the first connecting channel without entering the microflow channel. The reaction product entering the first connecting channel is further transferred to the test strip through the first connecting channel, which facilitates the reading of the detection result. That is, based on the design of the flow channel structure, the present application can realize complex fluid control in the detection process by simply controlling the placement direction of the chip, greatly reducing the difficulty and complexity of fluid control, and improving the portability of the microfluidic chip.

[0038] (2) In the preferred scheme of the present application, multiple test strips are arranged in the same detection module, and multiple first connecting channels are arranged correspondingly, so that different detections can be completed in parallel for the same sample, improving the detection efficiency.

[0039] (3) In the preferred scheme of the present application, an enhanced reagent chamber is further arranged in the detection module, wherein an enhanced reagent is arranged, the enhanced reagent chamber is connected with the top end of the test strip through a second connecting channel, and the flow resistance of the second connecting channel is greater than that 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 enhanced reagent in the enhanced reagent chamber cannot break through the second connecting channel, that is, the enhanced reagent does 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 enhanced reagent can be transferred to the test strip by applying a reverse force to the chip, so as to enhance the detection result on the test strip and improve the detection sensitivity.

[0040] (4) In the preferred scheme of the present application, a detection chamber for placing the test strip is specifically arranged in the detection module, and water-absorbing materials are arranged at both ends of the detection chamber, so as to improve the transfer efficiency of the reaction product in the reaction chamber or the enhanced reagent in the enhanced reagent chamber to the test strip.

[0041] (5) In some optional embodiments of the present application, the connecting point of the microchannel and the reaction chamber is higher than the bottom end of the reaction chamber, so as to facilitate the transfer of the sample in the quantitative chamber to the reaction chamber; in some optional embodiments of the present application, the connecting point of the microchannel and the reaction chamber is higher than the bottom end of the reaction chamber, so that the microchannel and the reaction chamber can be arranged on the same chip layer, simplifying the layered structure design of the chip.

[0042] (6) In the preferred scheme of the present application, the quantitative chamber is in the shape of a "V", and the top point of the "V" is the bottom end of the quantitative chamber, so as to facilitate the quantification of the sample and the transfer of the sample in the quantitative chamber to the reaction chamber. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 The structure schematic diagram of the portable microfluidic chip provided for the embodiment 1 of the present application is shown in the figure;

[0044] Figure 2 The layered structure schematic diagram of the portable microfluidic chip provided for the embodiment 1 of the present application is shown in the figure;

[0045] Figure 3 The structure schematic diagram of other portable microfluidic chips of the present application is shown in the figure; wherein (a) is the schematic diagram of a portable microfluidic chip containing three detection modules, and (b) is the schematic diagram of a portable microfluidic chip containing only one detection module;

[0046] Figure 4 The microfluidic detection method flowchart provided for the embodiment 2 of the present application is shown in the figure;

[0047] Figure 5 The structure diagram of the portable microfluidic chip provided for the embodiment 3 of the present application is shown in the figure; wherein, (a) is the detection module diagram in the chip, and (b) is the layered structure diagram of the chip;

[0048] Figure 6 The microfluidic detection method flowchart provided for the embodiment 4 of the present application is shown in the figure;

[0049] Figure 7 The structure diagram of the portable microfluidic chip provided for the embodiment 5 of the present application is shown in the figure; wherein, (a) is the detection module diagram in the chip, and (b) is the layered structure diagram of the chip;

[0050] Figure 8 The microfluidic detection method flowchart provided for the embodiment 6 of the present application is shown in the figure;

[0051] Figure 9 The structure diagram of the portable microfluidic chip provided for the embodiment 7 of the present application is shown in the figure; wherein, (a) is the detection module diagram in the chip, and (b) is the layered structure diagram of the chip;

[0052] Figure 10 The microfluidic detection method flowchart provided for the embodiment 8 of the present application is shown in the figure;

[0053] Figure 11 The microfluidic detection device diagram provided for the embodiment 9 of the present application is shown in the figure;

[0054] Figure 12 The experimental results of the temperature control provided by the present application are shown in the figure; wherein, (a) is the temperature change of the two chemical exothermic devices of the heating bag and the warming patch, (b) is the RPA and CRISPR results corresponding to the DNA released by the chemical cleavage agent combined with different heat sources, the gray value ratio of the detection line to the quality control line in the test strip is (T / C), (c) is the difference of RPA and CRISPR between the warming patch and the water bath, the gray value ratio of the detection line to the quality control line in the test strip is (T / C);

