CN111482207B - Disk-type microfluidic chip and use method thereof - Google Patents
Disk-type microfluidic chip and use method thereof Download PDFInfo
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- CN111482207B CN111482207B CN202010370201.6A CN202010370201A CN111482207B CN 111482207 B CN111482207 B CN 111482207B CN 202010370201 A CN202010370201 A CN 202010370201A CN 111482207 B CN111482207 B CN 111482207B
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- 238000001514 detection method Methods 0.000 claims abstract description 21
- 239000000523 sample Substances 0.000 claims description 65
- 241000894006 Bacteria Species 0.000 claims description 41
- 239000012472 biological sample Substances 0.000 claims description 22
- 239000011148 porous material Substances 0.000 claims description 22
- 238000004891 communication Methods 0.000 claims description 14
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- 238000011144 upstream manufacturing Methods 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 claims description 3
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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Abstract
The invention relates to a disk-type microfluidic chip, which comprises a sample adding port for adding a sample, a filtering area which is communicated with the sample adding port and is provided with a filter membrane, and at least one microfluidic channel path which is communicated with the filtering area, wherein the microfluidic channel path comprises at least one chamber for storing at least one reagent. The microfluidic chip has the advantages of low reagent consumption, high analysis speed, simple operation process, convenient integration and the like, and the flow of a sample on the chip is controllable, so that the microfluidic chip is particularly suitable for the rapid detection of pathogenic microorganisms, and can detect various pathogenic microorganisms simultaneously.
Description
Technical Field
The invention particularly relates to a disk-type microfluidic chip and a use method thereof.
Background
The traditional method for detecting bacteria in biological samples has complicated steps, long detection time and easy pollution of detection reagents. The micro-fluidic chip is mainly characterized in that fluid is controlled in a micrometer scale space, basic operation units such as biochemical reaction, separation, detection and the like of a sample can be integrated in a chip system, a network is formed by micro-channels, and controllable fluid penetrates through the whole system, so that various functions of a conventional laboratory are realized.
The microfluidic chip technology is combined with the pathogenic microorganism detection technology, a microfluidic chip which can be used for rapid quantitative detection of bacteria in biological samples is developed, a simple, rapid and effective solution is provided for rapid diagnosis of pathogenic microorganisms, and the microfluidic chip has important significance for treatment and prognosis of diseases.
Disclosure of Invention
The invention aims to provide the disk-type microfluidic chip which has the advantages of low reagent consumption, high analysis speed, simple operation process, convenience for integration, controllable flow of biological samples on the chip, and closed environment for ensuring that the inside of the chip is not polluted, and is particularly suitable for rapid quantitative detection of bacteria in the biological samples.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the first aspect of the invention provides a disc microfluidic chip comprising a sample addition port for adding a sample, a filtration zone in communication with the sample addition port and provided with a filter membrane, at least one microfluidic channel path in communication with the filtration zone, the microfluidic channel path comprising at least one chamber for storing at least one reagent. The disk-type microfluidic chip is particularly suitable for detecting bacteria in biological samples such as urine and the like.
Preferably, the filtering area comprises a first filtering area which is communicated with the sample adding port and provided with a first filter membrane, and a second filtering area which is communicated with the first filtering area and provided with a second filter membrane, and the second filtering area is communicated with the micro-fluid channel path.
Further preferably, the pore size of the first filter membrane is larger than the pore size of the second filter membrane.
Further preferably, the first filter membrane allows bacteria to pass therethrough and is capable of trapping impurities in a biological sample having a volume greater than the bacteria, and the second filter membrane is capable of trapping the bacteria.
Further preferably, the pore size of the first filter membrane is 1 to 100. Mu.m, preferably 1 to 50. Mu.m, more preferably 1 to 10. Mu.m; the pore size of the second filter membrane is 0.01-1 μm, preferably 0.01-0.5 urn, more preferably 0.1-0.3 μm. The biological sample is subjected to rough filtration by using the first filter membrane, so that impurities such as cells and crystals with the volume larger than that of bacteria in the biological sample are prevented from entering the flow channel, the flow channel is prevented from being blocked, the biological sample is subjected to fine filtration by using the second filter membrane, and the bacteria in the biological sample are trapped on the second filter membrane, so that the bacteria can be enriched on the second filter membrane.
