阅读说明：本技术 一种用于生物样本中细菌定量检测的微流控芯片及使用方法 (Micro-fluidic chip for quantitatively detecting bacteria in biological sample and use method ) 是由 颜菁 申炳阳 刘文佳 王磊 于 2020-04-30 设计创作，主要内容包括：本发明涉及一种用于生物样本中细菌定量检测的微流控芯片,包括用于加入所述的生物样本的加样区、与所述的加样区相连通且设置有第一滤膜的第一过滤区、与所述的第一过滤区相连通且设置有第二滤膜的第二过滤区、与所述的第二过滤区相连通的检测区、与所述的第二过滤区相连通且用于加入用于将所述的细菌自所述的第二滤膜上洗脱下来的试剂的第一试剂区、与所述的检测区相连通的第二试剂区。本发明的微流控芯片,试剂消耗量低、分析速度快,同时还具有操作过程简单、便于集成化等优点,生物样本在芯片上的流动可控,且封闭的环境证芯片内部不受污染,特别适合于病原微生物的快速检测。(The invention relates to a micro-fluidic chip for quantitatively detecting bacteria in a biological sample, which comprises a sample adding area for adding the biological sample, a first filtering area communicated with the sample adding area and provided with a first filtering membrane, a second filtering area communicated with the first filtering area and provided with a second filtering membrane, a detection area communicated with the second filtering area, a first reagent area communicated with the second filtering area and used for adding a reagent for eluting the bacteria from the second filtering membrane, and a second reagent area communicated with the detection area. The micro-fluidic chip has the advantages of low reagent consumption, high analysis speed, simple operation process, convenient integration and the like, the flow of a biological sample on the chip is controllable, the inside of the closed environmental card chip is not polluted, and the micro-fluidic chip is particularly suitable for the rapid detection of pathogenic microorganisms.)

1. A micro-fluidic chip for quantitative detection of bacteria in a biological sample is characterized in that: the device comprises a sample adding area for adding the biological sample, a first filtering area communicated with the sample adding area and provided with a first filter membrane, a second filtering area communicated with the first filtering area and provided with a second filter membrane, a detection area communicated with the second filtering area, a first reagent area communicated with the second filtering area and used for adding a reagent for eluting the bacteria from the second filter membrane, and a second reagent area communicated with the second filtering area and/or the detection area; wherein the first filter membrane allows bacteria to pass through and can retain impurities in a biological sample with a volume larger than that of the bacteria, and the second filter membrane can retain the bacteria.

2. The microfluidic chip for the quantitative detection of bacteria in a biological sample according to claim 1, wherein: the aperture of the first filter membrane is 1-100 mu m; the aperture of the second filter membrane is 0.01-1 μm.

3. The microfluidic chip for the quantitative detection of bacteria in a biological sample according to claim 1 or 2, wherein: the number of the first filtering areas is two or more, the two or more first filtering areas are arranged in series, and each first filtering area is provided with the first filtering membrane; or, the first filter membranes are two or more arranged along the flow direction of the biological sample passing through the first filtering area, and the two or more first filter membranes are arranged in parallel.

4. The microfluidic chip for the quantitative detection of bacteria in a biological sample according to claim 3, wherein: the pore sizes of the first filter membranes in two or more first filtering regions are different from each other, and the pore size of the first filter membrane located downstream in the biological sample flow direction is smaller than the pore size of the first filter membrane located upstream in the biological sample flow direction.

5. The microfluidic chip for the quantitative detection of bacteria in a biological sample according to claim 4, wherein: the aperture of the first filter membrane positioned at the upstream of the flow direction of the biological sample is 2-100 mu m; the aperture of the first filter membrane positioned at the downstream of the flow direction of the biological sample is 1-2 μm.

6. The microfluidic chip for the quantitative detection of bacteria in a biological sample according to claim 1, wherein: the ratio of the inner diameter of the first filtering area to the inner diameter of the second filtering area is 1.5-2.5: 1.