[0055] Figure 13 The experimental results related to the primer verification provided by the present application are shown in the figure; wherein, (a) is the detection principle of RPA, CRISPR and test strip, (b) is the single RPA verification result of the primers of four HPV subtypes (HPV 6, HPV 11, HPV 16 and HPV 18), (c) is the multiplex RPA verification result of the primers of four HPV subtypes (HPV 6, HPV 11, HPV 16 and HPV 18);

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

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

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

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

[0060] 1-chip body; 2-detection module;

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

[0062] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and not to limit the present application. In addition, the technical features involved in the various embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.

[0063] Those skilled in the art should understand that these embodiments are only used to explain the technical principles of the present application and not to limit the protection scope of the present application. It should be noted that in the description of the present application, the terms "upper", "lower", "left", "right", "inner", "outer" and other terms indicating the direction or positional relationship are based on the direction or positional relationship shown in the drawings, which is only for the convenience of description and does 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 on the present application. In addition, it should be noted that in the description of the present application, unless otherwise explicitly specified and limited, the terms "mounting", "connection", "connection" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium; it can be the communication between the two elements inside. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances. The terms "first", "second" and the like (if any) in the present application and the drawings are used to distinguish similar objects, and do not necessarily describe a specific order or sequence.

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

[0065] It is easy to understand that in the present application, the descriptions of "top end", "bottom end", "high", "low" and the like are all relative to the chip in the upright placement state, and in the upright placement state of the chip, the highest point of each structure is the top end of the corresponding structure, and the lowest point of each structure is the bottom end of the corresponding structure. "Positive force", "negative force" is relative to the top end and bottom end of the chip, the force from the top end of the chip to the bottom end of the chip is the positive force, and vice versa, the force from the bottom end of the chip to the top end of the chip is the negative force.

[0066] The following are embodiments.

[0067] Embodiment 1:

[0068] A portable microfluidic chip, such as Figures 1-2 As shown, comprising: a chip body 1, and 5 detection modules 2 on the chip body;

[0069] The detection module 2 comprises: 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-embedded with a reactant;

[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 of 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] Through the above flow channel structure design, the complex fluid control in the detection process can be realized by simply controlling the placement direction of the chip, which greatly reduces the difficulty and complexity of fluid operation and improves the portability of the microfluidic chip. Specifically, after the sample is quantified by the quantitative chamber, the sample can be transferred to the reaction chamber by applying a positive force by centrifugation or the like, and then the reaction chamber is inverted, so that the reaction product in the reaction chamber enters the first connecting channel and does not enter the microchannel. The reaction product entering the first connecting channel is further transferred to the test strip through the first connecting channel, which facilitates reading of the detection result.

[0074] The portable microfluidic chip provided in the 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 detection result is achieved by comprehensively interpreting the display results of the detection line 251 (T) and the quality control line 252 (C) on the test strip. Optionally, in the embodiment, the test strip used is a negative signal test strip. If the detection line 251 and the quality control line 252 simultaneously appear the detection result, it indicates that the detection result is negative; if only the quality control line 252 appears the detection result, it indicates that the detection result is positive; otherwise, it indicates that the detection result is invalid.

[0076] In actual application, the control of the flow channel flow resistance can be realized by designing the size of the flow channel, modifying the inner wall of the flow channel, or other ways.

[0077] Further as Figure 1 shown in the embodiment, the connecting point of the micro-channel 22 and the reaction chamber 24 is higher than the bottom end of the reaction chamber 23, and the bottom end of the micro-channel 22 is connected with the reaction chamber 23 through the first communicating hole 221, which can facilitate the sample in the quantitative chamber to be transferred to the reaction chamber.

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

[0079] Further as Figure 1 shown in the embodiment, the detection module 2 further comprises a detection chamber 26 for pre-embedding the test strip 25.

[0080] The detection chamber 26 is provided with a water-absorbing material 261 at both ends.

