CN117007800B - Dual-droplet microfluidic chip for escherichia coli detection and application thereof - Google Patents
Dual-droplet microfluidic chip for escherichia coli detection and application thereof Download PDFInfo
- Publication number
- CN117007800B CN117007800B CN202310999538.7A CN202310999538A CN117007800B CN 117007800 B CN117007800 B CN 117007800B CN 202310999538 A CN202310999538 A CN 202310999538A CN 117007800 B CN117007800 B CN 117007800B
- Authority
- CN
- China
- Prior art keywords
- sample
- double
- cross
- escherichia coli
- oil phase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 40
- 238000001514 detection method Methods 0.000 title claims abstract description 34
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 59
- 239000007788 liquid Substances 0.000 claims abstract description 41
- 238000003860 storage Methods 0.000 claims abstract description 17
- 239000002356 single layer Substances 0.000 claims abstract description 8
- 230000009977 dual effect Effects 0.000 claims description 23
- 239000000758 substrate Substances 0.000 claims description 12
- 239000000126 substance Substances 0.000 claims description 8
- HSHNITRMYYLLCV-UHFFFAOYSA-N 4-methylumbelliferone Chemical compound C1=C(O)C=CC2=C1OC(=O)C=C2C HSHNITRMYYLLCV-UHFFFAOYSA-N 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 238000006911 enzymatic reaction Methods 0.000 claims description 5
- 239000002131 composite material Substances 0.000 claims description 4
- 238000005336 cracking Methods 0.000 claims description 4
- 102000053187 Glucuronidase Human genes 0.000 claims description 3
- 108010060309 Glucuronidase Proteins 0.000 claims description 3
- 238000009826 distribution Methods 0.000 claims description 3
- WYTGDNHDOZPMIW-RCBQFDQVSA-N alstonine Natural products C1=CC2=C3C=CC=CC3=NC2=C2N1C[C@H]1[C@H](C)OC=C(C(=O)OC)[C@H]1C2 WYTGDNHDOZPMIW-RCBQFDQVSA-N 0.000 claims description 2
- ARQXEQLMMNGFDU-JHZZJYKESA-N 4-methylumbelliferone beta-D-glucuronide Chemical compound C1=CC=2C(C)=CC(=O)OC=2C=C1O[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O ARQXEQLMMNGFDU-JHZZJYKESA-N 0.000 claims 1
- ARQXEQLMMNGFDU-UHFFFAOYSA-N 4MUG Natural products C1=CC=2C(C)=CC(=O)OC=2C=C1OC1OC(C(O)=O)C(O)C(O)C1O ARQXEQLMMNGFDU-UHFFFAOYSA-N 0.000 claims 1
- 239000000839 emulsion Substances 0.000 abstract description 7
- 238000005516 engineering process Methods 0.000 abstract description 6
- 201000010099 disease Diseases 0.000 abstract description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 3
- 238000003911 water pollution Methods 0.000 abstract description 3
- 239000012071 phase Substances 0.000 description 44
- 238000000034 method Methods 0.000 description 6
- 239000004205 dimethyl polysiloxane Substances 0.000 description 4
- 235000013870 dimethyl polysiloxane Nutrition 0.000 description 4
- 239000006185 dispersion Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 description 4
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 description 4
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 239000008346 aqueous phase Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000010008 shearing Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 239000003651 drinking water Substances 0.000 description 2
- 235000020188 drinking water Nutrition 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
-
- 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
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/18—Water
- G01N33/1826—Organic contamination in water
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Urology & Nephrology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Virology (AREA)
- Biotechnology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Dispersion Chemistry (AREA)
- Clinical Laboratory Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Automatic Analysis And Handling Materials Therefor (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention discloses a double-liquid-drop micro-fluidic chip for escherichia coli detection and application thereof, wherein the chip comprises: the device comprises a sample inlet, an intermediate oil phase inlet, an outer water phase inlet, a sample flow channel, an oil phase flow channel, a water phase flow channel, a first cross-shaped port and a second cross-shaped port; a double liquid drop dispersing flow passage connected with the second cross-shaped port; a double droplet storage chamber connected with the tail end of the double droplet dispersing flow channel and used for single-layer arrangement of double droplets, wherein a first pillar and a second pillar are arranged, and a gap of 160-200 mu m is kept between adjacent first pillars for single droplets to pass through; and an outlet. The double emulsion technology is applied to the detection of the escherichia coli, so that the on-site, rapid, on-line and accurate detection requirements of the escherichia coli in water are realized, the forward movement of a water quality detection port is realized, the water quality safety is ensured at the source, the outbreak and the epidemic of the medium water pollution disease are effectively controlled, and the physical health of people is ensured.
