Gravity-driven microfluidic chip and portable detector

Abstract

The present invention relates to the field of biochemical analysis technology and provides a gravity-driven microfluidic chip and a portable detector. The gravity-driven microfluidic chip includes a housing, at least two reagent chambers, and at least one siphon pipe, each of which is provided with an air hole. The siphon pipe has at least one ascending section and one descending section, with the two ends of the siphon pipe connected to different reagent chambers, respectively. The highest point of the siphon pipe is higher than the liquid level in any of the reagent chambers connected to it. The connected reagent chambers are arranged in a stepped manner. By using the siphon pipe as a means of connecting the reagent chambers, the gravity-driven microfluidic chip can be tilted at a certain angle to achieve automatic liquid transfer using the siphon effect.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical analysis, and in particular to a gravity-driven microfluidic chip and a portable detector. Background Art

[0002] Gravity-driven microfluidic chips, an innovative application of microfluidic technology, utilize liquid gravity as the primary power source to achieve precise manipulation and processing of fluids at the microscale. Gravity-driven microfluidic chips do not rely on any external power devices, such as motors, but instead simply utilize the direction and magnitude of gravity to drive and control fluid dynamics at the microscale. This feature significantly simplifies system complexity and reduces chip cost. Gravity-driven microfluidic chips primarily rely on specially designed chambers and channel structures, or fluid density differences, to achieve automated fluid movement, transfer, and mixing. Fluids flow along pre-set paths under the influence of gravity, completing various biochemical reactions and analytical tasks. Compared to traditional microfluidic technologies, gravity-driven microfluidic chips do not require external pumps or valves to drive fluids, significantly reducing system complexity and operational difficulty. In recent years, a number of studies have proposed simple fluid control schemes based on gravity, channel inclination, fluid density, and channel design, as well as complex fluid control schemes based on valves, selective chambers, and other innovative structural designs. However, these designs suffer from complex structures, cumbersome fabrication, and the risk of cross-contamination. Summary of the Invention

[0003] The present invention provides a gravity-driven microfluidic chip to address the defects of existing microfluidic chips, such as high design complexity, inconvenient manufacturing, and easy chamber contamination. It realizes the flow of reagents between different reagent chambers by relying on gravity and siphon effect, is easy to operate, and is not prone to cross contamination.

[0004] The invention also provides a portable detector.

[0005] According to the first embodiment of the present invention, a gravity-driven microfluidic chip is provided, comprising:

[0006] A housing having at least two reagent chambers and at least one siphon line, each of the reagent chambers being provided with an air hole;

[0007] The siphon pipeline has at least one ascending section and one descending section, and the two ends of the siphon pipeline are respectively connected to different reagent chambers, and the highest point of the siphon pipeline is higher than the liquid level of any one of the reagent chambers connected to it;

[0008] The connected reagent chambers are distributed in a stepped manner.

[0009] The gravity-driven microfluidic chip of the embodiment of the present invention avoids the problem of liquid contamination in the reagent chamber by arranging the reagent chamber and the siphon pipeline in the shell; and adopts the siphon pipeline as a way to connect the reagent chamber, and applies the siphon effect to realize liquid transfer. The user only needs to tilt the gravity-driven microfluidic chip at a certain angle, and the operation is convenient and fast; and the siphon process of the siphon pipeline can automatically complete the liquid transfer after it is started. The user does not need to always control the gravity-driven microfluidic chip, and the operation experience is better. Moreover, the siphon pipeline can be directly opened in the shell and demolded without adding other components, making the overall production process of the gravity-driven microfluidic chip simpler and more convenient.

[0010] According to one embodiment provided by the present invention, the reagent chamber includes a cleaning liquid chamber, a sample chamber, an eluent chamber, a detection chamber, and a waste liquid chamber, wherein the cleaning liquid chamber and the eluent chamber are arranged higher than the sample chamber, and the detection chamber and the waste liquid chamber are arranged lower than the sample chamber;

[0011] The siphon pipeline includes a first siphon pipeline, a second siphon pipeline, a third siphon pipeline and a fourth siphon pipeline;

[0012] The first siphon line connects the bottom of the cleaning liquid chamber with the top of the sample chamber, the second siphon line connects the bottom of the eluent chamber with the top of the sample chamber, the third siphon line connects the bottom of the sample chamber with the top of the detection chamber, and the fourth siphon line connects the bottom of the sample chamber with the top of the waste liquid chamber.

[0013] The third siphon line, the fourth siphon line, and the outlet of the sample chamber are connected to each other; the first siphon line and the second siphon line are not located on the same side and coplanar with the sample chamber at the same time; the second siphon line and the fourth siphon line are not located on opposite sides and coplanar with the sample chamber at the same time.

[0014] According to an embodiment provided by the present invention, the first siphon line and the second siphon line are located on opposite sides of the sample chamber and are coplanar; the third siphon line and the fourth siphon line are located on opposite sides of the sample chamber and are coplanar.

[0015] According to an embodiment provided by the present invention, the first siphon pipeline, the second siphon pipeline, the third siphon pipeline and the fourth siphon pipeline are arranged in the same plane;

[0016] The reagent chamber further includes a temporary storage chamber, and the siphon pipeline further includes a fifth siphon pipeline; the temporary storage chamber is arranged higher than the sample chamber and lower than the cleaning liquid chamber;

[0017] The first siphon line is connected to the bottom of the cleaning liquid chamber and the top of the temporary storage chamber, and the fifth siphon line is connected to the bottom of the temporary storage chamber and the top of the sample chamber.

[0018] According to an embodiment provided by the present invention, a chamfered portion is provided between the bottom wall and the side wall of the shell along the direction of gravity, and the plane of the chamfered portion is perpendicular to the plane where the connected reagent chamber is located.

[0019] According to an embodiment provided by the present invention, a hydrophobic layer is provided on the surface of the reagent chamber, and / or a hydrophilic layer is provided on the inner wall surface of the siphon pipeline.

[0020] According to an embodiment provided by the present invention, absorbent cotton is provided in the waste liquid chamber for absorbing waste liquid.

[0021] A portable detector according to an embodiment of the second aspect of the present invention includes:

[0022] A gravity-driven microfluidic chip as described above;

[0023] The housing is provided with a placement space suitable for placing the gravity-driven microfluidic chip;

[0024] a heating assembly, disposed corresponding to the first side of the reagent chamber to be detected;

[0025] The detection component is correspondingly disposed on the second side of the reagent chamber to be detected, and the first side and the second side are not co-located.

[0026] The portable detector according to an embodiment of the present invention integrates a heating component, a detection component, and a gravity-driven microfluidic chip into a housing, thereby improving the integration of the portable detector, enhancing convenience, and realizing automated detection of target bacteria, thereby achieving instant and rapid detection of target bacteria.