[0081] By pre-embedding the test strip in the detection chamber and setting the water-absorbing material at both ends of the detection chamber, the efficiency of transferring the reaction product in the reaction chamber to the test strip through the first connecting channel can be improved under the attraction of the water-absorbing material. In actual application, the water-absorbing material can be water-absorbing paper, water-absorbing sponge, etc.

[0082] As a preferred embodiment, the quantitative chamber in the embodiment is in the shape of "V", and the vertex of the "V" shape is the bottom end 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. In addition, as Figure 1 shown in the embodiment, the quantitative chambers of the five detection modules integrated on the same chip body are connected with each other, so that the simultaneous quantification of the five quantitative chambers can be realized by adding sample to 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, the chambers in the embodiment are also provided with air holes to make the chambers communicate with the atmosphere.

[0084] In order to adapt to the above structure design, as an optional embodiment, the layered design scheme of the chip in the embodiment is as Figure 2 shown, the chip layers are sequentially from bottom to top: a bottom plate layer, a test strip layer, a water-absorbing layer, a chamber layer, a connecting layer, a quantification layer, a micro-channel layer and a top cover layer; the test strip is pre-embedded in the test strip layer, the water-absorbing material is arranged in the water-absorbing layer, the reaction chamber and the connecting channel are arranged in the chamber layer, the first quantitative chamber is arranged in the quantification layer, the micro-channel is arranged in the micro-channel layer; the connecting layer is provided with corresponding communicating holes. Optionally, in the embodiment, each chip layer is composed of PMMA.

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

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

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

[0088] Embodiment 2:

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

[0090] In practical application, the number of detection modules on the chip and the types of crRNA can be changed according to the requirements of target detection, so as to realize personalized customization of detection requirements.

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

[0092] S1: Place the portable microfluidic chip upright, add the sample to be detected into the quantitative chamber, divide it into 5 parts by means of the "V"-shaped quantitative chamber, realize quantification, and perform multiplexed recombinase polymerase amplification (RPA); during the multiplexed 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: Apply a forward force to the portable microfluidic chip, so that the sample after multiplexed RPA breaks through the microchannel and enters the reaction chamber, and reacts with the reactants in the reaction chamber to generate a reaction product;

[0094] S3: After the sample in the quantitative chamber is completely transferred to the reaction chamber and the reaction in the reaction chamber is completed, place the portable microfluidic chip 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 that 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 quantitative chamber through the microchannel;

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

[0096] Based on the portable microfluidic chip provided in Embodiment 1, the microfluidic detection method provided in this embodiment has the following advantages: (1) No need to extract and purify nucleic acids, simplifying the detection process, and the entire reaction (including sample preparation) can be completed within 1 hour; (2) The combination of chemical lysis reagent and chemical exothermic device enables the chip to stably perform rapid detection in an environment lacking professional heating instruments such as water baths and incubators; (3) Through the ingenious layout of the chip microchannel, without any external instrument, the chip microchannel is controlled by simply manipulating the direction of the chip and applying external force; (4) The combination of RPA and CRISPR significantly improves the detection sensitivity and specificity, and experiments show that the detection limit of unamplified and amplified samples based on LFA reading is 0.05 nM and 10 –18 M, and the diagnostic accuracy is 98.57%; (5) Strong universality, which can be used for various nucleic acid detection such as COVID-19, influenza A, and influenza B.

[0097] Overall, based on the proposed portable microfluidic chip, the challenges of traditional PCR in terms of portability, cost, and analysis performance can be overcome, and microfluidic detection can be realized quickly, at low cost, and conveniently.

[0098] Embodiment 3:

[0099] A portable microfluidic chip, such as Figure 5The embodiment is similar to the embodiment 1, but differs from the embodiment 1 in that, in order to further improve the enhanced color development effect of the test strip and improve the detection sensitivity, as shown in (a) of Figure 5 In the embodiment, each detection module 2 further comprises an enhancing reagent chamber 27 and a second connecting channel 28.

[0100] The enhancing reagent chamber 27 is provided with an enhancing reagent.

[0101] The bottom end of the second connecting channel 28 is in communication with the top end of the enhancing reagent chamber 27, and the top end of the second connecting channel 28 is in communication with the top end of the test strip 25.