Description
Technical Field
The invention relates to the field of microfluidic devices, in particular to a double-droplet microfluidic chip for escherichia coli detection and application thereof.
Background
Generally, the drinking water treated by a plurality of steps of a tap water plant is free of escherichia coli, and even if the drinking water is polluted, the content of the escherichia coli is extremely low, so that the requirements on the sensitivity and the reliability of an inspection method are high; existing test methods include traditional multitube fermentation and membrane filtration methods, and specific enzyme substrate methods. With the continuous occurrence of water source pollution events in various places, new demands are made on the technology of detecting escherichia coli in water, and the detection work of a large number of water samples is required to be completed more rapidly and efficiently on the premise of ensuring the reliability and accuracy of detection results. Meanwhile, because the escherichia coli test has high requirements on the condition of water sample preservation, the field test is often required to be carried out at a field water sample collection point. Therefore, the conventional detection method is difficult to realize the on-site, rapid and on-line detection requirement of the escherichia coli in water.
Therefore, the development of the online quick detector for the escherichia coli in the water based on the micro-flow control can effectively control the water quality safety problem, realize the forward movement of the water quality detection port, ensure the water quality safety at the source, greatly reduce the cost required by water quality detection, effectively control the outbreak and the epidemic of medium water pollution diseases and ensure the physical health of people.
For the above phenomenon, single droplet microfluidic technology has been studied for application to embedded E.coli. However, the wrapping effect of single liquid drops on the mixed liquid of the escherichia coli and the substrate is not good, the mixed liquid leakage phenomenon can occur, so that a large piece of fluorescence occurs in a fluorescence mode, and the quantity of the escherichia coli cannot be calculated according to the fluorescence quantity.
Disclosure of Invention
The invention aims to provide a double-liquid-drop micro-fluidic chip for detecting escherichia coli and application thereof, so as to solve the problem that the escherichia coli cannot be counted due to easy leakage caused by adopting a single-liquid-drop technology to detect the escherichia coli in the prior art.
According to a first aspect of the present invention there is provided a dual droplet microfluidic chip for detection of escherichia coli comprising: an external water phase inlet, an intermediate oil phase inlet, a sample inlet; the sample inlet is connected with a sample runner, the middle oil phase inlet is intersected with the sample runner at a first cross-shaped opening through two oil phase runners which are divided into two parts, and the outer water phase inlet is intersected with the runner from the first cross-shaped opening at a second cross-shaped opening through two water phase runners which are divided into two parts; the double liquid drop dispersing flow channels are connected with the second cross-shaped opening and are symmetrically arranged in a tree structure by taking the second cross-shaped opening as the center; the double-liquid-drop storage chamber is connected with the double-liquid-drop dispersing flow channel and is used for single-layer arrangement of double liquid drops, wherein a row of first struts close to the double-liquid-drop dispersing flow channel and a plurality of second struts dispersed in the double-liquid-drop storage chamber are arranged, gaps of 160-200 mu m are kept between the adjacent first struts for single liquid drops to pass through, and the second struts are used for supporting the double-liquid-drop storage chamber.
Preferably, the sample flow channel and the aqueous phase flow channel have surface hydrophilicity, and the oil phase flow channel has surface hydrophobicity.
Preferably, the sample flow channels are distributed in serpentine flow channels prior to entering the first cross-port.
Preferably, the first pillar comprises: the device comprises a centrally arranged first large support column and a plurality of first small support columns arranged along two sides of the first large support column, wherein the longitudinal dimension of the first large support column is 2-3 times of that of the first small support column.