[0027] According to an embodiment provided by the present invention, the housing is provided with a first through hole, and the first through hole is provided corresponding to the reagent chamber to be detected;

[0028] The side of the first through hole facing away from the reagent chamber is suitable for installing an image detection device.

[0029] According to an embodiment provided by the present invention, the detection assembly includes an excitation light source, a lens, a first filter, and a second filter;

[0030] The lens, the first filter, and the reagent chamber to be detected are sequentially arranged along the light direction of the excitation light source; the light direction and the direction of the reagent chamber to be detected pointing to the first through hole form a set angle;

[0031] The second filter is provided on one side of the first through hole close to the reagent chamber to be detected.

[0032] According to an embodiment provided by the present invention, the housing has a placement groove extending inward from the outer wall, and the placement groove is used to place the heating component.

[0033] According to an embodiment provided by the present invention, the housing includes a plurality of reagent placement positions, and the reagent placement positions are arranged corresponding to the number of reagents required by the reagent chamber.

[0034] According to an embodiment provided by the present invention, the housing has a light-shielding coating or is made of a light-shielding material.

[0035] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0037] Figure 1 Schematic diagram of the structure of a gravity-driven microfluidic chip provided by an embodiment of the present invention;

[0038] Figure 2 Schematic diagram of the working principle of the gravity-driven microfluidic chip provided by an embodiment of the present invention;

[0039] Figure 3 1 is a schematic structural diagram of a portable detector provided by an embodiment of the present invention;

[0040] Figure 4 Schematic diagram of the internal optical path of the portable detector provided by an embodiment of the present invention;

[0041] Figure 5 Schematic diagram of the optimization results of the nucleic acid cleavage method of the portable detector provided by an embodiment of the present invention;

[0042] Figure 6 2. It is a schematic diagram of the optimization result of the amount of silicon-based magnetic beads used in the portable detector provided by an embodiment of the present invention;

[0043] Figure 7 Schematic diagram of the optimization results of the reaction temperature and dosage of the portable detector provided by an embodiment of the present invention;

[0044] Figure 8This is a schematic diagram of the standard curve results of the portable detector provided by an embodiment of the present invention;

[0045] Figure 9 1 is a schematic diagram of the specificity evaluation results of the portable detector provided by an embodiment of the present invention;

[0046] Figure 10 This is a schematic diagram of the detection results of the portable detector provided by an embodiment of the present invention being used to detect Salmonella Typhimurium in spiked chicken samples.

[0047] Reference numerals:

[0048] 10. Reagent chamber; 11. Air hole; 101. Cleaning liquid chamber; 102. Sample chamber; 103. Eluent chamber; 104. Detection chamber; 105. Waste liquid chamber; 106. Temporary storage chamber;

[0049] 20, siphon pipeline; 201, first siphon pipeline; 202, second siphon pipeline; 203, third siphon pipeline; 204, fourth siphon pipeline; 205, fifth siphon pipeline;

[0050] 30. Shell;

[0051] 40. Housing; 401. Placement space; 402. First through hole; 403. Positioning portion; 404. Placement slot; 405. Reagent placement position;

[0052] 50, detection component; 501, excitation light source; 502, lens; 503, first filter; 504, second filter;

[0053] 60. Image detection equipment. DETAILED DESCRIPTION

[0054] The following embodiments of the present invention are described in further detail with reference to the accompanying drawings and examples. The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0055] In the description of the embodiments of the present invention, it should be noted that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the embodiments of the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore should not be understood as limiting the embodiments of the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only and should not be understood as indicating or implying relative importance.

[0056] In the description of the embodiments of the present invention, it should be noted that, unless otherwise specified or limited, the terms "connected" and "connection" should be understood in a broad sense. For example, they can refer to fixed connections, detachable connections, or integral connections; mechanical connections, electrical connections; and direct connections or indirect connections through an intermediary. Those skilled in the art will understand the specific meanings of the above terms in the embodiments of the present invention based on the specific circumstances.

[0057] In the embodiments of the present invention, unless otherwise expressly specified or limited, a first feature being "above" or "below" a second feature may mean that the first and second features are in direct contact, or that the first and second features are in indirect contact through an intermediate medium. Furthermore, a first feature being "above," "above," or "above" a second feature may mean that the first feature is directly above or diagonally above the second feature, or simply means that the first feature is at a higher level than the second feature. A first feature being "below," "below," or "below" a second feature may mean that the first feature is directly below or diagonally below the second feature, or simply means that the first feature is at a lower level than the second feature.

[0058] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0059] Combine Figure 1 As shown, the gravity-driven microfluidic chip provided in accordance with the embodiment of the first aspect of the present invention includes a shell 30, provided with at least two reagent chambers 10 and at least one siphon pipe 20, each reagent chamber 10 is provided with an air hole 11; the siphon pipe 20 has at least one ascending section and one descending section, and the two ends of the siphon pipe 20 are respectively connected to different reagent chambers 10, and the highest point of the siphon pipe 20 is higher than the liquid level of the liquid in any reagent chamber 10 connected thereto; the connected reagent chambers 10 are distributed in a stepped manner.

[0060] The gravity-driven microfluidic chip of the embodiment of the present invention controls the release of liquid through the dynamic balance between the siphon force and gravity on the liquid based on the siphon line 20 controlled by the tilt angle. Specifically, when the gravity-driven microfluidic chip is tilted (from the relatively higher reagent chamber 10 to the direction of the siphon line 20), the liquid in the reagent chamber 10 gradually transfers to the siphon line 20. At this time, the gravity of the liquid is greater than the siphon force on the liquid and remains in the siphon line 20 until the liquid level in the siphon line 20 exceeds the highest point of the siphon line 20. At this point, the gravity acting on the liquid is less than the siphon force, and the atmosphere entering the higher reagent chamber 10 through the air hole 11 squeezes the liquid. Then, the liquid in the higher reagent chamber 10 is transferred to the lower reagent chamber 10 through the siphon line 20 under the action of the siphon effect, completing the transfer of liquids in different reagent chambers 10. Moreover, when the reagent chamber 10 is connected to multiple different siphon lines 20, different siphon lines 20 can be selected by controlling the tilt direction to achieve the transfer of liquid to different reagent chambers 10.

[0061] The gravity-driven microfluidic chip of the embodiment of the present invention avoids the problem of liquid contamination in the reagent chamber 10 by arranging the reagent chamber 10 and the siphon line 20 in the shell 30; and adopts the siphon line 20 as a way to connect the reagent chamber 10, and applies the siphon effect to realize liquid transfer. The user only needs to tilt the gravity-driven microfluidic chip at a certain angle, and the operation is convenient and fast; and the siphon process of the siphon line 20 can automatically complete the liquid transfer after it is started. The user does not need to always control the gravity-driven microfluidic chip, and the operating experience is better. Moreover, the siphon line 20 can be directly opened in the shell 30 and demolded without adding other components, making the overall production process of the gravity-driven microfluidic chip simpler and more convenient.