Further preferably, the number of the first filtering areas is two or more, and the two or more first filtering areas are arranged in series, and each first filtering area is provided with the first filter membrane; or, the first filter membranes are two or more first filter membranes arranged in one first filter zone along the flow direction of the sample passing through the first filter zone, and the two or more first filter membranes are arranged in parallel. The first filter membrane with two or more filters is arranged, the biological sample is subjected to rough filtration twice, and impurities with the volume larger than that of bacteria in the biological sample are trapped as much as possible, so that the flow channel is further effectively prevented from being blocked, the purity of bacteria trapped on the second filter membrane is improved, and the accuracy of a detection result is prevented from being influenced due to excessive impurities on the second filter membrane.
More preferably, the pore size of the first filter membrane in two or more of the first filter regions or the pore size of the first filter membrane in two or more of the first filter regions is different, and the pore size of the first filter membrane located downstream of the sample flow direction is smaller than the pore size of the first filter membrane located upstream of the sample flow direction.
Because the biological sample, such as urine, has complex components, more impurities, such as cells and crystals, and the viscosity of the urine is higher than that of water, the problems of blockage and the like are easy to occur when the microfluidic chip is adopted to detect the biological sample, such as urine, and the blockage of a flow channel is well avoided by arranging the first filtering area with the first filtering film.
More preferably, the pore size of the first filter membrane located upstream of the sample flow direction is 2 to 100 μm; the aperture of the first filter membrane positioned at the downstream of the sample flow direction is 1-2 mu m, so that impurities with larger volume in the biological sample are removed firstly through the first filter membrane with larger aperture, and impurities with smaller volume are removed through the first filter membrane with smaller aperture, thereby further improving the rough filtration effect on one hand and better avoiding the blockage of the flow passage on the other hand.
Further preferably, the ratio of the cross-sectional area of the cross-section of the first filter membrane of the first filter area to the cross-sectional area of the cross-section of the second filter area is 3.5-4.5:1, so that enrichment of bacteria can be better realized, space can be saved, the size of the chip can be reduced, and the cost of the chip is further reduced.
Preferably, the disc microfluidic chip further comprises a first reagent zone in communication with the filtration zone and storing a first reagent.
Preferably, the microfluidic channel path includes a first chamber and a second chamber disposed radially outwardly from a center of the disc microfluidic chip, the first chamber being in communication with the filtration zone, the second chamber being in communication with the first chamber, at least one of the first chamber and the second chamber storing at least one reagent.
Further preferably, the sample is a biological sample, the first reagent is a culture medium, phage lyophilized powder is stored in the first chamber, and the detection reagent is stored in the second chamber, so that the reagent does not need to be prepared at present, and only the reagent stored in advance is added into a corresponding chamber when the reagent is required to be added, and the use is more convenient.
Preferably, the first chamber and the second chamber are each circular.
Preferably, the microfluidic channel path comprises a plurality of serially connected microfluidic channel paths, and the microfluidic channel paths are distributed along the circumferential direction of the disc-type microfluidic chip.
Preferably, the microfluidic channel path is communicated with the filtering area through a runner, the runner comprises a main runner, a split runner and a first chamber, one end of the split runner is communicated with the filtering area, the split runner is respectively communicated with the main runner and the first chamber of the microfluidic channel path, the main runner is arc-shaped, and the circle center of the main runner coincides with the circle center of the disc-type microfluidic chip in horizontal projection.
The microfluidic channel path only comprises the first chamber and the second chamber, so that the diameter of the chip can be reduced, the cost is saved, and meanwhile, one-time centrifugation step and energy consumption can be reduced. Further, the invention avoids liquid backflow into the path of the microfluidic channel by the design of the main flow channel and the shunt channel.
More preferably, the disc-type microfluidic chip further comprises an exhaust hole communicated with the other end of the main flow channel.