Preferably, the first large support and the first small support are rectangular, the longitudinal dimension of the first large support is 3840 μm, the transverse dimension of the first large support is 2500 μm, the longitudinal dimension of the first small support is 1770 μm, the transverse dimension of the first small support is 2500 μm, the second support is elliptical, the short axis dimension of the second large support is 1000 μm, and the long axis dimension of the second large support is 2500 μm.
Preferably, a 200 μm gap is maintained between adjacent first pillars to facilitate the passage of a single W/O/W type double droplet.
According to a second aspect of the present invention, there is provided an application of a dual droplet microfluidic chip in detection of escherichia coli, comprising the steps of: s1: adding PELB composite cracking reagent and enzymatic reaction substrate 4-CMUG into a water quality sample to be detected to prepare a sample, inputting the sample from the sample inlet, inputting a water phase through an external water phase inlet, inputting an oil phase through an intermediate oil phase inlet, shearing the sample by the oil phase at a first cross-shaped port to generate W/O single-weight inner liquid drops, then shearing the sample by the water phase at a second cross-shaped port to generate W/O/W double liquid drops, enabling the W/O/W double liquid drops to flow through a double liquid drop dispersing runner, and enabling the W/O/W double liquid drops to enter and be horizontally paved in a double liquid drop storage chamber in a single layer; s2: after incubating for 3-4 hours at 40 ℃, beta-glucuronidase secreted by the escherichia coli catalyzes and hydrolyzes a substrate 4-MUG into a specific fluorescent substance 4-MU, the specific fluorescent substance is observed and imaged under a fluorescence microscope, and the number of fluorescence is calculated by using poisson distribution, so that the escherichia coli in a water quality sample to be detected can be detected.
Preferably, the diameters of the generated W/O/W double droplets are between 100 and 150 μm by adjusting the flow rates of the sample, the oil phase and the water phase respectively.
According to the application of the method in the detection of the escherichia coli, through regulating and controlling the liquid flow rates of the three flow channels, liquid drops with different sizes and different shell thicknesses can be generated, and the size of the liquid drops is moderately regulated according to the concentration of a substrate, the intensity of fluorescence and the detection limit of a fluorescence machine. Preferably, the flow rate of the sample is 1.5-2 mu L/min, the flow rate of the oil phase is 4.5-6 mu L/min, the flow rate of the water phase is 7.5-10 mu L/min, and the W/O/W double emulsion with the diameter of about 120 mu m is finally obtained.
Preferably, the water quality sample to be detected is an escherichia coli enriched water quality sample.
Preferably, the concentration of the enzymatic reaction substrate 4-CMUG is 0.4mg/mL.
According to the application of the invention in the detection of the escherichia coli, the working principle is as follows: the mixture consisting of the test sample, the PELB composite cracking reagent and the enzymatic reaction substrate 4-CMUG is conveyed by a constant pressure pump to enter from a sample inlet, sheared by an intermediate oil phase at a first cross-shaped port to generate W/O type single inner liquid drops, sheared by an outer water phase to generate W/O/W type double liquid drops, and enters into a double liquid drop storage chamber of a chip through a double liquid drop dispersing runner; in the double liquid drop storage cavity, the double liquid drop is spread in a single layer, after incubation for 3 hours, beta-glucuronidase (GUS enzyme) secreted by escherichia coli catalyzes and hydrolyzes a substrate 4-MUG (4-methylumbelliferone-beta-D-galactoside) into a specific fluorescent substance 4-MU (4-methylumbelliferone), and finally, the detection is carried out under a fluorescent microscope, so that the detection of the quantity of escherichia coli in a water sample is realized.
It should be understood that according to the dual droplet microfluidic chip provided by the present invention, three incompatible liquid phases meet at the channel intersection of the pipeline, and a double emulsion is generated by a two-step method from the shearing force of the continuous relative dispersed phases. In the generation area of the liquid drop, the hydrophilic-hydrophobic correspondence of the corresponding flow channel is required to be met, and as the water-in-oil-in-water (W/O/W) double emulsion is required to be generated, the sample flow channel and the water phase flow channel are required to meet the surface hydrophilicity, and the oil phase flow channel is required to meet the surface hydrophobicity. According to the invention, the PDMS chip is used for experiments, and the PDMS is the hydrophobic material, so that the sample flow channel and the water phase flow channel can be modified into the hydrophilic material by combining plasma with the modifier.