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

[0103] In the embodiment, the enhancing reagent chamber is provided, the enhancing reagent chamber is in communication with the top end of the test strip through the second connecting channel, and the flow resistance of the second connecting channel is greater than that 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 enhancing reagent in the enhancing reagent chamber cannot break through the second connecting channel, that is, the enhancing reagent does 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 enhancing reagent can be transferred to the test strip by applying a reverse force to the chip to enhance the detection result on the test strip and improve the detection sensitivity.

[0104] Optionally, as shown in (b) of Figure 5 The embodiment adopts the same chip layering design scheme as the embodiment 1, and the enhancing reagent chamber, the second connecting channel, the reaction chamber and the first connecting channel are arranged in the same chip layer, i.e. the chamber layer.

[0105] The specific embodiments of the remaining structures in the embodiment are the same as those of the embodiment 1, and reference can be made to the description of the embodiment 1.

[0106] Embodiment 4:

[0107] A microfluidic detection method based on the portable microfluidic chip provided in the above embodiment 3. The embodiment is similar to the embodiment 2, but differs from the embodiment 2 in that, as shown in Figure 6 In the embodiment, in step S4, before reading the detection result of the test strip after a preset time, the following steps are further included:

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

[0109] The enhancing agent can enhance the color development effect of the test strip and improve the detection sensitivity.

[0110] Example 5:

[0111] A portable microfluidic chip, such as Figure 7 As shown. This embodiment is similar to Embodiment 1 above, except that, as 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 of the reaction chamber 23. Based on this structural design, in this embodiment, the microchannel 22 and the reaction chamber 23 can be disposed on the same chip layer.

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

[0113] Example 6:

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

[0115] Example 7:

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

[0117] Three test strips 25 are pre-embedded in three detection chambers 26, and absorbent material 262 is provided at both ends of the three detection chambers 26.

[0118] The bottom ends of the three first connecting channels 24 are 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 are evenly distributed into the three first channels to achieve secondary quantitative distribution. The products are then transferred to the three test strips through the three first connecting channels to complete the reading of the detection results.

[0120] By increasing the number of test strips, different test strips are set, and the embodiment can complete multiple detections on the same sample at a time.

[0121] The embodiment can also set an amplification reagent chamber in the detection module, at this time, the number of second connecting channels is 3, the bottom ends of the 3 second connecting channels are connected with the top ends of the amplification reagent chamber, and the top ends of the 3 second connecting channels are respectively connected with the top ends of the 3 test strips.

[0122] In the embodiment, the same layered design scheme as that in the above embodiment 1 is adopted, as shown in (b) of FIG. 1. Figure 9

[0123] Embodiment 8:

[0124] A microfluidic detection method based on the portable microfluidic chip provided in the above embodiment 7. As shown in (a) of FIG. 8, the detection process steps of the embodiment are the same as those in the above embodiment 2, and the specific implementation of each step can refer to the description in the above embodiment 2. Figure 10

[0125] Embodiment 9:

[0126] A microfluidic detection device, as shown in (a) of FIG. 9, comprising a base support and 5 portable microfluidic chips. Figure 11

[0127] The base support is provided with 5 slot positions, and the 5 portable microfluidic chips are respectively fixed in the 5 slot positions.

[0128] Among them, the portable microfluidic chip is the portable microfluidic chip provided in the above embodiment 1.

[0129] The embodiment can realize multiple detections at the same time by integrating multiple portable microfluidic chips in the same microfluidic detection device, thereby improving the detection throughput.

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

[0131] The following further analyzes and describes the technical effects that can be achieved by the application in combination with specific experimental results.

[0132] Figure 12 ​​​Figures for temperature control related results, where (a) is the temperature change of both chemical exothermic devices, heating pack and warm-up patch, (b) is the RPA and CRISPR results of chemical lysing reagent combined with different heat sources, the ratio of the gray value of the test line to the control line in the test strip is (T / C), (c) is the difference of RPA and CRISPR between warm-up patch and water bath, the ratio of the gray value of the test line to the control line in the test strip is (T / C).