Preferably, the filtering area comprises a first filtering area which is communicated with the sample adding port and is provided with a first filter membrane, and a second filtering area which is communicated with the first filtering area and is provided with a second filter membrane, and the second filtering area is communicated with the first chamber; the disk-type microfluidic chip further comprises a first reagent area which is communicated with the second filtering area and stores a first reagent; when the disk-type microfluidic chip is centrifuged in one direction, the first reagent of the first reagent zone flows into the second filtering zone; when the disk-type microfluidic chip is centrifuged in a direction opposite to the direction, the material in the second filtering area flows into the microfluidic channel path. According to a specific and preferred embodiment, the microfluidic channel path comprises a first chamber and a second chamber which are sequentially arranged radially outwards from the center of the disc-type microfluidic chip, the sample inlet is communicated with the first filtering area through a first flow channel, the bottoms of the first filtering area and the second filtering area are communicated through a second flow channel, the bottoms of the first reagent area and the second filtering area are communicated through a third flow channel, and the bottom of the second filtering area is communicated with the inner bottom of the first chamber of the microfluidic channel path through a fourth flow channel; in each microfluidic channel path, the outside top of the first chamber is in communication with the inside top of the second chamber via a fifth flow channel.
Preferably, the first reagent area is arc-shaped, the center of the circle coincides with the center of the disc-type microfluidic chip in horizontal projection, and the first filtering area, the second filtering area and the microfluidic channel path are positioned at the periphery of the first reagent area.
Preferably, the disc microfluidic chip further comprises a flow channel communicated with the top of the filtering area and an outlet communicated with the flow channel.
Preferably, the second filtering area comprises a mixing area which is positioned at the bottom and the cross section area of which is gradually reduced from bottom to top, and a filtering area which is positioned above the mixing area, and the second filtering film is arranged between the mixing area and the filtering area.
Preferably, at least the part of the disc-type microfluidic chip contacted with the sample or the reagent is treated by a hydrophobic material, so that the problems of flow rate reduction and incomplete sample reaction caused by adhesion of the sample or the reagent in a flow channel or each region can be effectively prevented, and the detection accuracy is improved.
Preferably, the thickness of the disc-type microfluidic chip is 8-12 mm, and the inner diameter of the runner on the disc-type microfluidic chip is 1-2 mm.
Preferably, the disc-type microfluidic chip further comprises a plurality of through holes for weight reduction and for controlling the center of gravity of the disc-type microfluidic chip. The center of gravity of the disc-type microfluidic chip is located at the center of a circle, centrifugal operation can be directly performed on the disc-type microfluidic chip, the balancing step is omitted, the disc-type microfluidic chip is safer and more convenient to centrifuge, the weight of the disc-type microfluidic chip is reduced, and the energy consumption can be reduced.
The disk-type microfluidic chip comprises a first soft film layer, a hard film layer and a second soft film layer which are sequentially laminated, wherein the hard film layer is made of a material which does not react with biological samples and reagents, and acrylic (PMMA) or Polystyrene (PA) and the like are preferably adopted; the soft film layer is made of a material which does not react with the biological sample and the reagent and can deform, and preferably rubber or silica gel is used.
In the invention, a quality control area can be arranged on the disk-type microfluidic chip.
The disk-type microfluidic chip can be used for centrifugation and signal detection by adopting a centrifuge and a luminescence detection instrument in the prior art.
The invention also provides a method for quantitatively detecting the bacterial content in a sample by adopting a disk-type microfluidic chip, which comprises the following steps of:
(1) Adding a sample to the sample addition port and flowing said sample through the filtration zone;
(2) Centrifuging the disc-type microfluidic chip to enable materials in the filtering area to enter a cavity of the microfluidic channel path, and enabling bacteria to react with the reagent to generate detection signals.
Preferably, the specific steps of the method are as follows:
(1) Adding a sample to the sample adding port, and enabling the sample to flow through a first filtering area to remove impurities in the sample, and then flowing through a second filtering area to entrap bacteria in the sample;
(2) Centrifuging the disc-type microfluidic chip in a certain direction to enable the first reagent in the first reagent zone to flow into the second filtering zone;
(3) Rotating the disk-type microfluidic chip in the forward and reverse directions to mix the first reagent with the bacteria;
(4) Centrifuging the disc-type microfluidic chip in the direction opposite to the direction of the step (2) to enable the mixed solution of the first reagent and the bacteria to flow to a first chamber and a second chamber of a microfluidic channel path in sequence, and detecting chemiluminescent signals after incubation.