The invention is mainly characterized in that the double emulsion technology is applied to the detection of the escherichia coli for the first time, and the inventor finds that in the detection of the escherichia coli wrapped by the single liquid drop, the generated fluorescent substance can leak, so that large fluorescence appears in a view field, and the calculation of the number of the fluorescent liquid drops is influenced. However, according to the double-droplet microfluidic chip and the application thereof provided by the invention, a water-in-oil-in-water (W/O/W) double emulsion is prepared, and the double-layer structure brings better wrapping performance, so that the accurate detection of escherichia coli is finally realized.
In summary, the dual-droplet microfluidic chip for escherichia coli detection and the application thereof provided by the invention have the following advantages compared with the prior art:
1) The double emulsion technology is applied to the detection of the escherichia coli, so that the on-site, rapid, on-line and accurate detection requirements of the escherichia coli in water are met;
2) The forward movement of the water quality detection port is realized, the water quality safety is ensured at the source, the outbreak and the epidemic of medium water pollution diseases are effectively controlled, and the physical health of people is ensured;
3) The liquid drops as small as nanoliter can increase the system concentration of the local fluorescent substances and exceed the detection limit of a detection instrument, so that the detection time is shortened;
4) The double-liquid drop micro-fluidic chip has low manufacturing cost, greatly reduces the cost required by water quality detection, is convenient to popularize and has wide application prospect.
Drawings
Fig. 1 is a schematic structural view of a dual droplet microfluidic chip according to a preferred embodiment of the present invention;
FIG. 2 is a schematic diagram of dual droplet generation;
FIG. 3 is a schematic illustration of dual drop dispersion;
FIG. 4 is a schematic illustration of dual droplet incubation within a chamber;
Wherein the reference numerals have the following meanings:
1: an external aqueous phase inlet; 2: an intermediate oil phase inlet; 3: a sample inlet; 4: a sample flow channel; 5: an oil phase flow channel; 6: a water phase flow channel; 7: a dual droplet dispersion flow channel; 8: a dual droplet storage chamber; 9: an outlet; 10: a first cross-shaped port; 11: a second cross-shaped port; 12: a first support column; 13: and a second pillar.
Detailed Description
The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The technical means used in the examples are, unless specified otherwise, conventional in the art or according to experimental methods recommended by the equipment manufacturer. Reagents and materials used in the examples were obtained commercially, unless otherwise specified.
Example 1
As shown in fig. 1, there is a dual droplet microfluidic chip according to a preferred embodiment of the present invention, comprising: an outer water phase inlet 1, an intermediate oil phase inlet 2, a sample inlet 3, a sample flow channel 4, an oil phase flow channel 5, an aqueous phase flow channel 6, a double droplet dispersion flow channel 7, a double droplet storage chamber 8 and an outlet 9.
As shown in fig. 2, the sample inlet 3 is connected with a serpentine sample runner 4 for inputting a sample to be detected, the middle oil phase inlet 2 is intersected with the sample runner 4 at a first cross-shaped port 10 through two oil phase runners 5 split into two, and the outer water phase inlet 1 is intersected with the runner from the first cross-shaped port 10 at a second cross-shaped port 11 through two water phase runners 6 split into two.
The double droplet dispersing flow channel 7 is connected with the second cross-shaped opening 11, and the double droplet dispersing flow channel 7 is symmetrically arranged in a tree structure (i.e. a step-by-step dispersing structure realized by dividing into two parts and dividing into two parts) by taking the second cross-shaped opening 11 as the center so as to facilitate the dispersion of single double droplets.
The dual droplet storage chamber 8 is connected to the end of the dual droplet dispersing channel 7 for single-layer arrangement of the dual droplets, wherein a row of first pillars 12 near the dual droplet dispersing channel and a plurality of second pillars 13 dispersed in the dual droplet storage chamber are provided.