[0062] In this embodiment, the shell 30 includes two opposing shell parts, on which at least part of the siphon line 20 and the reagent chamber 10 are provided. Preferably, the two shell parts can be made of equal thickness, and half of the siphon line 20 and the reagent chamber 10 are each formed on different shell parts, which facilitates the demolding of the shell parts.

[0063] In this embodiment, the housing 30 may be made of PDMS (polydimethylsiloxane). Of course, in other embodiments, other materials that do not react with the liquid in the reagent chamber 10 may be used.

[0064] In this embodiment, the air holes 11 are used to balance the air pressure, and the positions of the air holes 11 are all higher than the liquid level in the reagent chamber 10 to ensure that the reagent will not leak when the chip is tilted.

[0065] In this embodiment, the overall size of the housing 30 (in Figure 1For example, the height (along the up and down direction of the paper) is 70mm, the upper bottom length is 55mm, the lower bottom length is 40mm, and the thickness (perpendicular to the paper) is 4.5mm.

[0066] According to an embodiment provided by the present invention, Figure 1 and Figure 2 As shown, the reagent chamber 10 includes a cleaning liquid chamber 101, a sample chamber 102, an eluent chamber 103, a detection chamber 104 and a waste liquid chamber 105, and the cleaning liquid chamber 101 and the eluent chamber 103 are arranged higher than the sample chamber 102, and the detection chamber 104 and the waste liquid chamber 105 are arranged lower than the sample chamber 102.

[0067] Among them, the sample chamber 102 is pre-buried with D3 neodymium iron boron magnetic balls, which are suitable for capturing the nucleic acid-silicon-based magnetic bead complex in the nucleic acid-silicon-based magnetic bead complex solution. The eluent chamber 103 is suitable for placing nucleic acid eluent, the cleaning liquid chamber 101 is suitable for placing nucleic acid cleaning liquid, the waste liquid chamber 105 is suitable for collecting waste liquid, and the detection chamber 104 is pre-buried with freeze-dried RAA (recombinase-mediated isothermal nucleic acid) detection reagent.

[0068] The siphon line 20 includes a first siphon line 201, a second siphon line 202, a third siphon line 203, and a fourth siphon line 204. The first siphon line 201 connects the bottom of the cleaning liquid chamber 101 with the top of the sample chamber 102, the second siphon line 202 connects the bottom of the eluent chamber 103 with the top of the sample chamber 102, the third siphon line 203 connects the bottom of the sample chamber 102 with the top of the detection chamber 104, and the fourth siphon line 204 connects the bottom of the sample chamber 102 with the top of the waste liquid chamber 105.

[0069] The following is the specific working process of this embodiment: Figure 2 As shown, 500 μL of nucleic acid-silica-based magnetic bead complex solution is added to the sample chamber 102 , 300 μL of nucleic acid cleaning solution is added to the cleaning solution chamber 101 , and 100 μL of nucleic acid elution solution is added to the elution solution chamber 103 .

[0070] After the required reagents are loaded into the microfluidic chip, the chip is left to stand for 1 minute to allow the D3 NdFeB magnetic balls embedded in the sample chamber 102 to capture the nucleic acid-silicon-based magnetic bead complex.

[0071] During the first tilting, the gravity-driven microfluidic chip is tilted from the sample chamber 102 toward the fourth siphon line 204 . At this time, the liquid in the sample chamber 102 enters the waste liquid chamber 105 under the siphon effect of the fourth siphon line 204 .

[0072] During the second tilting, the gravity-driven microfluidic chip is tilted from the first siphon line 201 toward the sample chamber 102 until the cleaning liquid enters the sample chamber 102 through the first siphon line 201. It is then left horizontally for 5 minutes to allow the cleaning liquid to remove impurities on the silicon-based magnetic beads.

[0073] During the third tilting, the gravity-driven microfluidic chip is tilted from the sample chamber 102 toward the fourth siphon line 204 . At this time, the cleaning liquid containing the washed impurities enters the waste liquid chamber 105 through the fourth siphon line 204 .

[0074] The microfluidic chip was left to stand for 10 minutes to dry out any residual ethanol in the cleaning solution that would inhibit subsequent isothermal amplification.

[0075] The fourth tilt is performed, and the gravity-driven microfluidic chip is tilted from the second siphon line 202 to the sample chamber 102. At this time, the eluent enters the sample chamber 102 through the second siphon line 202. Thereafter, it is left horizontally for 10 minutes. The eluent elutes the nucleic acid from the silica-based magnetic beads and dissolves it in the eluent.

[0076] During the fifth tilt, the gravity-driven microfluidic chip is tilted from the sample chamber 102 toward the detection chamber 104 , and the eluent enters the detection chamber 104 through the third siphon line 203 ;

[0077] The eluent dissolves the freeze-dried RAA detection reagent pre-buried in the detection chamber 104 to form an amplification system, which is used for subsequent measurement and analysis.

[0078] It should be emphasized that the volumes of different reagents, experimental times, etc. mentioned in the above work process are not limitations of this application. In actual situations, the volumes and times of reagents vary according to experimental requirements, and staff should make adaptive adjustments based on actual needs.

[0079] It should be noted that food safety has always been a major issue of global concern, and Salmonella is one of the common causes of foodborne pathogen outbreaks. Therefore, the detection of Salmonella in food is an essential part of ensuring food safety.

[0080] Magnetic bead nucleic acid extraction continues to play a vital role in modern molecular biology laboratories and is widely used due to its ability to achieve high-throughput and automated nucleic acid extraction and purification. Compared with traditional centrifugal column technology, magnetic bead technology is superior due to its larger specific surface area, ability to move in a directional manner under the influence of a magnetic field, and ability to redisperse evenly after the magnetic field is removed. Currently, commercial magnetic beads for nucleic acid extraction mainly include hydroxyl (-COOH), amino (-NH2), and silica (SiO2) magnetic nanoparticles. SiO2 magnetic nanoparticles, in particular, are widely used in the industry because their surface functional groups can specifically bind to nucleic acids under specific chemical environments and release nucleic acids after changes in conditions. This method is easy to operate, has a fast extraction speed, high yield, and good purity, making it very suitable for high-throughput and automated nucleic acid extraction.

[0081] Recombinase-mediated isothermal amplification (RAA) is an advanced technology that rapidly amplifies target nucleic acid fragments at room temperature through the combined action of recombinase, single-stranded binding protein (SSB), and DNA polymerase. This process can be completed under isothermal conditions (approximately 37-42°C). Combined with fluorescent probes or pyrophosphatase, a detectable signal can be obtained in just 5-20 minutes, achieving single-digit copy-level detection sensitivity. The simplicity of RAA technology and its ability to avoid liquid evaporation and aerosol contamination under low temperature conditions further enhance its potential for rapid on-site diagnosis. In particular, in key areas such as food safety, environmental monitoring, and aquatic and animal disease detection, RAA technology, with its rapid response, high sensitivity, and specificity, provides a more efficient detection solution than traditional PCR (polymerase chain reaction) methods.

[0082] In this embodiment, different reagent chambers 10 are connected by different siphon pipes 20, and the reagents can be transferred to the reagent chambers 10 corresponding to the steps in sequence through tilt control, thereby realizing the automation of multiple steps such as magnetic bead nucleic acid extraction and nucleic acid detection, and realizing the integration of different experimental steps in the gravity-driven microfluidic chip, thereby achieving rapid detection of target bacteria.

[0083] Of course, it is understandable that based on different objects to be detected, the gravity-driven microfluidic chip of the present application can adaptively adjust the number of reagent chambers 10 and the coordinated communication relationship with the siphon line 20 to adapt to the experimental process of different detection objects.

[0084] Furthermore, to ensure that liquid transfer between steps in this embodiment does not interfere with each other (preventing cross-infection), in this embodiment, the third siphon line 203, the fourth siphon line 204, and the outlet of the sample chamber 102 are interconnected, so that liquid flowing out of the sample chamber 102 can selectively flow to the waste liquid chamber 105 or the detection chamber 104. The first siphon line 201 and the second siphon line 202 are not located on the same side and coplanar with the sample chamber 102 at the same time, so that during the first tilting process, the nucleic acid elution liquid will not flow into the sample chamber 102 at the same time. The second siphon line 202 and the fourth siphon line 204 are not located on opposite sides of the sample chamber 102 and coplanar with each other. Therefore, during the first tilting process, the cleaning liquid will not flow into the sample chamber 102.

[0085] Here, the definition of “same side” can be as follows Figure 2 As shown in , the second siphon line 202 and the fourth siphon line 204 are both located on the right side of the sample chamber 102, and the same applies to other directions; the definition of "coplanar" means that the multiple reagent chambers 10 located on the surface have the same tilt angle when tilted, so that liquids can flow into the sample chamber 102 at the same time; to this end, it is ensured that the reagent chambers 10 are not coplanar so that the two have a certain angle deviation. When the corresponding gravity-driven microfluidic chip is tilted, the two have different tilt angles, and thus the problem of cross contamination caused by the simultaneous flow of liquids into the sample chamber 102 will not occur. Of course, in other embodiments, when the experimental process of the detection object requires the simultaneous mixing of two liquids, the same-side and coplanar form can be adopted.

[0086] In this embodiment, the height of the first siphon line 201 (the distance from the connection point of the relatively higher reagent chamber 10 and the siphon line 20 to the highest point of the siphon line 20) is L1 = 22.5 mm, the height of the second siphon line 202 is L2 = 17.5 mm, and the heights of the third siphon line 203 and the fourth siphon line 204 are both L3 = 17.5 mm. Of course, as described above, the height of the siphon line 20 can be adaptively adjusted based on the gravity or viscosity of the liquid or reagent to be moved.

[0087] In this embodiment, the diameter of the D3 NdFeB magnetic ball is 3 mm. Of course, based on different concentrations of reagents, D3 NdFeB magnetic balls of different sizes can be used to meet adsorption requirements.

[0088] According to an embodiment provided by the present invention, Figure 1 As shown, the first siphon line 201 and the second siphon line 202 are located on opposite sides of the sample chamber 102 and are coplanar; the third siphon line 203 and the fourth siphon line 204 are located on opposite sides of the sample chamber 102 and are coplanar.

[0089] The first, third, and fifth tilts of the gravity-driven microfluidic chip are all swung in the same plane (first plane), while the second and fourth tilts are also swung in the same plane (second plane). The tilting process of the gravity-driven microfluidic chip is controlled on two planes, effectively simplifying the process of users controlling the tilting of the gravity-driven microfluidic chip, improving the user experience, and preventing cross-infection of reagents or liquids.

[0090] According to an embodiment provided by the present invention, Figure 1 As shown, the first siphon line 201, the second siphon line 202, the third siphon line 203 and the fourth siphon line 204 are arranged in the same plane, so that the tilting process of the gravity-driven microfluidic chip is controlled on a plane (on the paper shown in the figure), effectively simplifying the user's operation process.

[0091] In this case, the reagent chamber 10 further includes a temporary storage chamber 106, and the siphon line 20 further includes a fifth siphon line 205. The temporary storage chamber 106 is arranged above the sample chamber 102 and below the cleaning liquid chamber 101. The first siphon line 201 connects the bottom of the cleaning liquid chamber 101 with the top of the temporary storage chamber 106, and the fifth siphon line 205 connects the bottom of the temporary storage chamber 106 with the top of the sample chamber 102.

[0092] Then the specific working process of this embodiment is adjusted accordingly: Figure 2 As shown, 500 μL of nucleic acid-silica-based magnetic bead complex solution is added to the sample chamber 102 , 300 μL of nucleic acid cleaning solution is added to the cleaning solution chamber 101 , and 100 μL of nucleic acid elution solution is added to the elution solution chamber 103 .

[0093] After the required reagents are loaded into the microfluidic chip, the chip is left to stand for 1 minute to allow the D3 NdFeB magnetic balls embedded in the sample chamber 102 to capture the nucleic acid-silicon-based magnetic bead complex.

[0094] During the first tilt, the gravity-driven microfluidic chip is tilted 25° to the right. At this time, the liquid in the sample chamber 102 enters the waste liquid chamber 105 under the siphon effect of the fourth siphon line 204; at the same time, the cleaning liquid enters the temporary storage chamber 106 through the first siphon line 201.

[0095] During the second tilt, the gravity-driven microfluidic chip is tilted 45 degrees to the right, and the cleaning liquid in the temporary chamber 106 enters the sample chamber 102 through the fifth siphon line 205. It is then left horizontally for 5 minutes to allow the cleaning liquid to remove impurities on the silicon-based magnetic beads.

[0096] During the third tilting, the gravity-driven microfluidic chip is tilted 45° to the right. At this time, the cleaning liquid containing the washed impurities enters the waste liquid chamber 105 through the fourth siphon line 204 .

[0097] The microfluidic chip was left to stand for 10 minutes to dry out any residual ethanol in the cleaning solution that would inhibit subsequent isothermal amplification.

[0098] During the fourth tilt, the gravity-driven microfluidic chip is tilted 45° to the left. At this time, the eluent enters the sample chamber 102 through the second siphon line 202 and is then left horizontally for 10 minutes. The eluent elutes the nucleic acid from the silica-based magnetic beads and dissolves it in the eluent.

[0099] During the fifth tilt, the gravity-driven microfluidic chip is tilted 45 degrees to the left, and the eluent enters the detection chamber 104 through the third siphon line 203;

[0100] The eluent dissolves the freeze-dried RAA detection reagent pre-buried in the detection chamber 104 to form an amplification system, which is used for subsequent measurement and analysis.

[0101] It should be noted that the "left" and "right" inclinations in this embodiment are only the circumstances of the current embodiment and are not limitations on the implementation methods of this application. It can be understood that when the first siphon line 201, the second siphon line 202, the third siphon line 203 and the fourth siphon line 204 are coplanar with a surface perpendicular to the paper surface, the corresponding gravity-driven microfluidic chip also rotates and tilts on the plane perpendicular to the paper surface.

[0102] In this embodiment, the user's operation process of tilting the gravity-driven microfluidic chip is simplified, and a temporary storage chamber 106 is provided to prevent cross-infection of reagents or liquids, thereby effectively improving the accuracy of experimental results.

[0103] According to an embodiment provided by the present invention, Figure 1 As shown, along the gravity direction, a chamfered portion (not shown in the figure) is provided between the bottom wall and the side wall of the housing 30 , and the plane of the chamfered portion is perpendicular to the plane where the connected reagent chamber 10 is located.

[0104] In this embodiment, as described in the previous embodiments, different experimental steps (corresponding to different tilting processes) have different tilt angles. By setting a chamfered portion, when the plane of the chamfered portion is in contact with the plane of the original bottom wall, it indicates that the gravity-driven microfluidic chip has been tilted to a specific angle, meeting the requirements of the experimental steps.

[0105] It can be understood that when the inclination directions of multiple reagent chambers 10 are the same but the inclination angles are different, the chamfered portion may include multiple planes. For example, in the aforementioned embodiment, the first inclination is 25° to the right, and the second inclination is 45° to the right. The corresponding chamfered portion can be provided on the right side of the bottom wall, and the connected 25° chamfers and 45° chamfers are provided in sequence from the bottom wall to the top wall.

[0106] According to an embodiment provided by the present invention, a hydrophobic layer is provided on the surface of the reagent chamber 10 to prevent reagents or liquids from remaining in the reagent chamber 10 , which would cause incorrect ratios during the experiment and affect the accuracy of the test results.

[0107] In one embodiment, a hydrophilic layer is provided on the inner wall surface of the siphon line 20 , which facilitates the siphon line 20 to siphon the reagent or liquid in the reagent chamber 10 and improves the transfer efficiency.

[0108] According to an embodiment provided by the present invention, Figure 1 As shown, absorbent cotton (not shown) is provided in the waste liquid chamber 105 to absorb the waste liquid. Liquid entering the waste liquid chamber 105 from the sample chamber 102 is absorbed and fixed by the absorbent cotton, preventing the liquid in the waste liquid chamber 105 from sloshing back into the sample chamber 102 through the fourth siphon line 204 during subsequent tilting, thereby preventing cross-infection and affecting the experimental process.

[0109] A portable detector according to an embodiment of the second aspect of the present invention includes a gravity-driven microfluidic chip, a housing 40, a heating assembly, and a detection assembly 50 as described above. The housing 40 is provided with a placement space 401 suitable for placing the gravity-driven microfluidic chip. The heating assembly is disposed on a first side of a reagent chamber 10 to be detected. The detection assembly 50 is disposed on a second side of the reagent chamber 10 to be detected, and the first side and the second side are not co-located. This prevents the heating assembly from obstructing the detection assembly 50 and affecting the detection effect of the detection assembly 50.

[0110] In this embodiment, after the nucleic acid solution dissolves the freeze-dried RAA detection reagent pre-embedded in the detection chamber 104 to form an amplification system, the gravity-driven microfluidic chip is placed in the placement space 401, and the fluorescent signal of the liquid in the detection chamber 104 is stimulated by the detection component 50 and heated by the heating component to provide the set temperature required for the amplification reaction of the liquid in the detection chamber 104.

[0111] The portable detector according to an embodiment of the present invention integrates a heating component, a detection component 50, and a gravity-driven microfluidic chip by providing a housing 40, thereby improving the integration of the portable detector, enhancing convenience, and realizing automated detection of target bacteria, thereby achieving instant and rapid detection of target bacteria.

[0112] In this embodiment, the dimensions of the housing 40 are 120 mm (length d1 )×150 mm (width d2 )×110 mm (height d3 ).

[0113] In this embodiment, the heating component may be a constant temperature heating plate. Of course, other components with heating effects may also be used.

[0114] According to an embodiment provided by the present invention, Figure 3 As shown, the housing 40 is provided with a first through hole 402 , which is arranged corresponding to the reagent chamber 10 to be detected; a side of the first through hole 402 away from the reagent chamber 10 is suitable for installing the image detection device 60 .

[0115] In this embodiment, the image detection device 60 acquires image information of the reagent chamber 10 (e.g., the detection chamber 104) through the first through hole 402, and quantitatively analyzes the bacteria in the liquid in the reagent chamber 10 based on the image information. Mounting the image detection device 60 on the side of the first through hole 402 facing away from the reagent chamber 10 facilitates switching between different image detection devices 60, providing greater flexibility.

[0116] In this embodiment, the image detection device 60 can be a smart phone, which can be installed with image analysis software for analyzing image information to achieve quantitative detection and analysis of target bacteria; specifically, the smart phone takes a picture of the detection chamber 104 every 20 seconds, obtains its image information and analyzes it to establish a calibration curve, and finally calculates the corresponding time threshold of the amplification curve.

[0117] Of course, in other embodiments, the image detection device 60 may also be a camera, a microscope, or other device suitable for obtaining image information.

[0118] According to an embodiment provided by the present invention, Figure 3 As shown, the detection component 50 includes an excitation light source 501, a lens 502, a first filter 503 and a second filter 504; the lens 502, the first filter 503 and the reagent chamber 10 to be detected are arranged in sequence along the light direction of the excitation light source 501; the light direction and the direction of the reagent chamber 10 to be detected pointing to the first through hole 402 are at a set angle; the second filter 504 is arranged on the side of the first through hole 402 close to the reagent chamber 10 to be detected.

[0119] In this embodiment, the light emitted by the excitation light source 501 is formed into a parallel beam through the lens 502, and then filtered by the first filter 503 to form a light spot covering the reagent chamber 10 to be detected on the gravity-driven microfluidic chip, thereby exciting the fluorescence signal of the liquid in the reagent chamber 10. The fluorescence signal is reflected to the first through-hole 402, filtered by the second filter 504 provided at the first through-hole 402 to remove the stray light, and then captured by the image detection device 60 through the first through-hole 402. While ensuring the excitation of the fluorescence signal of the liquid, the interference of stray light on the image information ultimately captured by the image detection device 60 is reduced, thereby obtaining image information with higher contrast, and effectively improving the accuracy of the experimental detection results.

[0120] In this embodiment, a positioning portion 403 is further connected to the outer wall of the housing 40 , which is suitable for abutting against one end of the image detection device 60 to ensure that the lens of the image detection device 60 can be effectively aligned with the first through hole 402 .

[0121] In this embodiment, the first filter 503 is 470 mm in size and the second filter 504 is 510 mm in size; the excitation light source 501 adopts a 470 mm LED array light source; the angle between the excitation light source 501 and the gravity-driven micro-control flow chip is 45°, ensuring that the light spot is formed without blocking the first through hole 402; of course, the above size or angle parameters can be adaptively adjusted according to actual needs, and the excitation light source 501 can also adopt other devices that can generate light.

[0122] In this embodiment, the detection component 50 is also equipped with a 7.4V power adapter for power supply, and a 6V voltage is drawn out through the internal power module to power the LED array light source.

[0123] According to an embodiment provided by the present invention, Figure 3 As shown, the housing 40 has a placement slot 404 extending inward from the outer wall thereof, and the placement slot 404 is used to place the heating component. The heating component can be inserted and removed from the outside of the housing 40 and placed into the placement slot 404, making the installation of the heating component more convenient and reducing the maintenance and installation difficulty of the portable detector.

[0124] In this embodiment, the groove is prevented from being connected to the placement space 401 through the through hole, so that the heat generated by the heating component can be more directly transferred to the placement space 401, thereby improving the heating effect of the reagent chamber 10.

[0125] According to an embodiment provided by the present invention, Figure 3 As shown, the housing 40 includes a plurality of reagent placement positions 405, and the reagent placement positions 405 are set corresponding to the number of reagents required by the reagent chamber 10. When a user carries the portable detector, the reagents can be placed in the reagent chamber 10 to improve the convenience of use.

[0126] In this embodiment, the housing 40 includes a cover portion and a base portion that match each other. The reagent chamber 10 is disposed in the base portion and hidden between the cover portion and the base portion to prevent the reagent from being exposed and ensure the safety of the reagent.

[0127] According to an embodiment of the present invention, the housing 40 has a light-shielding coating or is made of a light-shielding material to prevent external light from affecting the collection of image information.

[0128] In this embodiment, the housing 40 is made of a light-proof black PLA (polylactic acid) material. Of course, it can also be made of other black opaque materials.

[0129] The present invention also provides a bacteria detection method based on a gravity-driven micro-control flow chip and a portable detector, comprising:

[0130] Step 100: Perform bacterial lysis on the sample to obtain a nucleic acid-silica-based magnetic bead complex. Specifically, first, place a centrifuge tube containing 500 μL of the Salmonella typhimurium solution sample to be tested in an ultrasonic cleaner for 5 minutes for ultrasonic lysis. After lysis is complete, add 15 μL of a silica-based magnetic bead suspension to the centrifuge tube and oscillate and mix for 2 minutes to form a nucleic acid-silica-based magnetic bead complex.

[0131] Step 200: Load the nucleic acid-silicon-based magnetic bead complex and the required reagents for the experiment into the gravity-driven microfluidic chip. Specifically, 500 μL of the nucleic acid-silicon-based magnetic bead complex solution is added to the sample chamber 102 of the gravity-driven microfluidic chip, 300 μL of nucleic acid cleaning solution is added to the cleaning solution chamber 101, and 100 μL of nucleic acid elution solution is added to the elution solution chamber 103.

[0132] Step 300 controls the tilting of the gravity-driven microfluidic chip to move reagents between different reagent chambers 10, thereby completing nucleic acid capture and cleaning. Specifically, after the gravity-driven microfluidic chip is loaded with the required reagents, it is allowed to stand for 1 minute to allow the D3 NdFeB magnetic balls embedded in the sample chamber 102 to capture the nucleic acid-silicon-based magnetic bead complex. During the first tilt, the gravity-driven microfluidic chip is tilted 25° to the right. At this time, the liquid in the sample chamber 102 enters the waste liquid chamber 105 under the siphon effect of the fourth siphon line 204; at the same time, the cleaning liquid enters the temporary storage chamber 106 through the first siphon line 201. During the second tilt, the gravity-driven microfluidic chip is tilted 45° to the right. The cleaning liquid in the temporary storage chamber 106 enters the sample chamber 102 through the fifth siphon line 205. Thereafter, it is allowed to stand horizontally for 5 minutes to allow the cleaning liquid to remove impurities on the silicon-based magnetic beads. During the third tilting, the gravity-driven microfluidic chip is tilted 45° to the right. At this time, the cleaning liquid containing the washed impurities enters the waste liquid chamber 105 through the fourth siphon line 204 .

[0133] Step 400, elute the captured nucleic acid. Specifically, after step 300, the microfluidic chip is allowed to stand for 10 minutes to dry the residual ethanol in the cleaning solution that will inhibit the subsequent isothermal amplification. During the fourth tilt, the gravity-driven microfluidic chip is tilted 45 degrees to the left. At this time, the eluent enters the sample chamber 102 through the second siphon line 202. Thereafter, it is allowed to stand horizontally for 10 minutes. The eluent elutes the nucleic acid from the silicon-based magnetic beads and dissolves it in the eluent. During the fifth tilt, the gravity-driven microfluidic chip is tilted 45 degrees to the left. The eluent enters the detection chamber 104 through the third siphon line 203; the eluent dissolves the freeze-dried RAA detection reagent pre-buried in the detection chamber 104 to form an amplification system, which is used for subsequent measurement and analysis.

[0134] Step 500: Heat amplify the eluted nucleic acid. Specifically, the nucleic acid solution dissolves the freeze-dried RAA detection reagent pre-embedded in the detection chamber 104 to form an amplification system. The microfluidic chip is placed in the storage space 401 of the accompanying portable detector. The excitation light source 501 and the heating component are turned on to stimulate the fluorescence signal in the detection chamber 104 and provide the required temperature for the amplification reaction.

[0135] In step 600, image detection equipment 60 captures image information after nucleic acid amplification for quantitative bacterial analysis. Specifically, a smartphone can be used to capture fluorescence images of detection chamber 104, and software can analyze changes in fluorescence intensity to achieve quantitative detection and analysis of target bacteria. The smartphone takes a photo of detection chamber 104 every 20 seconds, acquires the fluorescence image, and analyzes it to create a calibration curve. Ultimately, the corresponding time threshold for the amplification curve is calculated.

[0136] It should be emphasized that the volumes of different reagents, the tilt direction and angle of the gravity-driven microfluidic chip mentioned in the above working process are not limitations of this method. In actual situations, the volumes of reagents are different according to experimental requirements, and the relative orientations of different reagent chambers 10 make the tilt directions of the gravity-driven microfluidic chip different. Different reagents correspond to different liquid weights and viscosities, and the corresponding siphon forces required are different, and thus the highest points of the siphon pipeline 20 are different, resulting in different tilt angles for starting the siphon effect. Staff should make adaptive adjustments according to actual needs.

[0137] According to one embodiment of the present invention, step 100 further includes step 110, using ultrasonic lysis and thermal lysis to lyse bacterial samples of the same concentration, and evaluating the nucleic acid extraction content and purity, and the lysis method with higher nucleic acid extraction content and purity is used as the target lysis method. Specifically, Figure 5 As shown, this example selects and compares two nucleic acid lysis methods, ultrasonic lysis and thermal lysis, which are simple to operate and have portable supporting instruments.

[0138] This example uses 1.7×10 3 The nucleic acid extraction content and purity of Salmonella typhimurium samples with CFU / mL concentration were evaluated to select the most suitable extraction method for nucleic acid detection. The thermal lysis treatment method was to add 500 μL of 1.7×10 3 A sample of Salmonella Typhimurium at a CFU / mL concentration was added to a centrifuge tube and heated in a 95°C metal bath for 10 minutes to fully lyse the bacteria. Ultrasonic lysis was performed by adding the same Salmonella Typhimurium sample to a centrifuge tube, placing the tube in an ultrasonic cleaner, and sonicating for 10 minutes to fully lyse the bacteria. 100 μL of the nucleic acid solution obtained by each method was then washed with 300 μL of the wash buffer and then eluted with 100 μL of the elution buffer provided by the magnetic bead-based nucleic acid extraction kit, creating two new sample sets for evaluation. The nucleic acid solutions obtained using the four different extraction methods were then subjected to quantitative PCR. The Ct value represents the number of cycles during the PCR reaction at which the fluorescence signal exceeds the background noise and reaches the set threshold. Lower Ct values ​​indicate a higher concentration of target nucleic acid in the starting nucleic acid sample. The nucleic acid solution obtained by ultrasonic lysis, both before and after magnetic bead-based nucleic acid extraction, exhibited a higher nucleic acid content than the solution obtained by thermal lysis. Protein is the most common impurity in the nucleic acid extraction product. 280nm is the wavelength at which proteins have their highest absorption peak, while DNA has an absorption peak at 260nm. Therefore, the A260 / A280 ratio can be used as an indicator of the purity of the extracted nucleic acid solution. The nucleic acid solution obtained by ultrasonic lysis, both before and after magnetic bead extraction, was found to be purer than that obtained by thermal lysis. Based on the above content and purity performance, a comparison of the two nucleic acid extraction methods for Salmonella typhimurium revealed that the ultrasonic lysis method achieved superior extraction content and purity.

[0139] According to one embodiment of the present invention, step 300 further includes step 310, obtaining the amount of target silica-based magnetic beads based on the amounts of different silica-based magnetic beads and the Ct value of qPCR (fluorescence quantitative PCR) amplification detection.

[0140] Specifically, such as Figure 6 As shown, 500 μL of 1.7 × 10 5A Salmonella typhimurium sample with a CFU / mL concentration was added to a centrifuge tube. After ultrasonic lysis for 5 minutes, 5 to 20 μL of a silica-based magnetic bead suspension was added to the sample and shaken for 3 minutes. The tube was then placed on a magnetic rack for 30 seconds. Once the beads were fully adsorbed, the liquid was carefully aspirated. Then, 100 μL of elution buffer was added to the tube and the sample was placed on a magnetic rack for 10 minutes. This process simulated the nucleic acid elution step in a microfluidic chip. The nucleic acid solutions obtained by combining nucleic acids with four different volumes of silica-based magnetic bead suspension were then subjected to quantitative PCR. Lower Ct values ​​indicated a higher concentration of target nucleic acid in the sample. Increasing the amount of silica-based magnetic beads from 5 to 15 μL decreased the Ct value of the qPCR amplification assay from 15.736 to 10.583. Further increasing the amount of silica-based magnetic beads to 20 μL did not significantly decrease the Ct value of the qPCR amplification assay. Therefore, this example uses 15 μL as the optimal amount of silica-based magnetic beads required to capture nucleic acids.

[0141] According to one embodiment of the present invention, step 500 further includes step 510 of acquiring a target heating temperature based on a detected time threshold, and heating the nucleic acid at the target heating temperature.

[0142] Specifically, such as Figure 7 As shown, in this example, the real-time fluorescence RAA isothermal amplification technology was used to amplify and detect the extracted bacterial DNA. The amplification reaction temperature is crucial to the RAA detection effect. In order to determine the optimal amplification reaction temperature, this example used different reaction temperatures from 37°C to 52°C for 1.7×10 5 CFU / mL concentration of Salmonella typhimurium was detected. When the reaction temperature was set to 37°C, no fluorescent signal was generated during the entire 20-min detection process. When the reaction temperature was increased from 40°C to 49°C, the detection time threshold was reduced from 17.3min to 7.8min. When the reaction temperature was further increased to 52°C, the detection time threshold did not change significantly. Therefore, this embodiment adopts 49°C as the optimal reaction temperature for the RAA isothermal amplification reaction. The optimal RAA reaction temperature optimized in this embodiment is slightly higher than the commonly used reaction temperature range (37°C ~ 42°C). This is because the shell 30 of the gravity-driven microfluidic chip proposed in this application is made of PDMS material, which has relatively poor thermal conductivity. On the other hand, during amplification, only the bottom of the microfluidic chip is heated by the heating component, so the RAA detection system requires a higher heating temperature to achieve the best amplification effect.

[0143] According to one embodiment of the present invention, step 600 also includes step 610, where the image acquisition device acquires multiple images of the reagent chamber 10 and obtains the average G / (R+G+B) value and the sum of ten times the standard deviation of G / (R+G+B) to calculate the time threshold of the amplification curve.

[0144] Specifically, such as Figure 8 As shown, this figure is a standard curve. This embodiment uses software to image the reagent chamber 10 (detection chamber 104) and analyze the changes in fluorescence intensity to achieve quantitative detection and analysis of target bacteria. The image acquisition device takes a picture of the detection chamber 104 every 20 seconds, obtains its fluorescence image and analyzes it, and draws an amplification curve based on the average G / (R+G+B) value of the detection chamber 104 area. And following the Ct value calculation method of fluorescent quantitative PCR, the average G / (R+G+B) value (Average) of the 2nd to 10th images and the sum of ten times the standard deviation of G / (R+G+B) of the 2nd to 10th images are used as the fluorescence threshold (Threshold), and finally the time threshold Tt (Time threshold) of the amplification curve is calculated accordingly.

[0145]

[0146] Where k is the starting number, Gk, Rk, and Bk are the RGB values ​​of the kth image;

[0147] Under the optimal conditions, the concentration of this embodiment is 1.1×10 1 CFU / mL to 1.1×10 6 The bacteria with the highest CFU / mL were used as the target bacteria, and the experiment was repeated three times. 1 The CFU / mL increased to 1.1×10 6 CFU / mL, the detection time threshold decreased from 15.5min to 6.6min, and there was a good linear relationship between the time threshold and the logarithm of the bacterial concentration, with a determination coefficient as high as 0.99. Its mathematical expression can be expressed as T T =-1.727×lgC S +15.2215,

[0148] Among them, Cs refers to the bacterial concentration, and the detection sensitivity is as low as 1.1×10 1 CFU / mL.

[0149] In one embodiment, before step 100 , the method further includes step 700 of determining the specificity of the portable detector for Salmonella typhimurium.

[0150] Specifically, such as Figure 9 As shown, the concentration is the same as 1.1×106 CFU / mL of Salmonella typhimurium (ATCC14028); Escherichia coli O157:H7 (ATCC 43888); Listeria monocytogenes (ATCC 13932); Vibrio parahaemolyticus (ATCC 17802); Bacillus cereus (ATCC 11778) and Staphylococcus aureus (CICC 10001) were tested as non-target bacteria. Figure 9 As shown, among the six experimental groups, only the concentration of 1.1×10 6 The fluorescent signal was detected for Salmonella typhimurium (ATCC 14028) with a concentration of CFU / mL, while no fluorescence signal was detected for the other five groups, which indicates that the portable detector of the present application has good specificity.

[0151] In one embodiment, before step 100 , the method further includes step 800 of determining the practicability of the portable detection instrument.

[0152] Specifically, such as Figure 10 As shown in the example, the concentration of 1.1×10 2 The CFU / mL increased to 1.1×10 5 CFU / mL of Salmonella, and the biosensor and the gold standard culture method were used for simultaneous detection and verification. As the concentration of spiked Salmonella typhimurium increased from 1.1×10 2 The CFU / mL increased to 1.1×10 5 CFU / mL, and the recovery rates ranged from 91.54% to 125.45%, with an average recovery rate of 104.26%, indicating that the gravity-driven microfluidic chip and the portable detector have good feasibility and practicality in detecting contaminated real chicken samples.

[0153] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A gravity-driven microfluidic chip, characterized in that: include: A housing (30) is provided with at least two reagent chambers (10) and at least one siphon line (20), each of the reagent chambers (10) being provided with an air hole (11); The siphon pipeline (20) has at least one ascending section and one descending section, and the two ends of the siphon pipeline (20) are respectively connected to different reagent chambers (10), and the highest point of the siphon pipeline (20) is higher than the liquid level of any one of the reagent chambers (10) connected thereto; The connected reagent chambers (10) are distributed in a stepped manner; The reagent chamber (10) includes a cleaning liquid chamber (101), a sample chamber (102), an eluent chamber (103), a detection chamber (104), and a waste liquid chamber (105), wherein the cleaning liquid chamber (101) and the eluent chamber (103) are arranged higher than the sample chamber (102), and the detection chamber (104) and the waste liquid chamber (105) are arranged lower than the sample chamber (102); The siphon pipeline (20) comprises a first siphon pipeline (201), a second siphon pipeline (202), a third siphon pipeline (203), and a fourth siphon pipeline (204); The first siphon line (201) connects the bottom of the cleaning liquid chamber (101) and the top of the sample chamber (102), the second siphon line (202) connects the bottom of the elution liquid chamber (103) and the top of the sample chamber (102), the third siphon line (203) connects the bottom of the sample chamber (102) and the top of the detection chamber (104), and the fourth siphon line (204) connects the bottom of the sample chamber (102) and the top of the waste liquid chamber (105); The outlets of the third siphon line (203), the fourth siphon line (204) and the sample chamber (102) are interconnected; the first siphon line (201) and the second siphon line (202) are not located on the same side of the sample chamber (102) and are not coplanar; the second siphon line (202) and the fourth siphon line (204) are not located on opposite sides of the sample chamber (102) and are not coplanar; The surface of the reagent chamber (10) is provided with a hydrophobic layer, and / or the inner wall surface of the siphon pipeline (20) is provided with a hydrophilic layer.

2. The gravity-driven microfluidic chip according to claim 1, characterized in that: The first siphon line (201) and the second siphon line (202) are located on opposite sides of the sample chamber (102) and are coplanar; the third siphon line (203) and the fourth siphon line (204) are located on opposite sides of the sample chamber (102) and are coplanar.

3. The gravity-driven microfluidic chip according to claim 2, characterized in that: The first siphon pipeline (201), the second siphon pipeline (202), the third siphon pipeline (203) and the fourth siphon pipeline (204) are arranged in the same plane; The reagent chamber (10) further includes a temporary storage chamber (106), and the siphon pipeline (20) further includes a fifth siphon pipeline (205); the temporary storage chamber (106) is arranged higher than the sample chamber (102) and lower than the cleaning liquid chamber (101); The first siphon line (201) connects the bottom of the cleaning liquid chamber (101) and the top of the temporary storage chamber (106), and the fifth siphon line (205) connects the bottom of the temporary storage chamber (106) and the top of the sample chamber (102).

4. The gravity-driven microfluidic chip according to any one of claims 1 to 3, characterized in that: Along the direction of gravity, a chamfered portion is provided between the bottom wall and the side wall of the shell (30), and the plane of the chamfered portion is perpendicular to the plane where the connected reagent chamber (10) is located.

5. The gravity-driven microfluidic chip according to any one of claims 1 to 3, characterized in that: The waste liquid chamber (105) is provided with absorbent cotton for absorbing waste liquid.

6. A portable detector, characterized in that: include: The gravity-driven microfluidic chip according to any one of claims 1 to 5; The housing (40) is provided with a placement space (401) suitable for placing the gravity-driven microfluidic chip; A heating component is provided corresponding to a first side of the reagent chamber (10) to be detected; The detection component (50) is correspondingly arranged on the second side of the reagent chamber (10) to be detected, and the first side and the second side are not co-located.

7. The portable detector according to claim 6, characterized in that: The housing (40) is provided with a first through hole (402), and the first through hole (402) is arranged corresponding to the reagent chamber (10) to be detected; The side of the first through hole (402) facing away from the reagent chamber (10) is suitable for installing an image detection device (60).

8. The portable detector according to claim 7, characterized in that: The detection assembly (50) includes an excitation light source (501), a lens (502), a first filter (503), and a second filter (504); The lens (502), the first filter (503), and the reagent chamber (10) to be detected are sequentially arranged along the direction of light from the excitation light source (501); the direction of the light and the direction of the reagent chamber (10) to be detected pointing to the first through hole (402) form a set angle; The second filter (504) is provided on one side of the first through hole (402) close to the reagent chamber (10) to be detected.

9. The portable detector according to claim 6, characterized in that: The housing (40) has a placement groove (404) extending inward from the outer wall, and the placement groove (404) is used to place the heating component.

10. The portable detector according to claim 6, characterized in that: The housing (40) includes a plurality of reagent placement positions (405), and the reagent placement positions (405) are arranged corresponding to the number of reagents required by the reagent chamber (10).

11. The portable detector according to claim 6, characterized in that: The housing (40) has a light-shielding coating or is made of a light-shielding material.

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