7. The microfluidic chip for the quantitative detection of bacteria in a biological sample according to claim 1, wherein: the second filtering area is arranged separately from the detection area.

8. The microfluidic chip for the quantitative detection of bacteria in a biological sample according to claim 1, wherein: the thickness of the microfluidic chip is 8-12 mm, and the inner diameter of a flow channel on the microfluidic chip is 1-2 mm.

9. The microfluidic chip for the quantitative detection of bacteria in a biological sample according to claim 1, wherein: the microfluidic chip also comprises a third reagent area communicated with the second filtering area and/or the detection area.

10. The microfluidic chip for the quantitative detection of bacteria in a biological sample according to claim 1 or 9, wherein: the reagent zone is a sample addition port, or the reagent zone comprises a reagent cabin storing a reagent.

11. The microfluidic chip for the quantitative detection of bacteria in a biological sample according to claim 1, wherein: the microfluidic chip also comprises an injector for adding samples or reagents.

12. The microfluidic chip for the quantitative detection of bacteria in a biological sample according to claim 1, wherein: the microfluidic chip also comprises a waste liquid area communicated with the second filtering area and/or the detection area.

13. The microfluidic chip for the quantitative detection of bacteria in a biological sample according to claim 12, wherein: the waste liquid district be the appearance mouth that goes out, perhaps, the waste liquid district including be used for storing the waste liquid storehouse, be used for with waste liquid storehouse and external air vent that communicates.

14. The microfluidic chip for the quantitative detection of bacteria in a biological sample according to claim 1, wherein: the microfluidic chip comprises at least one hard film layer and at least one soft film layer which are arranged in a stacked mode.

15. The microfluidic chip for the quantitative detection of bacteria in a biological sample according to claim 14, wherein: the microfluidic chip comprises a first hard film layer, a second hard film layer, a first soft film layer and a second soft film layer which are sequentially stacked and fixedly connected through an adhesive; the thicknesses of the first hard film layer and the second hard film layer are independently 3-5 mm, and the thicknesses of the first soft film layer and the second soft film layer are independently 0.5-1.5 mm; or the microfluidic chip comprises a first hard film layer, a first soft film layer, a second hard film layer, a second soft film layer and a third hard film layer which are sequentially stacked and fixedly connected through a connecting piece; the thickness of first hard coating layer the thickness of third hard coating layer independently be 1 ~ 3mm, the thickness of second hard coating layer be 2 ~ 4mm, first soft coating layer the thickness of second soft coating layer independently be 0.5 ~ 1.5 mm.

16. The microfluidic chip for the quantitative detection of bacteria in a biological sample according to claim 14 or 15, wherein: the sample adding area, the first filtering area, the second filtering area, the detection area, the first reagent area, the second reagent area and the flow channel for communicating the areas are arranged on the hard film layer, and the flow channel is provided with an extrusion valve which can control the flow channel to be opened and closed.

17. The microfluidic chip for the quantitative detection of bacteria in a biological sample according to claim 16, wherein: the flow channels comprise a first flow channel respectively communicated with the bottom of the sample adding area and the bottom of the first filtering area, a second flow channel respectively communicated with the top of the first filtering area and the first filtering area, a third flow channel respectively communicated with the bottom of the first filtering area and the second filtering area, a fourth flow channel respectively communicated with the bottom of the second filtering area and the bottom of the detection area, a fifth flow channel respectively communicated with the bottom of the first reagent area and the top of the second filtering area, a sixth flow channel respectively communicated with the bottom of the second reagent area and the bottom of the detection area, and a seventh flow channel respectively communicated with the top of the second filtering area and the top of the waste liquid area, the waste liquid area is arranged on the second hard film layer, the first soft film layer and the second soft film layer.