According to the preferred embodiment, the first leg 12 comprises: a centrally arranged one of the large pillars, and a number of the small pillars arranged along both sides of the large pillar, each of the large and small pillars being rectangular, wherein the first large pillar preferably has a longitudinal dimension of 3840 μm, a transverse dimension of 2500 μm, the small pillar preferably has a longitudinal dimension of 1770 μm, a transverse dimension of 2500 μm, a 200 μm gap is maintained between adjacent first pillars 12 for the passage of a single double droplet, and the second pillar 13 is oval, preferably has a minor axis dimension of 1000 μm, and a major axis dimension of 2500 μm.
According to the preferred embodiment, the dual-droplet microfluidic chip is a PDMS chip, and the PDMS itself is made of a hydrophobic material, so that the oil phase flow channel 5 has surface hydrophobicity, and the sample flow channel 4 and the water phase flow channel 6 modify the corresponding channels by combining plasma with a modifying agent, and have surface hydrophilicity after modification.
Example 2
According to the embodiment, the application of the double-droplet microfluidic chip in escherichia coli detection is provided, and the method specifically comprises the following steps:
firstly, adding PELB composite cracking reagent and enzymatic reaction substrate 4-CMUG into a water quality sample to be detected to prepare a sample, wherein the concentration of 4-CMUG is 0.4mg/mL;
Then, 100. Mu.L of sample is fed into the sample flow channel 4 from the sample inlet 3 at a flow rate of 2. Mu.L/min, the middle oil phase is fed into the oil phase flow channel 5 from the middle oil phase inlet 2 at a flow rate of 6. Mu.L/min, the outer water phase is fed into the water phase flow channel 6 from the outer water phase inlet 1 at a flow rate of 10. Mu.L/min, the sample and the oil phase meet at the first cross-shaped opening 10, as shown in FIG. 2, the sample is sheared by the oil phase to form W/O type liquid drops, the liquid drops move to the downstream, and are sheared by the outer water phase to form W/O/W type double liquid drops when reaching the second cross-shaped opening 11, and the diameter of the liquid drops is about 120 μm;
Next, the generated double droplets are dispersed in the double droplet dispersing flow path 7 as shown in fig. 3, and the dispersed double droplets uniformly flow into the double droplet storage chambers 8 from the gaps of the first pillars 12, respectively, and are distributed therein in a single layer form as shown in fig. 4;
Then, the double liquid drops are kept for 3 hours at 40 ℃ in a double liquid drop storage chamber 8, the liquid drops are kept stable and not broken during the period, and GUS enzyme secreted by escherichia coli hydrolyzes a substrate and gradually accumulates fluorescent substances;
And then, the double-liquid-drop microfluidic chip is placed under a fluorescence microscope for observation, and the number of fluorescence is calculated by using poisson distribution, so that the number of escherichia coli in water is calculated.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and various modifications can be made to the above-described embodiment of the present invention. All simple, equivalent changes and modifications made in accordance with the claims and the specification of the present application fall within the scope of the patent claims. The present invention is not described in detail in the conventional art.
Claims (7)
1. The application of the double-liquid-drop microfluidic chip in the detection of escherichia coli in an aqueous sample is characterized by comprising the following steps:
S0: provided is a dual droplet microfluidic chip for E.coli detection, comprising:
The sample inlet is connected with one sample runner, the middle oil phase inlet is intersected with the sample runner at a first cross-shaped opening through two oil phase runners which are split into two, the outer water phase inlet is intersected with the runner from the first cross-shaped opening at a second cross-shaped opening through two water phase runners which are split into two, and the sample runners are distributed in a serpentine runner before entering the first cross-shaped opening;
The double liquid drop dispersing flow channels connected with the second cross-shaped opening are symmetrically arranged in a tree structure by taking the second cross-shaped opening as the center;
A dual droplet storage chamber connected to the end of the dual droplet dispersing channel, for single-layer arrangement of dual droplets, wherein a row of first pillars close to the dual droplet dispersing channel and a plurality of second pillars dispersed in the dual droplet storage chamber are provided, a gap of 160-200 μm is kept between adjacent first pillars for passing single dual droplets, the second pillars are used for supporting the dual droplet storage chamber, and the first pillars comprise: the device comprises a first large support column arranged in the middle and a plurality of first small support columns arranged along two sides of the first large support column, wherein the longitudinal dimension of the first large support column is 2-3 times that of the first small support column; and
An outlet;
s1: adding PELB composite cracking reagent and enzymatic reaction substrate 4-CMUG into a water quality sample to be detected to prepare a sample, inputting the sample from the sample inlet, inputting a water phase through an external water phase inlet, inputting an oil phase through an intermediate oil phase inlet, enabling the sample to be sheared by the oil phase at a first cross-shaped opening to generate W/O type single-weight inner liquid drops, then enabling the sample to be sheared by the water phase at a second cross-shaped opening to generate W/O/W type double liquid drops, enabling the W/O/W type double liquid drops to flow through a double liquid drop dispersing runner, and enabling the W/O/W type double liquid drops to enter and be flatly paved in a double liquid drop storage chamber in a single layer;
S2: after incubating for 3-4 hours at the temperature of 40 ℃, beta-glucuronidase secreted by the escherichia coli catalyzes and hydrolyzes a substrate 4-MUG into a specific fluorescent substance 4-MU, the specific fluorescent substance is observed and imaged under a fluorescence microscope, and the number of fluorescence is calculated by using poisson distribution, so that the escherichia coli in a water quality sample to be detected can be detected.