[0133] The present application uses clinical samples infected with HPV 16 as a proof of concept to verify the feasibility of chemical exothermic devices. First, record their temperature changes, the temperature of the heating pack can be maintained at 90℃ for about 10 minutes, which meets the temperature conditions required for cell lysis, as shown in the left graph of (a) in Figure 12 Similarly, the temperature of the warm-up patch can be maintained at 45℃ for about 60 minutes, which is enough to complete RPA amplification and CRISPR detection, as shown in the right graph of (a) in Figure 12 The cervical swab is added to the sample processing tube containing the chemical lysing reagent, mixed and divided into three parts, and then heated using room temperature, heating pack and water bath to release HPV DNA. Electrophoresis results show that there is no obvious target band in positive samples when left at room temperature, while the target is successfully amplified using chemical lysing reagent combined with heating pack or water bath. At the same time, the CRISPR detection results based on test strips show that there is no difference between negative and positive using chemical lysing reagent alone, while the other two methods will significantly reduce the signal intensity of the test line, as shown in (b) in Figure 12 Therefore, chemical lysing reagent combined with chemical exothermic device can replace physical heating devices driven by power. Finally, the present application uses warm-up patch and 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 (c) in Figure 12 The above data show that chemical exothermic devices can enable HID-MACRO to achieve home self-testing without power supply.

[0134] Figure 13 Figures for primer verification related results, where (a) is the detection principle of RPA, CRISPR and test strip, (b) is the single RPA verification results of four HPV subtypes (HPV 6, HPV 11, HPV 16 and HPV 18) primers, (c) is the multiplex RPA verification results of four HPV subtypes (HPV 6, HPV 11, HPV 16 and HPV 18) primers.

[0135] Figure 13(a) in FIG. 1 demonstrates the principle of RPA and test strip-based CRISPR / Cas12 detection. First, the sample is amplified exponentially by RPA, which can significantly improve the detection sensitivity. Subsequently, when the crRNA recognizes the HPV target in the RPA product, the transcleavage 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 complex captured by the test line. The performance of these primers was determined using singleplex RPA, and agarose gel electrophoresis showed that the four subtypes primers worked well, showing clear and dense bands, and there was no non-specific amplification to the negative control, as shown in (b) in FIG. 1. Subsequently, the RPA product was used for CRISPR-based test strip detection, and the results showed that all four HPV subtypes were significantly distinguished from the negative control, indicating that all targets were successfully amplified, as shown in (b) in FIG. 1. In addition, multiplex RPA analysis within the chip can save testing time and reduce reagent consumption, so the optimal primer structure and concentration are crucial to avoid primer dimerization and obtain sufficient amplicons for each target. The present application verified the four primers using multiplex RPA, as shown in (c) in FIG. 1, all targets were successfully amplified and no obvious primer dimer was formed. Therefore, this primer structure was used in the subsequent detection. Figure 13 Figure 13 Figure 13

[0136] Figure 14 The results of specificity and sensitivity detection of the four HPV subtypes are shown in FIG. 2, where (a) is a schematic diagram of specificity detection, (b) is the specificity detection result based on test strips, the left test strip result, and the right test strip detection line and quality control line ratio heatmap, (c) is the sensitivity of unamplified plasmids, from left to right are HPV 6, HPV 11, HPV 16, HPV 18, and (d) is the sensitivity of amplified plasmids, from left to right are HPV 6, HPV 11, HPV 16, HPV 18.

[0137] Due to the high homology between different HPV subtype sequences, crRNA designed for a specific subtype can recognize other subtypes. Therefore, a 4x4 matrix test (4 crRNAs x 4 HPV RPA amplicons) was designed to screen the cross-reactivity of crRNA. In addition, a control group without any target gene was also set up. Only when the target gene matches the crRNA, Cas12a will be activated and cleave the reporter probe, resulting in a decrease in the signal on the test line of the test strip, as shown in (a) in FIG. 3. The actual results of test strip-based CRISPR 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 disappeared, as shown in (a) in FIG. 3. Figure 14 Figure 14 ​​​​As shown in the left figure of (b) in the diagram, no extensive cross-activity was observed, as... Figure 14 The right-hand side of (b) in the diagram is shown. These highly specific crRNAs were subsequently used for multiplex nucleic acid detection.

[0138] Sensitivity determines the detection rate of a sample; therefore, this invention evaluates the detection sensitivity of direct CRISPR and RPA-based CRISPR. First, this invention uses plasmid samples of four HPV subtypes (without pre-amplification) to verify the sensitivity of CRISPR-based direct nucleic acid detection. For example... Figure 14 As shown in (c), the signal intensity of the detection line gradually decreases with increasing plasmid concentration. The sensitivities for HPV 6, HPV 11, HPV 16, and HPV 18 are 0.05 nM, 0.1 nM, 0.05 nM, and 0.05 nM, respectively. To further improve the detection sensitivity, this invention introduces RPA to amplify the plasmid sample before lysis detection. CRISPR detection based on the test strip shows that the sensitivities for HPV 6, HPV 11, HPV 16, and HPV 18 are 1 × 10⁻⁶. -16 M, 1×10 -16 M, 1×10 -18 M, 1×10 -18 M, such as Figure 14 As shown in (d), the performance was improved by a million-fold compared to 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 indicate that RPA can significantly improve detection sensitivity, and the HID-MACRO system can significantly improve the detection sensitivity of both non-amplified (0.05 nM) and RPA-amplified (1 × 10⁻⁶) targets. -18 HPV plasmids (M) have high detection sensitivity.

[0139] Figure 15 The results of 140 HPV clinical samples are shown. (a) shows the testing process, (b) shows images of some typical clinical samples, (c) shows the on-chip test results of 140 HPV clinical samples with 4 subtypes, (d) shows the ROC analysis results of the 4 HPV types, (e) shows the grouping scatter plot of the 4 HPV types, with a cutoff value of 0.54 (≤0.54 is positive; >0.54 is negative), and (f) shows the four-cell table of the on-chip and clinical test results of the 4 HPV subtypes.

[0140] The entire on-chip detection process takes approximately 60 minutes, including sample preparation, RPA, CRISPR detection, and readout. Figure 15 As shown in (a) in the figure. Figure 15(b) in FIG. 1 shows some typical results on the chip, from which it can be seen that the signal intensity of the detection line on the four subtypes detection strips corresponding to the negative control is obviously weaker than that of the negative control, indicating that the sample is positive for these subtypes. Figure 15 (c) in FIG. 1 shows the results of 140 samples on the chip for the detection of four HPV subtypes, and no false positive sample is found compared with the previous clinical results, which indicates that HID-MACRO has high specificity. Unfortunately, there are 8 false negative samples (indicated by green boxes), which may be caused by long storage time of the samples or low viral load leading to DNA degradation. In order to systematically analyze the performance of the HID-MACRO detection platform, the receiver operating characteristic (ROC) analysis was performed by comparing the chip and clinical detection results, as shown in Figure 15 (d) in FIG. 1, the area under the curve (AUC) is 0.9636 in a total of 560 detections (4 HPV subtypes x 140 clinical samples), indicating that the results of the two methods are highly consistent. When the critical value is set to 0.54 (≤0.54, positive; >0.54, negative), HID-MACRO can significantly distinguish between negative and positive, as shown in Figure 15 (e) in FIG. 1. Overall, HID-MACRO shows good performance in detecting patient samples, with a sensitivity and specificity of 92.73% and 100%, respectively, as shown in Figure 15 (f) in FIG. 1. These results show that the naked-eye reading-based HID-MACRO can be used for accurate identification of HPV in clinical samples, providing a low-cost, portable and reliable point-of-care diagnostic tool for cervical cancer screening.

[0141] Figure 16 FIG. 1 shows the interpretation results of 70 respiratory virus spiked samples, wherein (a) is the detection process, (b) is the image of some spiked samples, (c) is the on-chip detection results of three respiratory viruses in 70 spiked samples, (d) is the ROC analysis of three respiratory viruses on the chip, (e) is the grouped scatter plot of three respiratory viruses, and the critical value is set to 0.65 (≤0.65 is positive; >0.65 is negative), and (f) is the four-fold table of three respiratory viruses on the chip and spiked detection results.

[0142] The present application detects 70 spiked samples of three respiratory viruses, Figure 16 (a) in FIG. 1 shows the brief process of detection on the chip, which first amplifies the spiked samples by multiplex RPA, and then performs CRISPR detection based on test strips. Figure 16 (b) in FIG. 1 shows some typical detection results on the chip for some spiked samples, and when the sample contains a certain virus plasmid, the signal intensity of the detection line on the test strip is obviously reduced. Figure 16(c) in FIG. 6 shows the on-chip detection results of 70 spiked samples, and no false positive and false negative occurred, which indicates that HID-MACRO has high specificity and sensitivity. Through the receiver operating characteristic (ROC) analysis, as shown in Figure 16 (d) in FIG. 6, a total of 210 tests (3 respiratory viruses x 70 spiked samples) were performed, and the area under the curve of the 3 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 could significantly distinguish between negative and positive, as shown in Figure 16 (e) in FIG. 6. The sensitivity and specificity of the 3 respiratory viruses were 100% (n=98) and 100% (n=112), respectively, as shown in Figure 16 (f) in FIG. 6. The above data show that HID-MACRO can be used as a universal strategy for nucleic acid detection.

[0143] Those skilled in the art can easily understand that the above description is only a preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A portable microfluidic chip, characterized by, The reaction chamber is pre-embedded with reactants; a chip body, and a plurality of detection modules on the chip body M a plurality of detection modules The detection module comprises a quantitative chamber, a microchannel, a reaction chamber, N a first connecting channel, N a test strip, an enhanced reagent chamber and N a second connecting channel; The flow resistance of the first connecting channel is less than that of the micro-channel; One end of the microchannel is communicated with the bottom end of the dosing chamber, the other end is communicated with the reaction chamber, and the connection point of the microchannel and the reaction chamber is lower than the bottom end of the dosing chamber; the N bottom end of the first connecting channel is communicated with the top end of the reaction chamber, the top end of the first connecting channel is communicated with the top end of the test strip, respectively N The bottom end of the first connecting channel is communicated with the top end of the reaction chamber, the top end of the first connecting channel is communicated with the top end of the test strip, respectively N The bottom end of the first connecting channel is communicated with the top end of the reaction chamber, the top end of the first connecting channel is communicated with the top end of the test strip, respectively The enhancing reagent chamber is provided with an enhancing reagent; The flow resistance of the second connecting channel is greater than that of the first connecting channel; The N The bottom end of each of the second connection channels is connected to the top end of the enhanced reagent chamber. N The top of each second connection channel is respectively connected to N The tops of the test strips are connected; The detection chamber is provided with water-absorbing materials at both ends. wherein, M and N are each positive integers greater than or equal to 1.

2. The portable microfluidic chip of claim 1, wherein, N >1。 3. The portable microfluidic chip of claim 1 or 2, wherein, The detection module further comprises: N a detection chamber for embedding the N test strips respectively. The connecting point of the micro-channel and the reaction chamber is higher than the bottom end of the reaction chamber; 4. The portable microfluidic chip of claim 1 or 2, wherein, The chip body includes multiple chip layers, and the micro-channel and the reaction chamber are located in different chip layers. The connecting point of the micro-channel and the reaction chamber is located at the bottom end of the reaction chamber; 5. The portable microfluidic chip of claim 1 or 2, wherein, The chip body includes multiple chip layers, and the micro-channel and the reaction chamber are located in the same chip layer. The quantitative chamber is in the shape of "V", and the vertex of the "V" shape is the bottom end of the quantitative chamber.

6. The portable microfluidic chip of claim 1 or 2, wherein, The method comprises the following steps:

7. A microfluidic detection method for non-medical diagnostic purposes based on the portable microfluidic chip according to any one of claims 1 to 6, characterized in that, S1: Place the portable microfluidic chip upright, add the sample to be detected to the quantitative chamber of each detection module, and complete the sample quantification; S2: Apply a forward force to the portable microfluidic chip, so that the sample in each quantitative chamber breaks through the micro-channel and is transferred to the reaction chamber, reacts with the reactants in the reaction chamber, and obtains the 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, place the portable microfluidic chip upside down, so that the reaction product flows to the test strip through the first connecting channel; S4: After a preset time, apply a reverse force to the portable microfluidic chip, so that the enhancing reagent in the enhancing reagent chamber breaks through the second connecting channel and enters the test strip, and read the detection result of the test strip. The method comprises the following steps:

8. A microfluidic detection device, characterized in that ​ The base and K a portable microfluidic chip; The bottom support is provided with K a plurality of slot positions, and K a plurality of portable microfluidic chips are respectively fixed in the K slot positions. The portable microfluidic chip is the portable microfluidic chip according to any one of claims 1-6. K is a positive integer greater than or equal to 1.

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