CN111482207A - Disc type micro-fluidic chip and using method - Google Patents
Disc type micro-fluidic chip and using method Download PDFInfo
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- CN111482207A CN111482207A CN202010370201.6A CN202010370201A CN111482207A CN 111482207 A CN111482207 A CN 111482207A CN 202010370201 A CN202010370201 A CN 202010370201A CN 111482207 A CN111482207 A CN 111482207A
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- 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
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- 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 disc type microfluidic chip, which comprises a sample adding port for adding a sample, a filtering area communicated with the sample adding port and provided with a filter membrane, and at least one microfluidic channel path communicated with the filtering area, wherein the microfluidic channel path comprises at least one chamber for storing at least one reagent. The micro-fluidic chip has the advantages of low reagent consumption, high analysis speed, simple operation process, convenient integration and the like, and the sample flow on the chip is controllable, so the micro-fluidic chip is particularly suitable for the rapid detection of pathogenic microorganisms and can simultaneously detect various pathogenic microorganisms.
Description
Technical Field
The invention particularly relates to a disc type micro-fluidic chip and a using method thereof.
Background
The traditional method for detecting bacteria in a biological sample has the disadvantages of complicated steps, long detection time and easy pollution of detection reagents. The micro-fluidic chip is mainly characterized in that the fluid is controlled in a micron-scale space, basic operation units such as sample biochemical reaction, separation, detection and the like can be integrated in a chip system, and a network is formed by micro-channels so that the controllable fluid can penetrate through the whole system, thereby realizing various functions of a conventional laboratory.
The micro-fluidic chip technology and the pathogenic microorganism detection technology are combined to develop the micro-fluidic chip which can be used for rapidly and quantitatively detecting bacteria in a biological sample, a simple, rapid and effective solution is provided for rapidly diagnosing the pathogenic microorganism, and the micro-fluidic chip has important significance for the treatment and prognosis of diseases.
Disclosure of Invention
The invention aims to provide a disc type micro-fluidic chip which has the advantages of low reagent consumption, high analysis speed, simple operation process, convenient integration, controllable flow of a biological sample on the chip, no pollution in the chip due to a closed environment and particular suitability for rapid quantitative detection of bacteria in the biological sample.
In order to achieve the purpose, the invention adopts the technical scheme that:
in a first aspect, the present invention provides a disc microfluidic chip comprising a sample addition port for adding a sample, a filtration region in communication with the sample addition port and provided with a filter membrane, and at least one microfluidic channel pathway in communication with the filtration region, the microfluidic channel pathway comprising at least one chamber for storing at least one reagent. The disc microfluidic chip is particularly suitable for detecting bacteria in biological samples such as urine.
Preferably, the filtration zone comprises a first filtration zone in communication with the sample addition port and provided with a first filter membrane, and a second filtration zone in communication with the first filtration zone and provided with a second filter membrane, the second filtration zone being in communication with the microfluidic channel pathway.
Further preferably, the pore size of the first filter is larger than the pore size of the second filter.
Further preferably, said first filter allows the passage of bacteria and is capable of retaining impurities in a biological sample having a volume greater than that of said bacteria, and said second filter is capable of retaining said bacteria.
More preferably, the aperture of the first filter membrane is 1-100 μm, preferably 1-50 μm, and more preferably 1-10 μm; the aperture of the second filter membrane is 0.01-1 μm, preferably 0.01-0.5 urn, and more preferably 0.1-0.3 μm. The first filter membrane is used for roughly filtering the biological sample, 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 second filter membrane is used for finely filtering the biological sample, and the bacteria in the biological sample are intercepted 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 arranged in one first filtering area along the flow direction of the sample passing through the first filtering area, and the two or more first filter membranes are arranged in parallel. Set up two and above first filter membrane, carry out the coarse filtration twice to biological sample, the impurity that volume is greater than the bacterium in the biological sample is held back as much as possible, further effectively prevents that the runner from blockking up and improving the purity of the bacterium held back on the second filter membrane, avoids on the second filter membrane impurity too much and influences the accuracy of testing result.
More preferably, the pore size of the first filter membrane in two or more of the first filtration zones or the pore size of two or more of the first filter membranes in one of the first filtration zones is different, and the pore size of the first filter membrane located downstream in the sample flow direction is smaller than the pore size of the first filter membrane located upstream in the sample flow direction.
Because biological samples such as urine have 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 easily occur when the biological samples such as urine are detected by adopting the microfluidic chip.
More preferably, the pore size of the first filter membrane positioned at the upstream of the sample flowing direction is 2-100 μm; the aperture of the first filter membrane positioned at the downstream of the sample flowing direction is 1-2 μm, 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, therefore, on one hand, the coarse filtration effect can be further improved, and on the other hand, the blocking of a flow channel can be better avoided.
Further preferably, the ratio of the sectional area of the section where the first filter membrane of the first filter area is located to the sectional area of the section where the second filter membrane of the second filter area is located is 3.5-4.5: 1, so that the enrichment of bacteria can be better realized, the space can be saved, the size of the chip can be reduced, and the cost of the chip can be reduced.
Preferably, the disc microfluidic chip further comprises a first reagent zone communicated with the filtering zone and storing a first reagent.
Preferably, the microfluidic channel path comprises a first chamber and a second chamber arranged in sequence radially outward from the center of the disc microfluidic chip, the first chamber is communicated with the filtering region, the second chamber is communicated with the first chamber, and at least one of the first chamber and the second chamber stores at least one reagent.
Preferably, the sample is a biological sample, the first reagent is a culture medium, the phage freeze-dried powder is stored in the first chamber, and the detection reagent is stored in the second chamber.
Preferably, said first chamber and said second chamber are each circular.
Preferably, the microfluidic channel path includes a plurality of microfluidic channel paths connected in series, and the plurality of 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 region through a flow channel, the flow channel includes a main flow channel with one end communicated with the filtering region, and branch flow channels respectively communicated with the main flow channel and a first chamber of the microfluidic channel path, the main flow channel is arc-shaped, and the center of circle of the main flow channel coincides with the center of circle of the disc 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 simultaneously, one centrifugation step can be reduced, and the energy consumption is reduced. Further, the present invention avoids liquid back flow into the microfluidic channel path by the design of the primary and secondary channels.
More preferably, the disc microfluidic chip further comprises an exhaust hole communicated with the other end of the main channel.
Preferably, the filtration zone comprises a first filtration zone communicated with the sample addition port and provided with a first filter membrane, and a second filtration zone communicated with the first filtration zone and provided with a second filter membrane, wherein the second filtration zone is communicated with the first chamber; the disc type microfluidic chip also comprises a first reagent area which is communicated with the second filtering area and is stored with a first reagent; when the disc type microfluidic chip is centrifuged according to one direction, the first reagent in the first reagent area flows into the second filtering area; when the disc 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 arranged in sequence radially outward from the center of the disc microfluidic chip, the sample addition port is communicated with the first filtering region through a first flow channel, the bottoms of the first filtering region and the second filtering region are communicated through a second flow channel, the bottom of the first reagent region is communicated with the bottom of the second filtering region through a third flow channel, and the bottom of the second filtering region is communicated with the inner bottom of the first chamber of the microfluidic channel path through a fourth flow channel; in each of the microfluidic channel paths, the top of the outer side of the first chamber is communicated with the top of the inner side of the second chamber through a fifth flow channel.
Preferably, the first reagent area is arc-shaped, the center of the arc 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 located on 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 has a cross-sectional area which gradually decreases from bottom to top, and a filtering area which is positioned above the mixing area, and the second filtering membrane is arranged between the mixing area and the filtering area.
Preferably, at least the part of the disc microfluidic chip, which is in contact 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 the adhesion of the sample or the reagent in a flow channel or each area can be effectively prevented, and the detection accuracy is improved.
Preferably, the thickness of the disc type micro-fluidic chip is 8-12 mm, and the inner diameter of a flow channel on the disc type micro-fluidic chip is 1-2 mm.
Preferably, the disc microfluidic chip further comprises a plurality of through holes for weight reduction and for controlling the center of gravity of the disc microfluidic chip. The gravity center of the disc type micro-fluidic chip is positioned at the circle center, and the disc type micro-fluidic chip can be directly operated on a computer to carry out centrifugal operation, so that the balancing step is omitted, the disc type micro-fluidic chip is safer and more convenient to centrifuge, the weight of the chip is reduced, and the energy consumption can be reduced.
The disc type microfluidic chip comprises a first soft film layer, a hard film layer and a second soft film layer which are sequentially stacked, wherein the hard film layer is made of a material which does not react with a biological sample and a reagent, 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 adopted.
In the invention, the disc type micro-fluidic chip can also be provided with a quality control area.
The disc type microfluidic chip can adopt a centrifuge and a luminescence detector in the prior art to carry out centrifugation and signal detection.
Another aspect of the present invention provides a method for quantitatively detecting the content of bacteria in a sample by using a disc microfluidic chip, comprising the following steps:
(1) adding a sample to the sample addition port and allowing the sample to flow through the filtration zone;
(2) and centrifuging the disc microfluidic chip to enable the materials in the filtering area to enter the chamber of the microfluidic channel path and enable bacteria to react with the reagent to generate a detection signal.
Preferably, the method comprises the following specific steps:
(1) adding a sample to the sample port and allowing the sample to flow through the first filtration zone to remove impurities from the sample and then through the second filtration zone to retain bacteria from the sample;
(2) centrifuging the disc microfluidic chip in a certain direction to make the first reagent in the first reagent area flow into the second filtering area;
(3) rotating the disc microfluidic chip in the forward direction and the reverse direction to mix the first reagent with the bacteria;
(4) and (3) centrifuging the disc type microfluidic chip in the direction opposite to the direction in the step (2) to enable the mixed solution of the first reagent and the bacteria to sequentially flow to a first chamber and a second chamber of a microfluidic channel path, and detecting a chemiluminescent signal after incubation.
Further preferably, the first reagent is one or more of PBS buffer solution, MES buffer solution, L B culture medium, beef extract peptone culture medium and phage buffer solution, preferably L B culture medium and beef extract peptone culture medium.
Further preferably, the reagent stored in the first chamber is a lyophilized phage powder.
Further preferably, the reagent stored in the second chamber comprises a luminescent substrate.
In the invention, the phage is a phage with a gene capable of expressing fluorescent protein; the method for introducing the specific gene into the phage is a conventional method in the field, wherein the specific gene can express a specific fluorescent protein and react with a specific luminescent substrate, so that the luminescence of the catalytic reaction solution is realized; for example, the lux gene can express 1ux protein, catalyze decanal to generate oxidation-reduction reaction, and further generate optical signals; while the nanoluc gene reacts with the substrate Furimazine to emit light. For the selection of phage, it is determined according to the species of bacteria to be detected, for example, K1F phage exclusively harbors E.coli, and therefore, when E.coli is to be detected, K1F phage is used; the Felix O1 phage is specific for Salmonella, and thus can be used to detect Salmonella.
In the invention, the phage comprises K1F phage, Felix O1 phage and the like, specific phage is determined according to bacteria to be detected, the fluorescent protein comprises L ux protein, GFP protein, Nano L uc protein and the like, and the luminescent substrate comprises aldehyde such as decanal, aromatic aldehyde and the like, imidazopyrazine (such as Furimazine) and the like.
Further preferably, the incubation time of the bacteriophage and the bacteria is 10-60 min. The use method of the invention adopts the disc type micro-fluidic chip to detect bacteria.
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages: the disc type microflow chip provided by the invention has the advantages of low reagent consumption, high analysis speed, simple operation process, convenient integration and the like, the flow of a sample on the chip is controllable, the diameter of the chip is small, the cost of the chip is low, the disc type microflow chip is particularly suitable for the rapid detection of pathogenic microorganisms, and various pathogenic microorganisms can be detected simultaneously.
Drawings
Fig. 1 is a perspective view of a disc-type microfluidic chip of example 1;
fig. 2 is a top view of the disc-type microfluidic chip of example 1.
In the above drawings: 1. a sample addition 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 channel; 11. a shunt channel; 12. a first chamber; 13 a fifth flow channel; 14. a second chamber; 15. a quality control region; 16. a first reagent zone; 17. a third flow path; 18. a sixth flow path; 19. a through hole; 20. and (4) a bayonet.
Detailed Description
The invention will be further described with reference to examples of embodiments shown in the drawings to which the invention is attached. The invention will be further described with reference to examples of embodiments shown in the drawings to which the various features may be combined as required, unless the context clearly dictates otherwise.
Example 1
This embodiment is defined in the orientation of fig. 1, wherein the sample addition zone has its opening facing downward.
As shown in FIGS. 1 and 2, a disk type microfluidic chip comprises a sample addition port 1 for adding a sample, a first filtering section 3 communicating with the sample addition port 1 through a first flow channel 2 and provided with a first filter 5 and a second first filter 4, a second filtering section 8 communicating with the first filtering section 3 through a second flow channel 6 and provided with a second filter 9, the disc-type micro-fluidic chip comprises a first filtering area 8, a first reagent area 16 communicated with the second filtering area 8 through a third flow channel 17 and storing a first reagent, and at least one micro-fluidic channel path communicated with the second filtering area 8, wherein each micro-fluidic channel path comprises a first chamber 12 and a second chamber 14 which are sequentially arranged from the center of the disc-type micro-fluidic chip to the outside in the radial direction, the first chamber 12 in each micro-fluidic channel path is communicated with the second filtering area 8 through a fourth flow channel, and the second chamber 14 is communicated with the first chamber 12 through a fifth flow channel 13.
Specifically, as shown in fig. 1 and fig. 2, the sample addition port 1 is located on the lower surface of the disc microfluidic chip, the first flow channel 2 includes a first portion extending in the up-down direction and having a lower end communicating with the sample addition port 1, a second portion extending in the left-right direction and having an inner end communicating with the upper end of the first portion, a third portion extending in the up-down direction and having an upper end communicating with the outer end of the second portion, and a fourth portion extending in the left-right direction and having an inner end communicating with the lower end of the third portion, and the outer end of the fourth portion communicates with the bottom of the first filtering region 3; through setting first flow channel 2 to the structure of so tortuous, can be so that when the application of sample to application of sample port 1, can directly not cause too big local pressure to first filter membrane.
The second flow channel 6 comprises a fifth part of which the outer end is communicated with the top of the first filtering area 3 and extends along the left-right direction, a sixth part of which the upper end is communicated with the inner end of the fifth part and extends along the up-down direction, and a seventh part of which one end is communicated with the lower end of the sixth part and extends along the left-right direction, and the other end of the seventh part is communicated with the bottom of the second filtering area 8.
Two 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, and the third flow channel 17 is arc-shaped and the extension direction of the arc is consistent with that of the first reagent area 16.
The bottom of the second filter zone 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 comprises a main flow channel 10 and a plurality of branch flow channels 11 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 and the circle center of the disc type micro-fluidic chip are coincident when viewed from a horizontal projection, and the branch flow channels 11 are uniformly distributed along the length direction of the main flow channel 10.
The outer side of the branch flow passage 11 communicates with the inner bottom of the first chamber 12, and the outer top of the first chamber 12 communicates with the inner top of the second chamber 14 through a fifth flow passage 13.
The top of the second filtering section 8 is also communicated with one end of a sixth flow passage 18, and the other end of the sixth flow passage 18 is communicated with an outlet.
The first filter 5 and the second filter 4 are of a pore size such as to allow the passage of bacteria and to retain impurities in a biological sample having a volume greater than that of the bacteria, and the second filter 9 is of a pore size such as to retain said bacteria. In this embodiment, the first filtering area 3 is provided with a second first filter membrane 4 and a first filter membrane 5 respectively along the up-down direction, the pore diameters of the second first filter membrane 4 and the first filter membrane 5 may be the same or different, the pore diameters of the second first filter membrane 4 and the first filter membrane 5 are independently 1-100 μm, for further improving the rough filtering effect, the pore diameter of the first filter membrane 5 located below is preferably 2-100 μm, in this embodiment 2-5 μm, and the pore diameter of the second first filter membrane 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 sectional area of the section where the first filter membrane of the first filter area 3 is located is 3.5-4.5 times of the sectional area of the section where the second filter membrane of the second filter area 8 is located.
The second filtering area 8 comprises a mixing area 7 with a sectional area gradually reduced from bottom to top and a filtering area positioned above the mixing area 7, the second filtering membrane 9 is arranged between the mixing area 7 and the filtering area, bosses for fixing the second filtering membrane 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 membrane 9, the aperture of the second filtering membrane 9 is preferably 0.01-1 mu m, the second filtering membrane is 0.22 mu m in the embodiment, bacteria can be intercepted and enriched to the greatest extent, and the accuracy of detection is improved.
The microfluidic channel path comprises 5 microfluidic channel paths which are connected in series in sequence, 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 a detection reagent is stored in the second chamber 14, wherein different phage freeze-dried powder can be stored in the first chamber 12 of each microfluidic channel path, for example, the first chamber 12 can store K1F phage introduced with L ux gene, the first second chamber 14 can store decanal solution so as to detect Escherichia coli, the second first chamber 12 can store Felix O1 phage introduced with L ux gene, and the second chamber 14 can store decanal solution so as to detect Salmonella.
The first reagent area 16 is arc-shaped, the center of the circle coincides with the center of the disc-type microfluidic chip on the horizontal projection, and the first reagent stored in the first reagent area 16 is a culture medium. The first filter zone 3, the second filter zone 8 and the microfluidic channel path are located at the periphery of the first reagent zone 16. And 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 micro-fluidic chip is located in the circle center, the disc type micro-fluidic chip can be directly operated on a computer to carry out centrifugal operation, a balancing step is omitted, the disc type micro-fluidic chip is safer and more convenient to centrifuge, the weight of the chip is reduced by arranging 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 micro-fluidic chip is preferably 8-12 mm, the inner diameter of a flow channel on the disc type micro-fluidic chip is preferably 1-2 mm, the thickness setting in the embodiment is the most preferable thickness obtained according to the setting of the internal structure of the micro-fluidic chip, the disc type micro-fluidic chip has the smallest volume while achieving the best detection effect, and the manufacturing cost is saved.
At least the part of the disc type micro-fluidic chip, which is in contact with the sample or the reagent, is treated by a hydrophobic material, and in order to simplify the operation, the hard film layer of the disc type micro-fluidic chip can also be subjected to hydrophobic treatment, so that the flow rate reduction and incomplete sample reaction caused by the adhesion of the sample or the reagent in a flow channel or each area can be prevented, and the detection accuracy is improved.
In this embodiment, a plurality of bayonets 20 for fixing during centrifugation are further provided on the disc-type microfluidic chip.
In this embodiment, the disc-type microfluidic chip is disc-shaped.
When the disc microfluidic chip is centrifuged in one direction, the first reagent in the first reagent zone 16 flows into the second filter zone 8; when the disc microfluidic chip is centrifuged in the direction opposite to the above direction, the material in the second filter region 8 flows into the microfluidic channel path. If the layout of the parts on the chip shown in fig. 1 and fig. 2 of this embodiment is adopted, a syringe can be inserted into the sample port 1 for sample application, a 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 microfluidic chip is centrifuged on the disc microfluidic chip, the culture medium is firstly centrifuged counterclockwise to flow through the second filtering film 9 and flow into the mixing area 7, the bacteria are eluted from the second filtering film 9, then the bacteria are mixed by shaking at low speed forward and reverse rotation, then the culture medium containing the bacteria is centrifuged clockwise to flow into the first chamber 12 to contact and react the bacteria and the phage, the reactant of the bacteria and the phage is centrifuged clockwise again to flow into the second chamber 14 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 a 10m L urine sample is added through the syringe inserted into the sample addition port 1, the sample flows through the first filtering area 3 and the second filtering area 8 and then flows out from the outlet;
2. centrifuging counterclockwise to cause the medium to flow into the mixing zone 7;
3. vibrating and mixing the bacteria and the culture medium at low speed in positive and negative rotation;
4. centrifuging clockwise to allow the liquid containing the bacteria in the medium to flow into the first chamber 12, allowing the bacteria to react with the phage for 15 min;
5. the clockwise centrifugation causes the bacterial and phage reactants to flow into the second chamber 14 and react with the detection reagent, and the chemiluminescent signal is detected.
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (26)
1. A disc microfluidic chip is characterized in that: the device comprises a sample adding port (1) for adding a sample, a filtering area which is communicated with the sample adding port (1) and is provided with a filter membrane, and at least one microfluid channel path which is communicated with the filtering area, wherein the microfluid channel path comprises at least one chamber for storing at least one reagent.
2. The disc microfluidic chip of claim 1, wherein: the filtration zone comprises a first filtration zone (3) which is communicated with the sample adding port (1) and is provided with a first filter membrane, and a second filtration zone (8) which is communicated with the first filtration zone (3) and is provided with a second filter membrane (9), wherein the second filtration zone (8) is communicated with the micro-fluid channel path.
3. The disc microfluidic chip of claim 2, wherein: the pore size of the first filter membrane is larger than that of the second filter membrane (9).
4. The disc microfluidic chip according to claim 2 or 3, wherein: the aperture of the first filter membrane is 1-100 mu m; the aperture of the second filter membrane (9) is 0.01-1 μm.
5. The disc microfluidic chip of claim 2, 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 membranes are two or more arranged along the flow direction of the sample passing through the first filtering area (3), and the two or more first filter membranes are arranged in parallel.
6. The disc microfluidic chip of claim 5, wherein: the pore size of the first filter membrane in two or more of the first filtration zones (3) is different or the pore size of two or more of the first filter membranes is different, and the pore size of the first filter membrane located downstream in the sample flow direction is smaller than the pore size of the first filter membrane located upstream in the sample flow direction.
7. The disc microfluidic chip of claim 6, wherein: the pore diameter of the first filter membrane positioned at the upstream of the sample flowing direction is 2-100 mu m; the aperture of the first filter membrane positioned at the downstream of the sample flowing direction is 1-2 μm.
8. The disc microfluidic chip of claim 2, wherein: the ratio of the sectional area of the section where the first filter membrane of the first filtering area (3) is located to the sectional area of the section where the second filter membrane (9) of the second filtering area (8) is located is 3.5-4.5: 1.
9. The disc microfluidic chip of claim 1, wherein: the disc type microfluidic chip also comprises a first reagent area (16) which is communicated with the filtering area and stores a first reagent.
10. The disc microfluidic chip according to claim 1 or 9, wherein: the microfluidic channel path comprises a first chamber (12) and a second chamber (14) which are arranged in sequence from the center of the disc microfluidic chip to the outside in the radial direction, 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) is stored with at least one reagent.
11. The disc microfluidic chip of claim 10, wherein: the sample is a biological sample, the first reagent is a culture medium, phage freeze-dried powder is stored in the first chamber (12), and a detection reagent is stored in the second chamber (14).
12. The disc microfluidic chip of claim 1, wherein: the microfluidic channel path comprises a plurality of microfluidic channel paths which are connected in series in sequence, and the plurality of microfluidic channel paths are distributed along the circumferential direction of the disc type microfluidic chip.
13. The disc microfluidic chip according to claim 1 or 12, wherein: the micro-fluid channel path is communicated with the filtering area through a flow channel, the flow channel comprises a main flow channel (10) and a branch flow channel (11), one end of the branch flow channel is communicated with the filtering area, the branch flow channel is respectively communicated with the main flow channel (10) and a first chamber (12) of the micro-fluid channel path, the main flow channel (10) is arc-shaped, and the circle center of the main flow channel (10) is coincided with the circle center of the disc type micro-fluid control chip.
14. The disc microfluidic chip of claim 13, wherein: the disc type micro-fluidic chip also comprises an exhaust hole communicated with the other end of the main runner (10).
15. The disc microfluidic chip of claim 1, wherein: 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, and a second filtering area (8) which is communicated with the first filtering area (3) and is provided with a second filter membrane (9), and the second filtering area (8) is communicated with the first chamber (12); the disc type microfluidic chip also comprises a first reagent area (16) which is communicated with the second filtering area (8) and stores a first reagent; when the disc type microfluidic chip is centrifuged according to one direction, the first reagent in 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 filter region (8) flows into the microfluidic channel path.
16. The disc microfluidic chip of claim 15, wherein: the microfluidic channel path comprises a first chamber (12) and a second chamber (14) which are sequentially arranged from the center of the disc microfluidic chip to the outside in the radial direction, 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 bottom of the first reagent area (16) is communicated with the bottom of the second filtering area (8) 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 of the microfluidic channel paths, the outer top of the first chamber (2) communicates with the inner top of the second chamber (14) via a fifth flow channel (13).
17. The disc microfluidic chip of claim 15, wherein: the first reagent area (16) is arc-shaped, the circle center of the first reagent area coincides with the center of the disc type microfluidic chip, and the first filtering area (3), the second filtering area (8) and the microfluidic channel path are positioned on the periphery of the first reagent area (16).
18. The disc microfluidic chip of claim 1, wherein: the disc type micro-fluidic chip also comprises a flow channel communicated with the top of the filtering area and an outlet communicated with the flow channel.
19. The disc microfluidic chip of claim 2, wherein: the second filtering area (8) comprises a mixing area (7) which is positioned at the bottom and the cross-sectional area of which is gradually reduced from bottom to top and a filtering area which is positioned above the mixing area (7), and the second filtering membrane is arranged between the mixing area (7) and the filtering area.
20. The disc microfluidic chip of claim 1, wherein: at least the part of the disc type microfluidic chip, which is in contact with the sample or the reagent, is treated by a hydrophobic material.
21. The disc microfluidic chip of claim 1, wherein: the thickness of the disc type micro-fluidic chip is 8-12 mm, and the inner diameter of a flow channel on the disc type micro-fluidic chip is 1-2 mm.
22. The disc microfluidic chip of claim 1, wherein: the disc microfluidic chip also comprises a plurality of through holes (19) for weight reduction and for controlling the gravity center of the disc microfluidic chip.
23. A method for quantitatively detecting the content of bacteria in a sample by adopting a disc type microfluidic chip is characterized by comprising the following steps: the method comprises the following steps:
(1) adding a sample to the sample addition port (1) and allowing the sample to flow through the filtration zone;
(2) and centrifuging the disc microfluidic chip to enable the materials in the filtering area to enter the chamber of the microfluidic channel path and enable bacteria to react with the reagent to generate a detection signal.
24. The method of claim 23, wherein: the method comprises the following specific steps:
(1) adding a sample to the sample addition port (1) and allowing the sample to flow through the first filtration zone (3) to remove impurities from the sample and then through the second filtration zone (8) to entrap bacteria in the sample;
(2) centrifuging said disc microfluidic chip in a direction to cause said first reagent in said first reagent zone (16) to flow into said second filtration zone (8);
(3) rotating the disc microfluidic chip in the forward direction and the reverse direction to mix the first reagent with the bacteria;
(4) and (3) centrifuging the disc type microfluidic chip in the direction opposite to the direction in the step (2) to enable the mixed solution of the first reagent and the bacteria to sequentially flow to a first chamber (12) and a second chamber (14) of a microfluidic channel path, and detecting a chemiluminescent signal after incubation.
25. The method according to claim 24, wherein the first reagent is one or more of PBS buffer, MES buffer, L B medium, beef extract peptone medium, and phage buffer, the reagent stored in the first chamber (12) is phage lyophilized powder, the reagent stored in the second chamber (14) comprises a luminescent substrate, and the phage is a phage having a gene capable of expressing a fluorescent protein.
26. The method of claim 25, wherein: the incubation time of the phage and the bacteria is 10-60 min.
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