2. The use of claim 1, wherein the sample flow channel and the aqueous flow channel have surface hydrophilicity and the oil flow channel has surface hydrophobicity.
3. The use of claim 1, wherein the first large struts and the first small struts are each rectangular and the second struts are elliptical.
4. The use according to claim 3, wherein a 200 μm gap is maintained between adjacent first pillars to facilitate the passage of a single W/O/W type double droplet.
5. The use according to claim 1, wherein the diameters of the generated W/O/W type double droplets are 100-150 μm by adjusting the flow rates of the sample, the oil phase and the water phase, respectively.
6. The use according to claim 5, wherein the flow rate of the sample is 1.5-2 μl/min, the flow rate of the oil phase is 4.5-6 μl/min, and the flow rate of the water phase is 7.5-10 μl/min.
7. The use according to claim 1, wherein the water quality sample to be detected is an e.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310999538.7A CN117007800B (en) | 2023-08-09 | 2023-08-09 | Dual-droplet microfluidic chip for escherichia coli detection and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310999538.7A CN117007800B (en) | 2023-08-09 | 2023-08-09 | Dual-droplet microfluidic chip for escherichia coli detection and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117007800A CN117007800A (en) | 2023-11-07 |
| CN117007800B true CN117007800B (en) | 2024-05-10 |
Family
ID=88574085
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310999538.7A Active CN117007800B (en) | 2023-08-09 | 2023-08-09 | Dual-droplet microfluidic chip for escherichia coli detection and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117007800B (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150293102A1 (en) * | 2013-04-13 | 2015-10-15 | Jung-Uk Shim | Detecting low-abundant analyte in microfluidic droplets |
| CN110016429A (en) * | 2018-01-09 | 2019-07-16 | 四川大学华西医院 | A kind of double droplet containing cells and preparation method thereof |
| CN110804531A (en) * | 2019-11-15 | 2020-02-18 | 深圳市第二人民医院 | A microdroplet-based detection system for gut microbes |
| CN110819507A (en) * | 2019-11-15 | 2020-02-21 | 深圳市第二人民医院 | Micro-droplet preparation chip for detecting intestinal microorganisms |
| CN111468200A (en) * | 2020-04-22 | 2020-07-31 | 东莞市东阳光诊断产品有限公司 | Microfluidic chip and biochemical analysis and detection device |
| CN115350733A (en) * | 2022-07-13 | 2022-11-18 | 中国科学院上海微系统与信息技术研究所 | A microfluidic chip with a single-layer inclined structure droplet storage cavity and its preparation method |
| JP2022175985A (en) * | 2021-05-14 | 2022-11-25 | 東ソー株式会社 | METHOD AND APPARATUS FOR GENERATING EMULSION IN MICROCHANNEL CHIP |
| CN116144742A (en) * | 2021-11-18 | 2023-05-23 | 韩国科学技术院 | Method for providing quantitative information on a target and device using the method |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106132544A (en) * | 2013-11-11 | 2016-11-16 | 阿卜杜拉国王科技大学 | High power capacity manufacture and the micro-fluidic device of process for monodispersed emulsion |
| US11465148B2 (en) * | 2018-04-16 | 2022-10-11 | Pattern Bioscience, Inc. | Methods and apparatus for forming 2-dimensional drop arrays |
-
2023
- 2023-08-09 CN CN202310999538.7A patent/CN117007800B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150293102A1 (en) * | 2013-04-13 | 2015-10-15 | Jung-Uk Shim | Detecting low-abundant analyte in microfluidic droplets |
| CN110016429A (en) * | 2018-01-09 | 2019-07-16 | 四川大学华西医院 | A kind of double droplet containing cells and preparation method thereof |