18. The microfluidic chip for the quantitative detection of bacteria in a biological sample according to claim 14, wherein: the microfluidic chip comprises a first hard film layer, a first soft film layer, a second hard film layer, a second soft film layer and a third hard film layer which are sequentially stacked and fixedly connected through a connecting piece; the thicknesses of the first hard film layer and the third hard film layer are independently 1-3 mm, the thickness of the second hard film layer is 2-4 mm, and the thicknesses of the first soft film layer and the second soft film layer are independently 0.5-1.5 mm; when the microfluidic chip is not added with samples or reagents, the first soft membrane layer and the second soft membrane layer are respectively arranged on the flow channel in a blocking manner to close the flow channel; when the microfluidic chip is used for adding samples or reagents, the first soft membrane layer and the second soft membrane layer deform under the action of pressure to open the flow channel.

19. The microfluidic chip for the quantitative detection of bacteria in a biological sample according to claim 18, wherein: the sample adding area is a first through hole formed in the first hard film layer, the first filtering area is a second through hole formed in the second hard film layer, the first through hole is located above the second through hole, the diameter of the first through hole is smaller than that of the second through hole, a plurality of third through holes are formed in the first soft film layer between the first through hole and the second through hole, and the third through holes and the first through hole are arranged in a staggered mode; the flow channel comprises a first flow channel which is arranged on the second hard film layer and/or the third hard film layer and one end of which is communicated with the bottom of the first filtering area, a second flow channel which is arranged on the second hard film layer and the lower end of which is communicated with the other end of the first flow channel, a third flow channel which is arranged on the first hard film layer and/or the second hard film layer and one end of which is communicated with the upper end of the second flow channel, the second filtering area is a fourth through hole which is arranged on the second hard film layer, the other end of the third flow channel is communicated with the top of the fourth through hole, the flow channel also comprises a fourth flow channel which is arranged on the second hard film layer and/or the third hard film layer and one end of which is communicated with the bottom of the fourth through hole, and a fifth flow channel which is arranged on the second hard film layer and the lower end of which is communicated with the other end of the fourth flow channel, the microfluidic chip also comprises a waste liquid area, wherein the waste liquid area is a fifth through hole formed in the first hard film layer, the fifth through hole is communicated with the fifth flow channel, a plurality of sixth through holes are formed in the first soft film layer between the fifth through hole and the fifth flow channel, and the sixth through holes and the fifth flow channel are arranged in a staggered mode; the flow channel also comprises a sixth flow channel which is arranged on the second hard film layer and the upper end of which is communicated with the first reagent area, and a seventh flow channel which is arranged on the second hard film layer and/or the third hard film layer and one end of which is communicated with the lower end of the sixth flow channel, wherein the other end of the seventh flow channel is communicated with the bottom of the fourth through hole; the flow channel also comprises an eighth flow channel which is arranged on the second hard film layer and the upper end of which is communicated with the second reagent area, a ninth flow channel which is arranged on the second hard film layer and one end of which is communicated with the lower end of the eighth flow channel, a tenth flow channel which is arranged on the third hard film layer and one end of which is communicated with the other end of the ninth flow channel, and an eleventh flow channel which is arranged on the second hard film layer and one end of which is communicated with the other end of the tenth flow channel, the other end of the eleventh flow channel is communicated with the bottom of the fourth through hole, the microfluidic chip also comprises a third reagent area which is arranged on the second hard film layer and is used for storing reagents, and when no reagent is added in the second reagent area, the part of the second soft film layer is positioned between the third reagent area and the tenth flow channel to prevent the reagent in the third reagent area from entering the tenth flow channel, when reagent is added into the second reagent zone, the second soft membrane layer deforms to enable the ninth flow channel, the tenth flow channel, the eleventh flow channel and the third reagent zone to be communicated.

20. The microfluidic chip for the quantitative detection of bacteria in a biological sample according to claim 19, wherein: the first reagent area is a seventh through hole formed in the second hard coating layer, a first reagent stored in the seventh through hole and a first piston connected with the seventh through hole in a sliding manner; the second reagent area is an eighth through hole formed in the second hard film layer, a second reagent stored in the eighth through hole, and a second piston connected with the eighth through hole in a sliding mode.

21. A method for quantitatively detecting the content of bacteria in a biological sample by adopting a microfluidic chip is characterized by comprising the following steps: the method comprises the following steps:

(1) adding the biological sample from the sample adding area of the microfluidic chip, allowing the biological sample to flow through a first filtering area to remove impurities in the biological sample, and then flowing through a second filtering area to trap bacteria in the biological sample;

(2) reacting the reagent with said bacteria and generating a detection signal.

22. The method of claim 21, wherein: the specific steps of the step (2) are as follows:

(a) adopting a cleaning solution or a buffer solution to flow through the second filtering area and enabling the bacteria trapped in the second filtering area to flow into the detection area together with the cleaning solution or the buffer solution;

(b) if the step (a) adopts a cleaning solution, adding a buffer solution and a reaction solution into the detection area; if a buffer solution is adopted in the step (a), adding a reaction solution into the detection area; and detecting a chemiluminescence signal after incubation.

23. The method of claim 21, wherein: the method further comprises a cleaning step between the step (1) and the step (2), wherein the cleaning step specifically comprises the following steps: and cleaning fluid flows through the first filtering area and the second filtering area in sequence.

24. The method of claim 22, wherein: the biological sample is urine; the cleaning solution is one or more of PBS buffer solution, MES buffer solution, LB culture medium and beef extract peptone culture medium; the buffer solution is a phage solution with genes capable of expressing fluorescent proteins; the reaction solution is a mixture of a luminescent substrate and the cleaning solution.

25. The method of claim 24, wherein: in the reaction solution, the volume ratio of the luminescent substrate to the cleaning solution is 1: 1-50.

26. The method of claim 21, wherein: the adding amount of the biological sample is 8-15 mL, the adding amount of the buffer solution is 10-200 mu L, and the adding amount of the reaction solution is 60-2000 mu L.

27. The method of claim 21, wherein: the incubation time of the buffer solution and the bacteria is 10-60 min.

Technical Field

The invention particularly relates to a micro-fluidic chip for quantitatively detecting bacteria in a biological sample and a using method thereof.

Background

The traditional method for detecting bacteria in a biological sample has the problems of complicated steps, long detection time, requirement of professional operation and the like, and the detection requirement of carrying out on-site, rapid, micro-quantitative and portable detection on additives is difficult to meet. 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 micro-fluidic chip which has the advantages of low reagent consumption, high analysis speed, simple operation process, convenient integration and controllable flow of a biological sample on the chip, and is particularly suitable for the rapid quantitative detection of bacteria in the biological sample.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides a micro-fluidic chip for quantitatively detecting bacteria in a biological sample, which comprises a sample adding area for adding the biological sample, a first filtering area communicated with the sample adding area and provided with a first filtering membrane, a second filtering area communicated with the first filtering area and provided with a second filtering membrane, a detection area communicated with the second filtering area, a first reagent area communicated with the second filtering area and used for adding a reagent for eluting the bacteria from the second filtering membrane, and a second reagent area communicated with the second filtering area and/or the detection area; wherein the first filter membrane has a pore size configured to allow bacteria to pass therethrough and to retain impurities in a biological sample having a volume greater than that of the bacteria, and the second filter membrane has a pore size configured to retain the bacteria.

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 μm, 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 in the detection area, and the accuracy of the detection result is ensured.

Preferably, the number of the first filtering areas is two or more, the two or more first filtering areas are arranged in series, and each first filtering area is provided with the first filtering membrane; or, the first filter membranes are arranged in two or more first filter areas along the flow direction of the biological sample passing through the first filter areas, 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.

Further preferably, the pore size of the first filter membrane in two or more of the first filter regions or the pore size of two or more of the first filter membranes in one of the first filter regions is different, and the pore size of the first filter membrane located downstream of the biological sample flow direction is smaller than the pore size of the first filter membrane located upstream of the biological 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 a microfluidic chip.

Further preferably, the pore size of the first filter membrane positioned at the upstream of the flowing direction of the biological sample is 2-100 μm; the aperture of the first filter membrane positioned at the downstream of the flow direction of the biological sample 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 second filter membrane with smaller aperture, thereby further improving the coarse filtration effect on one hand, and better avoiding the blocking of a flow channel on the other hand.

Preferably, the ratio of the inner diameter of the first filtering area to the inner diameter of the second filtering area is 1.5-2.5: 1, so that the enrichment of bacteria can be better realized, the space can be saved, the volume of the chip can be reduced, and the cost of the chip can be reduced.

Preferably, said second filtration zone coincides with said detection zone; alternatively, the second filtration zone is disposed separately from the detection zone. The detection area is a chamber for detecting the chemiluminescence intensity in the solution after the buffer solution and the reaction solution are added. If set up second filtering area and detection zone coincidence, can reduce the setting of part runner and detection pond, reduce chip inner structure complexity, nevertheless the interference that filter membrane etc. can be received to the testing result. If set up second filtering area and detection zone separately, then can avoid measuring time, because the second filtering area has the background interference that other debris such as filter membrane lead to, improve the accuracy of measuring to, owing to separately set up, can make the chip do thinly, thereby reduce cost.

Preferably, the thickness of the microfluidic chip is 8-12 mm, and the inner diameter of a flow channel on the microfluidic chip is 1-2 mm.

Preferably, the sample application region coincides with the first filtering region; or, the sample adding region and the first filtering region are separately arranged.

Preferably, the microfluidic chip further comprises a third reagent region communicated with the second filtering region and the detection region. By the arrangement of the third reagent zone, the washing solution, the buffer solution or the reaction solution can be stored separately.

According to one embodiment, the reagent zone is a sample port, and in this embodiment, the reagent is added through the sample port.

According to another embodiment, the reagent zone comprises a reagent bin for storing the reagent, so that the reagent is not required to be prepared for use on site, and only the reagent pre-stored in the reagent bin is required to be added into the corresponding chamber when the reagent is required to be added, and the use is more convenient.

The reagent chamber is a region for storing reagents required in the test process on the chip in advance, such as cleaning solution, buffer solution, reaction solution and the like.

Preferably, the microfluidic chip further comprises a syringe for loading or adding a reagent, so that the chip can be used in kit with the syringe without additional syringe dispensing by a user.

Preferably, the microfluidic chip further comprises a waste liquid region communicated with the second filtering region and/or the detection region.

Further preferably, the waste liquid area be outlet, perhaps, the waste liquid area including be used for storing the waste liquid storehouse of waste liquid, be used for with the waste liquid storehouse with the external air vent of intercommunication.

Preferably, the microfluidic chip comprises at least one hard film layer and at least one soft film layer which are arranged in a stacked manner.

In the invention, the hard coat layer is made of a material which does not react with the biological sample and the 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.

According to one embodiment, the microfluidic chip comprises a first hard film layer, a second hard film layer, a first soft film layer and a second soft film layer which are sequentially stacked and fixedly connected through an adhesive; the thicknesses of the first hard film layer and the second hard film layer are independently 3-5 mm, and the thicknesses of the first soft film layer and the second soft film layer are independently 0.5-1.5 mm; in this way, the microfluidic chip cannot be reused.

According to another embodiment, the microfluidic chip comprises a first hard film layer, a first soft film layer, a second hard film layer, a second soft film layer and a third hard film layer which are sequentially stacked and fixedly connected through a connecting piece; the thicknesses of the first hard film layer and the third hard film layer are independently 1-3 mm, the thickness of the second hard film layer is 2-4 mm, and the thicknesses of the first soft film layer and the second soft film layer are independently 0.5-1.5 mm; therefore, after the micro-fluidic chip is used, all layers can be disassembled and cleaned, and then the micro-fluidic chip is assembled again, so that the aim of recycling is fulfilled, and the cost can be saved.

Further preferably, the sample addition zone, the first filtering zone, the second filtering zone, the detection zone, the first reagent zone, the second reagent zone and a flow channel communicating the zones are arranged on the hard coat layer, and a squeezing valve capable of controlling the flow channel to be opened and closed is arranged on the flow channel.

According to a specific and preferred embodiment, the flow channels include a first flow channel respectively communicating with the bottom of the sample addition zone and the bottom of the first filtration zone, a second flow channel respectively communicating with the top of the first filtration zone and the top of the second filtration zone, a third flow channel respectively communicating with the bottom of the second filtration zone and the bottom of the first filtration zone, a fourth flow channel respectively communicating with the bottom of the second filtration zone and the bottom of the detection zone, a fifth flow channel respectively communicating with the bottom of the first reagent zone and the top of the second filtration zone, a sixth flow channel respectively communicating with the bottom of the second reagent zone and the bottom of the detection zone, and a seventh flow channel respectively communicating with the top of the second filtration zone and the top of the waste liquid zone, wherein the waste liquid zone is opened on the second hard film layer, the first flow channel, the second flow channel, the third flow channel, the fourth flow channel, the fifth flow channel, The first soft film layer and the second soft film layer.

According to a specific and preferred embodiment, the microfluidic chip comprises a first hard film layer, a first soft film layer, a second hard film layer, a second soft film layer and a third hard film layer which are sequentially stacked and fixedly connected through a connecting piece; the thickness of first hard coating layer the thickness of third hard coating layer independently be 1 ~ 3mm, the thickness of second hard coating layer be 2 ~ 4mm, first soft coating layer the thickness of second soft coating layer independently be 0.5 ~ 1.5 mm.

Preferably, when the microfluidic chip is not loaded with a sample or is not loaded with a reagent, the first soft membrane layer and the second soft membrane layer are respectively arranged on the flow channel in a blocking manner to close the flow channel; when the microfluidic chip is used for adding samples or reagents, the first soft membrane layer and the second soft membrane layer deform under the action of pressure to open the flow channel. The control flow channel is kept in a closed state in the normal state of the chip and after the chip is used, so that the reagent or the reaction liquid after reaction can be prevented from volatilizing into the air, the inside of the chip can be ensured to be in a closed environment, and the chip can be ensured not to be easily polluted.

Preferably, the sample adding region is a first through hole formed in the first hard film layer, the first filtering region is a second through hole formed in the second hard film layer, the first through hole is located above the second through hole, the diameter of the first through hole is smaller than that of the second through hole, a plurality of third through holes are formed in the first soft film layer between the first through hole and the second through hole, and the third through holes are arranged in a staggered manner with the first through holes; the flow channel comprises a first flow channel which is arranged on the second hard film layer and/or the third hard film layer and one end of which is communicated with the bottom of the first filtering area, a second flow channel which is arranged on the second hard film layer and the lower end of which is communicated with the other end of the first flow channel, a third flow channel which is arranged on the first hard film layer and/or the second hard film layer and one end of which is communicated with the upper end of the second flow channel, the second filtering area is a fourth through hole which is arranged on the second hard film layer, the other end of the third flow channel is communicated with the top of the fourth through hole, the flow channel also comprises a fourth flow channel which is arranged on the second hard film layer and/or the third hard film layer and one end of which is communicated with the bottom of the fourth through hole, and a fifth flow channel which is arranged on the second hard film layer and the lower end of which is communicated with the other end of the fourth flow channel, the microfluidic chip also comprises a waste liquid area, wherein the waste liquid area is a fifth through hole formed in the first hard film layer, the fifth through hole is communicated with the fifth flow channel, a plurality of sixth through holes are formed in the first soft film layer between the fifth through hole and the fifth flow channel, and the sixth through holes and the fifth flow channel are arranged in a staggered mode; the flow channel also comprises a sixth flow channel which is arranged on the second hard film layer and the upper end of which is communicated with the first reagent area, and a seventh flow channel which is arranged on the second hard film layer and/or the third hard film layer and one end of which is communicated with the lower end of the sixth flow channel, wherein the other end of the seventh flow channel is communicated with the bottom of the fourth through hole; the flow channel also comprises an eighth flow channel which is arranged on the second hard film layer and the upper end of which is communicated with the second reagent area, a ninth flow channel which is arranged on the second hard film layer and one end of which is communicated with the lower end of the eighth flow channel, a tenth flow channel which is arranged on the third hard film layer and one end of which is communicated with the other end of the ninth flow channel, and an eleventh flow channel which is arranged on the second hard film layer and one end of which is communicated with the other end of the tenth flow channel, the other end of the eleventh flow channel is communicated with the bottom of the fourth through hole, the microfluidic chip also comprises a third reagent area which is arranged on the second hard film layer and is used for storing reagents, and when no reagent is added in the second reagent area, the part of the second soft film layer is positioned between the third reagent area and the tenth flow channel to prevent the reagent in the third reagent area from entering the tenth flow channel, when reagent is added into the second reagent zone, the second soft membrane layer deforms to enable the ninth flow channel, the tenth flow channel, the eleventh flow channel and the third reagent zone to be communicated.

Still further preferably, the first reagent zone is a seventh through hole formed in the second hard coat layer, the first reagent stored in the seventh through hole, and a first piston slidably connected to the seventh through hole; the second reagent area is an eighth through hole formed in the second hard film layer, a second reagent stored in the eighth through hole, and a second piston connected with the eighth through hole in a sliding mode.

The invention also provides a method for quantitatively detecting the content of bacteria in a biological sample by adopting a microfluidic chip, which comprises the following steps:

(1) adding the biological sample from the sample adding area of the microfluidic chip, allowing the biological sample to flow through a first filtering area to remove impurities in the biological sample, and then flowing through a second filtering area to trap bacteria in the biological sample;

(2) reacting the reagent with said bacteria and generating a detection signal.

Preferably, the specific steps of step (2) are:

(a) adopting a cleaning solution or a buffer solution to flow through the second filtering area and enabling the bacteria trapped in the second filtering area to flow into the detection area together with the cleaning solution or the buffer solution;

(b) if the step (a) adopts a cleaning solution, adding a buffer solution and a reaction solution into the detection area; if a buffer solution is adopted in the step (a), adding a reaction solution into the detection area; and detecting a chemiluminescence signal after incubation.

Preferably, the method further comprises a cleaning step between the step (1) and the step (2), wherein the cleaning step specifically comprises: the cleaning liquid flows through the first filtering area and the second filtering area in sequence, so that the bacteria remained in the flow channel or the first filtering area can be better enriched by the second filtering area, and the impurities remained in the second filtering area can be better removed, thereby further improving the detection accuracy.

Preferably, the biological sample is urine.

Preferably, the cleaning solution is one or more of PBS buffer solution, MES buffer solution, LB culture medium and beef extract peptone culture medium.

Preferably, the buffer solution is a phage solution with genes capable of expressing fluorescent proteins, and the method for introducing specific genes into the phage is a conventional method in the field, wherein the specific genes can express specific fluorescent proteins and react with specific luminescent substrates to realize the luminescence of the catalytic reaction solution; for example, the lux gene can express lux 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.

Preferably, the reaction solution is a mixture of a luminescent substrate and the cleaning solution.

In the invention, the phage comprises K1F phage, Felix O1 phage and the like, and the specific phage is determined according to the bacteria to be detected; the fluorescent protein comprises a Lux protein, a GFP protein, a 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.

More preferably, in the reaction solution, the volume ratio of the luminescent substrate to the cleaning solution is 1: 1-50.

Preferably, the adding amount of the biological sample is 8-15 mL, the adding amount of the buffer solution is 10-200 muL, and the adding amount of the reaction solution is 60-2000 muL.

Preferably, the incubation time of the buffer solution and the bacteria is 10-60 min.

The flow of quantitative detection of bacteria in a biological sample by using the microfluidic chip of the present invention is shown in fig. 15 to 22, wherein fig. 15 to 18 are corresponding flow charts of a method of flowing a cleaning solution through the second filtering region and making bacteria trapped in the second filtering region flow into a detection region together with the cleaning solution; FIGS. 19 to 22 are corresponding flow charts of the method of flowing a buffer solution through the second filtration zone and allowing the bacteria trapped in the second filtration zone to flow into the detection zone along with the buffer solution. Fig. 15 and 19 are sample introduction flows, after a biological sample is added to a sample addition region, the biological sample is driven to flow through a first filtering region and a second filtering region in sequence and then flows into a waste liquid region, the first filtering region can retain substances such as cells and crystals with larger sizes in the sample, and the second filtering region retains and enriches bacteria in the biological sample. FIGS. 16 and 20 show a cleaning process in which the cleaning liquid flows into the waste liquid zone after passing through the first filtering zone and the second filtering zone in this order. FIGS. 17 and 21 show a washing and/or incubation sequence, wherein FIG. 17 uses a washing solution to wash the bacteria trapped on the second filter membrane of the second filtration zone to the detection zone, and a buffer solution and a reaction solution are added to the detection zone, as shown in FIG. 18; FIG. 21 shows the procedure of FIG. 22, in which the bacteria trapped on the second filter of the second filtration zone are washed with a buffer solution to the detection zone, and the reaction solution is added to the detection zone.

In the invention, the sample and the reagent can be driven to pass through the flow channel by positive pressure, negative pressure or a squeezing valve and the like.

Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:

the micro-fluidic chip has the advantages of low reagent consumption, high analysis speed, simple operation process, convenient integration and the like, and the flow of the biological sample 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.

Drawings

Fig. 1 is a perspective view of a microfluidic chip of example 1 of the present invention;

fig. 2 is a bottom view of the microfluidic chip of example 1 of the present invention;

FIG. 3 is a cross-sectional view of the microfluidic chip of example 1 of the present invention taken along A-A of FIG. 2;

FIG. 4 is a cross-sectional view of the microfluidic chip of example 1 of the present invention taken along line B-B of FIG. 2;

FIG. 5 is a cross-sectional view of the microfluidic chip of example 1 of the present invention taken along C-C of FIG. 2;

FIG. 6 is a cross-sectional view of the microfluidic chip of example 1 of the present invention taken along D-D in FIG. 2;

fig. 7 is a schematic structural view of a microfluidic chip according to example 2 of the present invention;

fig. 8 is a perspective view of a microfluidic chip of example 3 of the present invention;

fig. 9 is a top view of a microfluidic chip according to example 3 of the present invention;

fig. 10 is a bottom view of a microfluidic chip of example 3 of the present invention;

fig. 11 is a front view of a microfluidic chip of example 3 of the present invention;

fig. 12 is a rear view of a microfluidic chip of example 3 of the present invention;

fig. 13 is a right side view of a microfluidic chip of example 3 of the present invention;

fig. 14 is a left side view of a microfluidic chip of example 3 of the present invention;

FIG. 15 is a flow chart of a sample injection process when the content of bacteria is quantitatively detected by a microfluidic chip according to an embodiment;

FIG. 16 is a flow chart of the cleaning process of the microfluidic chip for quantitatively detecting the content of bacteria according to an embodiment;

FIG. 17 is a flow chart of an elution process using a cleaning solution in quantitative determination of bacterial content using a microfluidic chip according to an embodiment;

FIG. 18 is a reaction flow chart of a microfluidic chip for quantitatively detecting bacteria content according to an embodiment;

FIG. 19 is a flow chart of the sample injection in the case of quantitative determination of bacteria content by a microfluidic chip according to another embodiment;

FIG. 20 is a flow chart of a cleaning process of a microfluidic chip for quantitatively detecting bacteria content according to another embodiment;

FIG. 21 is a flow chart of elution with a buffer solution in quantitative determination of bacterial content using a microfluidic chip according to another embodiment;

FIG. 22 is a reaction flow chart of the microfluidic chip for quantitative determination of bacterial content according to another embodiment.

Detailed Description

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.