Further preferably, the first reagent is one or more of PBS buffer, MES buffer, LB medium, beef extract peptone medium and phage buffer; preferably LB medium and beef extract peptone medium.
Further preferably, the reagent stored in the first chamber is phage lyophilized powder.
Further preferably, the reagent stored in the second chamber comprises a luminescent substrate.
In the invention, the phage is phage with genes capable of expressing fluorescent proteins; the method of introducing a specific gene into phage is a conventional method in the art, wherein the specific gene can express a specific fluorescent protein, and react with a specific luminescent substrate, thereby realizing luminescence of a catalytic reaction solution; for example, the lux gene expresses 1ux protein, catalyzes the oxidation-reduction reaction of decanal, and thus generates an optical signal; whereas the nanolu gene reacts with the substrate Furimazine to emit light. For phage selection, the determination is made according to the type of bacteria to be detected, for example, K1F phage specifically hosts E.coli, so K1F phage is used when E.coli detection is desired; the Felix O1 phage specifically hosts salmonella, and thus can be used to detect salmonella.
In the invention, the phage comprise K1F phage, felix O1 phage and the like, and the specific phage is determined according to bacteria to be detected; the fluorescent protein comprises Lux protein, GFP protein, nanoLuc protein and the like; the luminescent substrate comprises aldehydes such as decanal, aromatic aldehyde and the like, imidazopyrazinones (such as Furimazine) and the like.
Further preferably, the incubation time of the phage with the bacteria is 10 to 60 minutes. The use method of the invention adopts the disc-type microfluidic chip to detect bacteria.
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages: the disk-type microfluidic chip provided by the invention has the advantages of low reagent consumption, high analysis speed, simple operation process, convenience in integration and the like, and the flow of a sample on the chip is controllable, the chip diameter is small, the chip cost is low, and the disk-type microfluidic chip is particularly suitable for the rapid detection of pathogenic microorganisms and can detect various pathogenic microorganisms simultaneously.
Drawings
Fig. 1 is a perspective view of a disk-type microfluidic chip of example 1;
fig. 2 is a top view of the disk-type microfluidic chip of example 1.
In the above figures: 1. a sample adding port; 2. a first flow passage; 3. a first filtration zone; 4. a second first filter membrane; 5. a first filter membrane; 6. a second flow passage; 7. a mixing zone; 8. a second filtration zone; 9. a second filter membrane; 10. a main flow passage; 11. a sub-runner; 12. a first chamber; 13 a fifth flow path; 14. a second chamber; 15. a quality control region; 16. a first reagent zone; 17. a third flow passage; 18. a sixth flow passage; 19. a through hole; 20. a bayonet.
Detailed Description
The invention will be further described with reference to examples of embodiments shown in the drawings. The invention will be further described with reference to the embodiments shown in the drawings, wherein the various features may be combined as desired unless the context clearly indicates otherwise.
Example 1
This embodiment is defined by the orientation of FIG. 1, wherein the sample application area is open downward.
As shown in fig. 1 and 2, a disc microfluidic chip includes a sample addition port 1 for adding a sample, a first filtration zone 3 communicating with the sample addition port 1 through a first flow channel 2 and provided with a first filtration membrane 5 and a second first filtration membrane 4, a second filtration zone 8 communicating with the first filtration zone 3 through a second flow channel 6 and provided with a second filtration membrane 9, a first reagent zone 16 communicating with the second filtration zone 8 through a third flow channel 17 and storing a first reagent, at least one microfluidic channel path communicating with the second filtration zone 8, each microfluidic channel path including a first chamber 12 and a second chamber 14 disposed radially outwardly in sequence from the center of the disc microfluidic chip, the first chamber 12 in the microfluidic channel path communicating with the second filtration zone 8 through a fourth flow channel, and the second chamber 14 communicating with the first chamber 12 through a fifth flow channel 13.
Specifically, as shown in fig. 1 and 2, the sample port 1 is located on the lower surface of the disk-type microfluidic chip, the first flow channel 2 includes a first portion extending in the up-down direction and having a lower end in communication with the sample port 1, a second portion extending in the left-right direction and having an inner end in communication with the upper end of the first portion, a third portion extending in the up-down direction and having an upper end in communication with the outer end of the second portion, and a fourth portion extending in the left-right direction and having an inner end in communication with the lower end of the third portion, the outer end of the fourth portion being in communication with the bottom of the first filtering section 3; by providing the first flow channel 2 with such a meandering structure, it is possible to prevent excessive local pressure from being directly applied to the first filter membrane when the sample is applied to the sample application port 1.
The second flow passage 6 includes a fifth portion having an outer end communicating with the top of the first filtering section 3 and extending in the left-right direction, a sixth portion extending in the up-down direction and having an upper end communicating with the inner end of the fifth portion, a seventh portion extending in the left-right direction and having one end communicating with the lower end of the sixth portion, and the other end of the seventh portion communicating with the bottom of the second filtering section 8.
Both ends of the third flow channel 17 are respectively communicated with the bottom of the second filtering area 8 and the bottom of the first reagent area 16, the third flow channel 17 is arc-shaped, and the extending direction of the arc shape is consistent with that of the first reagent area 16.
The bottom of the second filtering section 8 communicates with the bottom of the first chamber 12 of the plurality of microfluidic channel paths through a fourth flow channel; the fourth flow channel includes a main flow channel 10 and a plurality of sub-flow channels 11 that are communicated with the main flow channel 10, one end of the main flow channel 10 is communicated with the bottom of the second filtering area 8, the other end of the main flow channel 10 is communicated with the exhaust port, the main flow channel 10 is arc-shaped, the circle center of the main flow channel 10 coincides with the circle center of the disc-type micro-fluidic chip in view of horizontal projection, the sub-flow channels 11 are uniformly distributed along the length direction of the main flow channel 10, in this embodiment, the number of the sub-flow channels 11 is 6, wherein 5 sub-flow channels 11 respectively correspond to one micro-flow channel path, and one sub-flow channel 11 is communicated with the quality control area 15.
The outside of the sub flow channel 11 communicates with the inside bottom of the first chamber 12, and the outside top of the first chamber 12 and the inside top of the second chamber 14 communicate with each other through the fifth flow channel 13.
The top of the second filter zone 8 is also in communication with one end of a sixth flow passage 18, the other end of the sixth flow passage 18 being in communication with the outlet.
The first filter 5 and the second filter 4 have a pore size arranged to allow bacteria to pass and to be able to entrap impurities in a biological sample having a volume larger than the bacteria, and the second filter 9 has a pore size arranged to be able to entrap said bacteria. In this embodiment, the first filtering area 3 is provided with a second first filtering film 4 and a first filtering film 5 along the up-down direction, the pore diameters of the second first filtering film 4 and the first filtering film 5 may be the same or different, the pore diameters of the second first filtering film 4 and the first filtering film 5 are independently 1-100 μm, in order to further improve the rough filtering effect, the pore diameter of the first filtering film 5 located below is preferably 2-100 μm, in this embodiment 2-5 μm, and the pore diameter of the second first filtering film 4 located above is preferably 1-2 μm, in this embodiment 1 μm. In order to enable the second filter membrane 9 to better enrich bacteria, the cross-sectional area of the cross-section of the first filter membrane of the first filter zone 3 is 3.5-4.5 times of the cross-sectional area of the cross-section of the second filter membrane of the second filter zone 8.
The second filtering area 8 comprises a mixing area 7 with the sectional area gradually decreasing from bottom to top and a filtering area positioned above the mixing area 7, the second filtering film 9 is arranged between the mixing area 7 and the filtering area, bosses for fixing the second filtering film 9 between the mixing area 7 and the filtering area are respectively arranged at the upper end and the lower end of the second filtering film 9, the aperture of the second filtering film 9 is preferably 0.01-1 mu m, the aperture of the second filtering film 9 is preferably 0.22 mu m, bacteria can be trapped and enriched to the greatest extent, and the detection accuracy is improved.
The microfluidic channel paths comprise 5 microfluidic channel paths which are sequentially connected in series, and the 5 microfluidic channel paths are distributed along the circumferential direction of the disc-type microfluidic chip. The first chamber 12 and the second chamber 14 are respectively circular, phage freeze-dried powder is stored in the first chamber 12, and detection reagent is stored in the second chamber 14. Wherein, the first chamber 12 of each microfluidic channel path can store different phage lyophilized powder, for example, the first chamber 12 can store K1F phage into which Lux gene is introduced, and the first second chamber 14 stores decanal solution, thereby detecting Escherichia coli; the second first chamber 12 can store Felix O1 phage into which Lux gene is introduced, and the second chamber 14 stores decanal solution, so that Salmonella can be detected.
The first reagent area 16 is arc-shaped, and the center of the circle coincides with the center of the disk-type microfluidic chip in horizontal projection, and the first reagent stored in the first reagent area 16 is a culture medium. The first filtering section 3, the second filtering section 8 and the microfluidic channel path are located at the periphery of the first reagent section 16. The disc-type microfluidic chip is also provided with a through hole 19 for reducing weight and controlling the gravity center of the disc-type microfluidic chip. Through the arrangement, the center of gravity of the disc-type microfluidic chip is located at the center of a circle, centrifugal operation can be directly performed on the disc-type microfluidic chip, the balancing step is omitted, the disc-type microfluidic chip is safer and more convenient during centrifugation, the weight of the disc-type microfluidic chip is reduced through the through holes 19, the required centrifugal speed and time can be reduced, and the energy consumption is reduced.
The thickness of the disc-type microfluidic chip is preferably 8-12 mm, the inner diameter of the runner on the disc-type microfluidic chip is preferably 1-2 mm, and the thickness setting in the embodiment is the most preferred thickness obtained according to the setting of the internal structure of the microfluidic chip, so that the disc-type microfluidic chip achieves the best detection effect, has the smallest volume and saves the manufacturing cost.
The part of the disc-type microfluidic chip, which is at least contacted with the sample or the reagent, is treated by adopting a hydrophobic material, and in order to simplify the operation, the hard film layer of the disc-type microfluidic chip can be subjected to hydrophobic treatment, so that the phenomenon that the flow rate is reduced and the sample reaction is incomplete due to the fact that the sample or the reagent is adhered to a flow channel or each region can be prevented, and the detection accuracy is improved.
In this embodiment, a plurality of bayonets 20 for fixing during centrifugation are also provided on the disk microfluidic chip.
In this embodiment, the disc-type microfluidic chip is disc-shaped.
When the disk microfluidic chip is centrifuged in one direction, the first reagent of the first reagent zone 16 flows into the second filtration zone 8; when the disc microfluidic chip is centrifuged in a direction opposite to the above direction, the material of the second filter zone 8 flows into the microfluidic channel path. If the layout of each part on the chip shown in fig. 1 and 2 in this embodiment is adopted, a syringe can be inserted into the sample inlet 1 to perform sample feeding, the sample flows out from the outlet through the first filtering area 3 and the second filtering area 8, bacteria in the sample are enriched below the second filtering film 9, the disc-type microfluidic chip is centrifuged in a centrifugal way, the culture medium flows through the second filtering film 9 into the mixing area 7, the bacteria are eluted from the second filtering film 9, then the low-speed positive and negative rotation vibration mixing is performed, then the culture medium containing the bacteria flows into the first chamber 12 by centrifugation in a clockwise way, the bacteria are contacted and reacted with phage, the reactants of the bacteria and phage flow into the second chamber 14 by centrifugation in a clockwise way again to react with the detection reagent, and finally the chemiluminescent signal is detected.
Example 2: detection Using the microfluidic chip of example 1
1. After 10mL of urine sample is added through the syringe insertion sample addition port 1, the sample flows through the first filtering area 3 and the second filtering area 8, and then flows out of the outlet;
2. centrifuging counterclockwise to allow the culture medium to flow into the mixing zone 7;
3. low-speed forward and backward rotation shake mixed bacteria and culture medium;
4. centrifuging clockwise to allow the liquid containing the culture medium of bacteria to flow into the first chamber 12, and allowing the bacteria to react with phage for 15min;
5. the clockwise centrifugation causes the bacterial and phage reactants to flow into the second chamber 14 and react with the detection reagent, and finally the chemiluminescent signal is detected.
The above embodiments are provided to illustrate the technical concept and features of the present invention and are intended to enable those skilled in the art to understand the content of the present invention and implement the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made in accordance with the spirit of the present invention should be construed to be included in the scope of the present invention.
Claims (19)
1. A disk-type microfluidic chip, characterized in that: the disk-type microfluidic chip is used for detecting bacteria in biological samples, and comprises a sample adding port (1) for adding samples, a filtering area which is communicated with the sample adding port (1) and is provided with a filter membrane, at least one microfluidic channel path which is communicated with the filtering area, wherein the microfluidic channel path comprises at least one cavity which is used for storing at least one reagent, the filtering area comprises a first filtering area (3) which is communicated with the sample adding port (1) and is provided with a first filter membrane, a second filtering area (8) which is communicated with the first filtering area (3) and is provided with a second filter membrane (9), the second filtering area (8) is communicated with the microfluidic channel path, the aperture of the first filter membrane is larger than that of the second filter membrane (9), the first filter membrane allows bacteria to pass through and can retain impurities in biological samples larger than the bacteria, the second filter membrane (9) can retain the bacteria, the cross section area of the first filter membrane (3) is in the shape of the area which is in the shape of a curve, the cross section of the first filter area (4) is in the section of the first filter area (4) which is communicated with the flow channel (1) of the first filter area (16), the cross section of the second filter area (8) is in the section of the flow channel is in the shape of the curve-shaped section of the area (1) which is communicated with the area (1), the flow channel comprises a main flow channel (10) with one end communicated with the filtering area, and a shunt channel (11) respectively communicated with the main flow channel (10) and a first chamber (12) of the microfluidic channel path, wherein the main flow channel (10) is arc-shaped, the circle center of the main flow channel (10) coincides with the circle center of the disc-type microfluidic chip, the disc-type microfluidic chip further comprises a plurality of through holes (19) for reducing weight and controlling the gravity center of the disc-type microfluidic chip, the first reagent is one or more of PBS buffer solution, MES buffer solution, LB medium, beef extract protein medium and phage buffer solution, and when the disc-type microfluidic chip is centrifuged in one direction, the first reagent of the first reagent area (16) flows into the second filtering area (8); when the disc microfluidic chip is centrifuged in a direction opposite to the direction, the material in the second filtering area (8) flows into the microfluidic channel path.
2. The disk-type microfluidic chip according to claim 1, wherein: the aperture of the first filter membrane is 1-100 mu m; the pore diameter of the second filter membrane (9) is 0.01-1 mu m.
3. The disk-type microfluidic chip according to claim 1, wherein: the number of the first filtering areas (3) is two or more, the two or more first filtering areas (3) are arranged in series, and each first filtering area (3) is provided with the first filter membrane; or, the first filter membrane is two or more than two filter membranes arranged along the flow direction of the sample passing through the first filter zone (3), and the two or more than two first filter membranes are arranged in parallel.
4. A disk-type microfluidic chip according to claim 3, wherein: the pore diameters of the first filter membranes in the two or more first filter areas (3) or the pore diameters of the two or more first filter membranes are different, and the pore diameter of the first filter membrane positioned at the downstream of the sample flow direction is smaller than the pore diameter of the first filter membrane positioned at the upstream of the sample flow direction.
5. The disk microfluidic chip according to claim 4, wherein: the pore diameter of the first filter membrane positioned at the upstream of the sample flow direction is 2-100 mu m; the pore size of the first filter membrane positioned downstream of the sample flow direction is 1-2 mu m.
6. The disk-type microfluidic chip according to claim 1, wherein: the microfluidic channel path comprises a first chamber (12) and a second chamber (14) which are sequentially arranged radially outwards from the center of the disc-type microfluidic chip, the first chamber (12) is communicated with the filtering area, the second chamber (14) is communicated with the first chamber (12), and at least one of the first chamber (12) and the second chamber (14) stores at least one reagent.
7. The disk microfluidic chip according to claim 6, wherein: the sample is a biological sample, phage freeze-dried powder is stored in the first chamber (12), and a detection reagent is stored in the second chamber (14).
8. The disk-type microfluidic chip according to claim 1, wherein: the microfluidic channel path comprises a plurality of microfluidic channel paths which are sequentially connected in series, and the microfluidic channel paths are distributed along the circumferential direction of the disc-type microfluidic chip.
9. The disk-type microfluidic chip according to claim 1, wherein: the disk-type microfluidic chip also comprises an exhaust hole communicated with the other end of the main runner (10).
10. The disk-type microfluidic chip according to claim 1, wherein: the microfluidic channel path comprises a first chamber (12) and a second chamber (14) which are sequentially arranged radially outwards from the center of the disc-type microfluidic chip, the sample adding port (1) is communicated with the first filtering area (3) through a first flow channel (2), the bottoms of the first filtering area (3) and the second filtering area (8) are communicated through a second flow channel (6), the bottoms of the first reagent area (16) and the second filtering area (8) are communicated through a third flow channel (17), and the bottom of the second filtering area (8) is communicated with the inner bottom of the first chamber (12) of the microfluidic channel path through a fourth flow channel; in each microfluidic channel path, the outer top of the first chamber (12) is in communication with the inner top of the second chamber (14) via a fifth flow channel (13).
11. The disk-type microfluidic chip according to claim 1, wherein: the first filtering area (3), the second filtering area (8) and the micro-fluid channel path are positioned at the periphery of the first reagent area (16).
12. The disk-type microfluidic chip according to claim 1, wherein: the disk-type microfluidic chip also comprises a runner communicated with the top of the filtering area and an outlet communicated with the runner.
13. The disk-type microfluidic chip according to claim 1, wherein: the second filtering area (8) comprises a mixing area (7) which is arranged at the bottom and the cross section area of which is gradually reduced from bottom to top, and a filtering area which is arranged above the mixing area (7), and the second filtering film is arranged between the mixing area (7) and the filtering area.
14. The disk-type microfluidic chip according to claim 1, wherein: at least the part of the disk-type microfluidic chip, which is contacted with the sample or the reagent, is treated by adopting a hydrophobic material.
15. The disk-type microfluidic chip according to claim 1, wherein: the thickness of the disc-type micro-fluidic chip is 8-12 mm, and the inner diameter of a runner on the disc-type micro-fluidic chip is 1-2 mm.
16. A method for quantitatively detecting the bacterial content in a sample by using the disk-type microfluidic chip according to any one of claims 1 to 15, wherein: the method comprises the following steps:
(1) Adding a sample to the sample addition port (1) and flowing said sample through the filtration zone;
(2) Centrifuging the disc-type microfluidic chip to enable materials in the filtering area to enter a cavity of the microfluidic channel path, and enabling bacteria to react with the reagent to generate detection signals.
17. The method according to claim 16, wherein: the method comprises the following specific steps:
(1) Adding a sample to the sample adding port (1), and allowing the sample to flow through the first filtering area (3) to remove impurities in the sample, and then flowing through the second filtering area (8) to entrap bacteria in the sample;
(2) Centrifuging the disc microfluidic chip in a certain direction to enable the first reagent in the first reagent zone (16) to flow into the second filtering zone (8);
(3) Rotating the disk-type microfluidic chip in the forward and reverse directions to mix the first reagent with the bacteria;
(4) Centrifuging the disc-type microfluidic chip in the direction opposite to the direction of the step (2) to enable the mixed solution of the first reagent and the bacteria to flow to a first chamber (12) and a second chamber (14) of a microfluidic channel path in sequence, and detecting chemiluminescent signals after incubation.
18. The method according to claim 17, wherein: the reagent stored in the first chamber (12) is phage freeze-dried powder; the reagent stored in the second chamber (14) comprises a luminescent substrate; wherein the phage is phage with gene capable of expressing fluorescent protein.
19. The method according to claim 18, wherein: the incubation time of the phage and the bacteria is 10-60 min.
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