| CN110804531A (en) * | 2019-11-15 | 2020-02-18 | 深圳市第二人民医院 | A microdroplet-based detection system for gut microbes |
| CN110819507A (en) * | 2019-11-15 | 2020-02-21 | 深圳市第二人民医院 | Micro-droplet preparation chip for detecting intestinal microorganisms |
| CN111468200A (en) * | 2020-04-22 | 2020-07-31 | 东莞市东阳光诊断产品有限公司 | Microfluidic chip and biochemical analysis and detection device |
| JP2022175985A (en) * | 2021-05-14 | 2022-11-25 | 東ソー株式会社 | METHOD AND APPARATUS FOR GENERATING EMULSION IN MICROCHANNEL CHIP |
| CN116144742A (en) * | 2021-11-18 | 2023-05-23 | 韩国科学技术院 | Method for providing quantitative information on a target and device using the method |
| CN115350733A (en) * | 2022-07-13 | 2022-11-18 | 中国科学院上海微系统与信息技术研究所 | A microfluidic chip with a single-layer inclined structure droplet storage cavity and its preparation method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117007800A (en) | 2023-11-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103226127B (en) | Multi-channel micro-fluidic chip and mass spectrum combined device | |
| CN104849111B (en) | The forming method of gradient microlayer model array based on sequential injection and microflow control technique | |
| CN101718795B (en) | Microfluidic chip liquid drop control method based on pneumatic micro valve | |
| US9895690B2 (en) | Microfluidic chip and application thereof | |
| Yang et al. | Mass transfer characteristics of bubbly flow in microchannels | |
| Kong et al. | Integrated microfluidic immunoassay for the rapid determination of clenbuterol | |
| CN110787851B (en) | Pressure-driven multi-channel droplet quantitative measuring device and measuring method | |
| CN101486004A (en) | Automatic device for quantitatively distributing microfluid and using method | |
| CN108273576A (en) | The micro-fluidic chip system that adjustable lotion is formed | |
| Zhang et al. | Paper spray mass spectrometry-based method for analysis of droplets in a gravity-driven microfluidic chip | |
| CN117007800B (en) | Dual-droplet microfluidic chip for escherichia coli detection and application thereof | |
| CN111272887A (en) | Chromatographic analysis device based on multifunctional integrated probe and using method | |
| Sun et al. | High-throughput sample introduction for droplet-based screening with an on-chip integrated sampling probe and slotted-vial array | |
| CN105647789A (en) | Real-time cell metabolite detection device based on micro-fluidic chip | |
| Zheng et al. | Bubble generation rules in microfluidic devices with microsieve array as dispersion medium | |
| US20140147908A1 (en) | Method for splitting droplets on demand in microfluidic junction | |
| WO2013014216A1 (en) | Device and method for high-throughput, on-demand generation and merging of droplets | |
| Mavrakis et al. | Chip-based microfluidics on-line with inductively coupled plasma-mass spectrometry for standard dilution analysis | |
| CN103165389B (en) | A kind of sampling device | |
| CN114931988B (en) | Micro-fluidic chip for generating nano-scale molecular concentration gradient liquid drops and application method thereof | |
| CN208194420U (en) | The micro-fluidic chip system that adjustable lotion is formed | |
| CN112857956B (en) | Capillary pressure difference enhanced concentration permeation chip | |
| WO2014141861A1 (en) | Microchannel device and method pertaining thereto | |
| CN207567249U (en) | A kind of micro-fluidic chip for nucleic acid amplification detection | |
| CN115932133B (en) | Migration spectrum detection device and method with multiple injection modes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |