CN114669337A - Micro-fluidic device and application thereof in bacteria separation - Google Patents
Micro-fluidic device and application thereof in bacteria separation Download PDFInfo
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Abstract
The invention provides a micro-fluidic chip, a micro-fluidic device comprising the chip and application of the micro-fluidic device in bacteria separation. Furthermore, after the bacteria are cultured, a target flora can be screened and enriched, for example, potential anti-obesity probiotics interacted with engineering probiotics can be screened, and a rich and reliable source is provided for the functional research of the bacteria.
Description
Technical Field
The invention belongs to the technical field of genetic engineering and microorganism separation equipment, and particularly relates to a micro-fluidic device and application thereof in bacteria separation.
Background
Recently, ultra-high throughput droplet microfluidic platforms for functional screening of valuable microbial populations have become an attractive alternative. It allows packaging of single bacteria in double emulsion droplets and screening of these microdroplets by Fluorescence Activated Cell Sorter (FACS) (Terekhov et al, 2018; Terekhov et al, 2017). However, the implementation of FACS-based methods is limited by their high cost, complex double milk generation and multiple limitations of fluorescence signal discrimination. Furthermore, droplet-based microfluidic methods enable low abundance bacterial growth in some samples compared to traditional culture methods. (VillaMax et al; Watterson et al, 2020).
Obesity increases the risk of cardiovascular and metabolic diseases, osteoarthritis, dementia, depression and certain cancers (bliher, 2019). Numerous studies have shown that various probiotics can play a role in combating obesity by reducing intestinal bacterial lipopolysaccharides, altering the composition of the intestinal microbiota and reducing fat stores (barathikann et al, 2019; Dahiya et al, 2017). For example, Lactobacillus acidophilus improves obesity by regulating gut microbiota dysbiosis (Kang et al, 2022). It is clear that if these strains are obtained and demonstrated to have anti-obesity effects, they are of great importance for the treatment of obesity. In our previous studies, butyric acid producing engineered bacteria (EBPB) were constructed and demonstrated to have potential anti-obesity effects in mice (baietal.,2020), probably due to the enrichment of the engineered probiotic with a fraction of the beneficial anti-obesity probiotic.
To date, droplet-based microfluidic technology has not been applied to the high abundance screening of populations of interest, particularly for potential anti-obesity probiotics.
Disclosure of Invention
The present invention aims to overcome the above-mentioned deficiencies of the prior art and to provide a microfluidic device and its use for isolating cells. In particular, the amount of the solvent to be used,
in a first aspect of the invention, a microfluidic chip is provided, which comprises two inlets, one outlet and several channels.
Preferably, one of the two inlets is a continuous phase inlet; one is a dispersed phase inlet; the outlet is a droplet outlet.
The channel comprises a rectangular continuous phase liquid inlet channel and a linear disperse phase liquid inlet channel, the rectangular continuous phase liquid inlet channel and the linear disperse phase liquid inlet channel are respectively connected with a continuous phase inlet and a disperse phase inlet, are intersected with a cross-shaped Flow focusing structure, and extend forwards from the channel to be connected with an outlet at the cross-shaped Flow focusing structure (Flow-Focus).
More preferably, the cross-shaped flow focusing structure has a channel size of 10 μm (width) × 20 μm (height) of intersecting channels.
More preferably, the chip structure is symmetrical up and down.
Preferably, the raw material of the chip is selected from one or more of Polydimethylsiloxane (PDMS), polymethyl methacrylate and glass. More preferably, the material is selected from Polydimethylsiloxane (PDMS).
In a second aspect of the present invention, a method for preparing a microfluidic chip is provided, wherein the method comprises the following steps (1) and template preparation: obtaining a template with a chip channel by using a soft lithography technology, (2) preparing the mold: mixing the raw material and the curing agent, pouring the mixture into a template, vacuumizing the template by using a vacuum device, and communicating the vacuum device with the atmosphere to eliminate bubbles in the template; 3) bonding the mold and the glass slide: and carrying out surface treatment on the mold and the glass slide, and bonding the mold and the glass slide to obtain the chip.
Preferably, the step 1) comprises surface treatment of the template with trichlorosilane.
Preferably, the step 2) includes mixing a raw material, such as Polydimethylsiloxane (PDMS), with a curing agent to prepare the chip, and more preferably, the mass ratio of the PDMS: the curing agent is 10: 1.
Preferably, the surface treatment in step 3) includes oxygen plasma surface treatment.
In a third aspect of the invention, a microfluidic device is provided, which comprises the above microfluidic chip.
In a fourth aspect of the invention, a microfluidic system is provided, comprising the above microfluidic device and a continuous phase.
Preferably, the continuous phase comprises mineral oil and Span80, and more preferably, the Span80 is present in the continuous phase in an amount of 2% to 5%, and can be in any range within the above range, or any value such as: 3% -5%, 4% -5%, etc.
More preferably, the continuous phase comprises a medium suitable for the growth of bacteria.
Preferably, the system comprises a culture unit for bacteria.
More preferably, the culture unit of the bacteria comprises a medium for screening the flora of interest.
It is further preferable that the medium is a conditioned medium for screening and enriching the target flora, and more preferably, the conditioned medium is any conditioned medium, such as butyric acid producing conditioned medium, anti-obesity conditioned medium, probiotic conditioned medium, etc., for screening and enriching the flora that can be used for producing butyric acid, anti-obesity, probiotic, etc.
In a specific embodiment, the conditioned medium is a conditioned medium comprising a metabolite of an engineered butyrate-producing bacterium.
Preferably, the system further comprises an identification unit for bacteria. More preferably, the identification unit of the bacteria comprises a reagent for amplifying and/or identifying the 16srRNA of the bacteria, and further preferably, the reagent comprises a universal primer aiming at the V3-V4 region of the bacteria. In a specific embodiment, the primer sequences are shown as SEQ ID NO. 1 and SEQ ID NO. 2.
In a fifth aspect of the invention, there is provided the use of the above microfluidic chip, microfluidic device or microfluidic system in bacterial separation.
In a sixth aspect of the present invention, a bacteria separation method is provided, where the bacteria separation method includes separating bacteria by using the above microfluidic chip, microfluidic device or microfluidic system.
Preferably, the separation method comprises the steps of: 1) the bacterial suspension enters through a dispersed phase inlet; 2) the continuous phase enters through a continuous phase inlet, and 3) a droplet of a continuous phase packaging dispersed phase is formed in the microfluidic chip; 4) a single droplet was collected.
Preferably, the droplets encapsulate individual cells, forming single-cell droplets.
More preferably, of the droplets, the droplets encapsulating more than one cell do not exceed 10%, even more preferably not exceed 5%, such as not exceed 4%, not exceed 3%, not exceed 2%, not exceed 1% of the total droplets, and in a specific embodiment, the droplets do not include droplets encapsulating more than one cell.
More preferably, the bacteria are capable of growing in the droplets.
Preferably, the bacterial sample in the bacterial suspension in step 1) is derived from a tissue sample of an organism, such as epidermis, milk, blood, sputum, saliva, urine, feces, and other tissue samples, or an environmental sample, such as air, soil, water, and the like.
More preferably, the bacteria are prepared as a bacterial suspension following anaerobic culture.
Preferably, said step 1) comprises adjusting the OD of the bacterial suspension600The value is such that the bacteria are encapsulated by single cells into single cell droplets.
Preferably, the OD of the bacterial suspension in said step 1)600From 0.01 to 0.04 and can be any range within the above range, or any value, for example: 0.01-0.03, 0.01-0.02, etc.
In a specific embodiment, the sample is from feces and the OD of the bacterial suspension of the sample600From 0.01 to 0.04 and can be any range within the above range, or any value, for example: 0.01-0.03, 0.01-0.02, etc.
Preferably, the flow rate of the dispersed phase in the step 1) is 0.15 to 1 μ L/min.
Preferably, the flow rate of the continuous phase in the step 2) is 0.15-1 μ L/min.
More preferably, the flow rate of the dispersed phase in step 1) is kept the same as the flow rate of the continuous phase in step 2).
Preferably, the continuous phase in step 2) comprises mineral oil and Span80, and more preferably, the Span80 is present in the continuous phase in an amount of 2% to 5%, and can be in any range within the above range, or any value, such as: 3% -5%, 4% -5%, etc.
More preferably, the continuous phase comprises a medium suitable for the growth of bacteria.
Preferably, the droplets in step 3) comprise single cells.
Preferably, the step 4) collects the droplets by using a sealed tube.
Preferably, the method further comprises step 5): and (3) bacterial culture: the droplets are cultured.
More preferably, the bacterial culturing in step 5) comprises culturing the liquid drop with a conditioned medium to screen and enrich the target flora.
Further preferably, the conditioned medium is any conditioned medium, such as a butyric acid-producing conditioned medium, an anti-obesity conditioned medium, a probiotic conditioned medium, and the like, for screening and enriching for a flora that can be used for butyric acid production, anti-obesity, probiotic, and the like.
More preferably, the conditioned medium comprises a conditioned medium of a metabolite of butyric acid-producing engineered bacteria. Further preferably, the conditioned medium comprises a supernatant after EBPB culture for screening for enrichment of potential anti-obesity bacteria. Preferably, the supernatant after EBPB culture: the volume ratio of the basic culture medium is (1-3): (3-1), in a specific embodiment, the basal medium is YCFA medium.
Preferably, the method further comprises the step 6) of identifying the bacteria: identifying the isolated or cultured bacteria.
More preferably, in step 6), the bacteria are identified using 16 srna sequencing.
Further preferably, the step 6) comprises designing a universal primer aiming at the V3-V4 region to identify the bacteria.
In a specific embodiment, the primer sequences are shown as SEQ ID NO. 1 and SEQ ID NO. 2.
In a seventh aspect, the present invention provides a bacterial flora prepared by any of the above isolation methods.
Preferably, the flora enrichment comprises one or more of bacteria of the genus lactobacillus, bifidobacterium, enterococcus and bacillus, more preferably lactobacillus and/or bifidobacterium, further preferably one or more of bacillus cereus, bacillus naersonii, bacillus umbelliferus, bacillus circulans, enterococcus faecalis, lactobacillus murinus, lactobacillus enteric, lactobacillus vaginalis, lactobacillus reuteri, lactobacillus johnsonii and bifidobacterium pseudocatenulatum, and more preferably the bacterial flora is enriched in lactobacillus murinus, lactobacillus enteric, lactobacillus vaginalis, lactobacillus reuteri, lactobacillus johnsonii and/or bifidobacterium pseudocatenulatum.
The enrichment refers to a higher abundance of the bacteria relative to a method without droplet encapsulation.
The invention has the good technical effects that:
1. the present invention creates a platform for the microfluidic system to isolate, screen and/or enrich bacteria with a total throughput of 5500 droplets per second and a diameter of about 14.20 ± 0.27 μm (mean ± sd), which allows isolation of strains by encapsulating individual strains in each droplet. Our platform has higher droplet throughput and produces smaller, more uniform droplets, which is more advantageous for screening strains than other methods.
2. The platform of the present invention can isolate strains in the fecal microbiota and wrap each strain in a single droplet, and then collect these empty droplets and droplets with single cells through a sealed tube for culture. The bacteria can maintain good activity and even proliferate in the droplets. In addition, the droplets eliminate growth competition between species, so that when propagated onto the conditioned medium, as many species can be screened as possible.
3. The bacteria separated by the method of the invention have higher abundance, can enrich lactobacillus, bifidobacterium, enterococcus and bacillus, and provide good, sufficient and reliable sources for further researching the functions of the flora.
4. The present invention creatively combines droplet-based microfluidics, bacterial fermentation and 16 srna gene sequencing technologies to screen populations of interest, such as potential anti-obesity probiotics in the gut microbiota that can interact with engineered probiotics. The EBPB fermentation product is taken as a screening condition, strains of four genera of lactobacillus, bifidobacterium, enterococcus and bacillus mouse are indeed enriched, and the strains are possible to be anti-obesity strains.
5. The invention utilizes the liquid drop micro-fluidic system to separate bacteria, and the processes of culture and identification have integrality and universality, so that the application of the liquid drop micro-fluidic system is simplified and practical.
The documents cited in the present invention are as follows:
1、Bai L.,Gao M.,Cheng X.,Kang G.,Cao X.,and Huang H.(2020).Engineered butyrate- producing bacteria prevents high-fat diet-induced obesity in mice.Microbial Cell Factories 19.94. doi:10.1186/s12934-020-01350-z。
2、Janda JM.,and Abbott SL.(2007).16S rRNA Gene Sequencing for Bacterial Identification in the Diagnostic Laboratory:Pluses,Perils,and Pitfalls.Journal of Clinical Microbiology 45.2761- 2764.doi:10.1128/jcm.01228-07。
3、Pearson WR.(2013).An Introduction to Sequence Similarity(“Homology”)Searching. Current Protocols in Bioinformatics 42.3.1.1-3.1.8.doi:10.1002/0471250953.bi0301s42。
4、Chung HJ.,et al.(2016).Intestinal removal of free fatty acids from hosts by Lactobacilli for the treatment of obesity.FEBS Open Bio 6.64-76.doi:10.1002/2211-5463.12024.
5、Lee CS.,et al.(2021).Antiobesity Effect of Novel Probiotic Strains in a Mouse Model of High-Fat Diet–Induced Obesity.Probiotics and Antimicrobial Proteins13.1054-1067. doi:10.1007/s12602-021-09752-0.
6、Sanchis-ChordàJ.,d et al.(2019).Bifidobacterium pseudocatenulatum CECT7765 supplementation improves inflammatory status in insulin-resistant obese children.European Journal of Nutrition 58.2789-2800.doi:10.1007/s00394-018-1828-5.
these documents are incorporated by reference in their entirety for the present invention.
Drawings
Fig. 1 is a schematic workflow diagram of a droplet-based microfluidic platform for screening potential probiotics.
Fig. 2 is a channel geometry of a microfluidic chip for generating droplets.
FIG. 3 shows encapsulation and OD of single cells in droplets600Graph of relationship between, wherein 3A represents different OD600Fluorescence micrograph of droplet encapsulation at the horizontal. 3B is at different OD600The percentage of x cells was found in each droplet of the level. 3C represents the percentage of droplets containing different cell numbers and OD600The relationship between them.
Fig. 4 is a light field micrograph of droplets produced from mineral oil containing 2% Span80 and a diameter distribution, where fig. 4A is the light field micrograph and fig. 4B is the diameter distribution.
Fig. 5 is a light field micrograph of droplets produced from mineral oil containing 5% Span80 and a diameter distribution, where fig. 5A is the light field micrograph and fig. 5B is the diameter distribution.
FIG. 6 is a comparison of fecal bacteria incubated on the plate with (D) or without (P) droplet encapsulation, three samples, P1, P2, P3, D1, D2, D3 in order. Wherein (A) the relative abundance of fecal bacteria (in order lactococcus, Streptococcus, Pseudomonas, enterococcus, Lactobacillus, Bacillus, Proteus, Escherichia) cultured on plates with (D) or without (P) droplet encapsulation. (B) Relative abundance of probiotic populations with (D) or without (P) droplet encapsulation (./P <0.05) were cultured on plates. (C) Comparison of indices of Chao1 for fecal bacteria cultured on plates with or without droplet encapsulation. (D) Comparison of shannon index of fecal bacteria cultured on plates with or without droplet encapsulation.
FIG. 7 shows a bacterium obtained by screening after EBPB conditioned medium culture, wherein (A) shows genus-related bacteria; (B) is a related bacterium; (C) is a comparison between different genera with the obese group.
FIG. 8 is the relative percentage of strains screened at the genus level.
FIG. 9 is a graph of the relationship between EBPB, enriched beneficial anti-obesity strains and obesity.
Detailed Description
The technical solution of the present invention is further described with reference to the following specific examples. It will be understood that the specific embodiments described herein are shown by way of example and not as limitations of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.
1. The main reagents and equipment involved in the examples are as follows:
escherichia coli BL 21: beijing Quanjin Biotechnology Ltd, Trans BL21(DE 3).
Butyric acid producing engineered bacteria (EBPB): constructed according to reference 1.
EBPB conditioned medium: EBPB was inoculated from a freshly transformed single colony on LB (Luria-Bertani, LB) agar plate into 5mL of liquid medium and cultured at 37 ℃ to OD600The value reaches 1. Then, 2mL of the strain was transformed into 200mL of fresh LB liquid medium and cultured at 37 ℃ and 220rpm for 24 hours. The fermentation supernatant was collected by centrifugation at 12000rpm for 10 minutes and then filtered through a 0.22- μm PES filter. Similar to the addition of antibiotics to the medium, when the temperature of the YCFA medium containing 3% agar, which can be sterilized by high temperature and high pressure, reaches 60 ℃, the supernatant and the YCFA medium are distributed into disposable plastic plates as Conditioned Medium Plates (CMPs) according to the ratio of 1: 1. These plates were stored at 4 ℃ for later use after agar solidification.
YCFA medium (1L): casein peptone, 10.0 g; yeast extract, 2.5 g; the concentration of glucose in the aqueous solution is controlled,2.0 g; 2.0g of soluble starch; cellobiose, 2.0 g; NaHCO 234.0 g; 1.0g of L-cysteine; k2HPO4,0.45g; KH2PO4,0.45g;NaCl,0.9g;MgSO4·7H2O,0.09g;CaCl20.09 g; resazurin, 1.0 mg; 1.0mg of heme; biotin, 1.0 μ g; cobalamin, 1.0 μ g; 3.0 mu g of p-aminobenzoic acid; folic acid, 5.0 μ g; pyridoxamine, 15.0 μ g; autoclaving at 115 deg.C for 15 min. Thiamine, 5.0 μ g; riboflavin, 5.0. mu.g, was added after filtration sterilization.
LB liquid medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, 1L ultrapure water preparation. 2. And (3) data analysis:
all statistical analyses were performed using GraphPad Prism 8.3.0(538) (GraphPad Software, San Diego, California USA, www.graphpad.com).
Example 1: manufacture of microfluidic chip
The schematic flow chart of the working process of the droplet-based microfluidic platform for screening potential probiotics is shown in fig. 1: after the bacterial sample is suspended, the microfluidic device encapsulates individual bacterial cells into droplets. The stable and uniform droplets were collected into Eppendorf tubes containing mineral oil. It is important to allow the droplets to be evenly distributed on the growth medium plate. All droplets burst and bacteria in the droplets can continue to grow on the plate. The species of the growing colonies were identified using 16s sequencing technology.
The structure of the chip comprises:
the device comprises two inlets, an outlet and a plurality of channels, wherein one of the two inlets is a continuous phase inlet and is positioned on the left side of a chip; one is a dispersed phase inlet and is positioned in the middle of the chip; one is a droplet outlet, located on the right side of the chip. The channels are respectively a rectangular continuous phase liquid inlet channel and a linear disperse phase liquid inlet channel, are respectively connected with a continuous phase inlet and a disperse phase inlet, are intersected with the cross-shaped flow focusing structure, extend forwards from the channels to be connected with an outlet from the cross-shaped flow focusing structure, and are vertically symmetrical in the whole chip structure.
The steps of preparing the chip include:
1) preparing a template: AutoCAD2016 software (AutoDesk) was used to design microfluidic device masks. The silicon molds used to make chips of this structure are performed in a clean room using common soft lithography techniques. Sequentially immersing a silicon plate into acetone, ultrasonically cleaning methanol and isopropanol for 5 minutes, drying by using nitrogen, performing surface treatment such as adding a correction mark and the like on the silicon plate, firstly spreading a layer of SU-8 photoresist on the silicon plate, then adding a photomask above the photoresist, performing ultraviolet exposure, then placing the photoresist in a developing solution, curing the SU-8 photoresist covered by the photomask, dissolving other regions, repeating the steps to obtain a chip channel, and finally performing surface treatment on the treated silicon wafer by using trichlorosilane to prolong the service life of the silicon wafer to obtain a template.
2) Preparing a PDMS mold: according to the weight ratio of 10:1, respectively weighing PDMS silica gel and a curing agent (Dow Corning SYLGARD 184), stirring to mix the two uniformly, pouring the uniformly mixed gel into a template to a proper height, putting the template into a vacuum apparatus for vacuumizing, and slowly connecting the vacuum apparatus with the atmosphere to ensure that all bubbles in the gel are broken and disappear.
Placing the foam-removed PDMS template into an oven, drying for more than 2h at 80 ℃ until PDMS is solidified, cutting off a PDMS chip containing a channel by using a scalpel, punching a hole at the inlet and the outlet of the channel of the PDMS chip by using a puncher with the outer diameter of 1.5mm, sequentially immersing a glass slide (the size of the glass slide is 25 multiplied by 75mm) into acetone, methanol and isopropanol, ultrasonically cleaning for 5min respectively, blow-drying by using nitrogen, placing the PDMS chip and the glass slide into the oven, drying for 15min at 80 ℃, and further removing the surface solvent residues.
3) Bonding PDMS to glass slide: and (3) carrying out surface treatment on the PDMS chip and the glass slide by using an oxygen plasma surface treatment instrument, wherein the vacuum degree of oxygen is set to be 0.4mbar, the treatment time is set to be 30s, bonding the treated PDMS chip and the glass slide, putting the bonded PDMS chip and the glass slide into an oven after bubbles automatically disappear, and drying the bonded PDMS chip and the glass slide for more than 8h at the temperature of 80 ℃ to obtain a crude product of the microfluidic chip.
And observing whether the channel of the crude microfluidic chip is complete under a microscope, particularly the cross-shaped flow focusing structure part, and selecting the crude microfluidic chip with the complete structure for further processing.
And removing the front and back of the needle head with the diameter of 1.6mm, filing and flattening, inserting into the inlet and outlet of the crude product of the microfluidic chip, and gluing the inlet and outlet needle head to prevent liquid leakage to obtain the microfluidic chip.
The microfluidic chip was a microfluidic chip with 10 μm (width) by 20 μm (height) cross channels (see fig. 2). The size of the chip channel is designed for the study of bacteria with small body types, so that the single bacteria packaging probability is improved, and the capacity is higher. Droplets are generated at the flow focus. Flow-focused droplet formation devices used in microfluidic devices are generally a passive formation method involving the flow of a dispersed phase meeting a continuous phase at an angle (indicated by the arrows) and then being constrained to produce droplets.
Example 2: dispersion phase condition optimization
Coli BL21 expressing Green Fluorescent Protein (GFP) was used to determine the most suitable fecal microbiota at 600nm (OD)600) To ensure single loading of each droplet. Construction of E.coli BL21 expressing Green Fluorescent Protein (GFP): and connecting the exogenous DNA fragment for expressing the GFP gene fragment to a pet vector, transforming Escherichia coli BL21, and then carrying out positive screening to obtain Escherichia coli BL21 for expressing GFP.
Coli BL21 was routinely cultured in LB liquid medium and LB agar plates at 37 ℃.
The GFP fluorescent E.coli BL21 cell suspension was diluted in PBS as a dispersion, and the cells were randomly packed in droplets (1.41-1.59 pL). The flow rates were all 0.5. mu.L/min.
All strains selected from the fecal samples were anaerobically cultured in YCFA broth at 37 ℃ and stored in YCFA medium containing 24% glycerol at-80 ℃.
3. As a result:
cell loading density is an effective method to control the number of encapsulated cells in a droplet, thus evaluating various ODs600Values to determine the optimal conditions for single cell encapsulation. The bacteria are difficult to be in the open field due to small sizeMicroscopic observation, therefore, GFP-expressing e.coli BL21 was used as a model strain to aid in the detection of individual bacterial cells in the droplets by fluorescence imaging. To ensure that there is no more than one cell per droplet, we set different loading densities (OD)600Values) of 0.01, 0.03, 0.06, 0.1, 0.6 and 1.0, respectively. As shown in FIG. 3A and Table 1, except that OD600Outside the value equal to 0.01, two cells are present in one droplet. In addition, we also make statistics of different ODs in detail600Percentage of cells in the droplet at the level. At OD600At a value equal to 0.01, 8% of the droplets contained one cell (fig. 3B). Albeit at OD600The percentage of droplets containing one cell at a value equal to 0.01 is significantly less than at OD600Values equal to 0.06, 0.1 and 0.6, but only the number of cells in the droplet at OD600The value is not more than one at 0.01, while at OD600At values equal to 0.03, there are droplets that encapsulate more than one cell less than 5%. We further compared the percentage of droplets containing different cell numbers with OD600The relationship between them. The results show that with OD600The number of droplets encapsulated by single cells increased and then decreased, and the number of droplets encapsulated by multiple cells gradually increased (fig. 3C). Measurement of intestinal flora OD600Diluting to 0.01-0.04, and regulating OD of intestinal flora600To ensure as much as possible that only one cell is encapsulated in the droplet, e.g., no more than 10% of droplets encapsulating more than one cell, and most preferably to ensure that each droplet contains no more than one cell, which is important for the formation of single-cell colonies.
Table 1: different OD600Corresponding drop cell number ratio example
Example 3: bacteria separation and continuous phase condition optimization based on micro-fluidic chip
The stool samples we used were previously collected from animal experiments (Bai et al, 2020). The day before the experiment the anaerobic incubator was opened and filled with an anaerobic gas mixture. Anaerobic incubator supplied with 85% N2/10%O2/5%H2A gas mixture. The fecal samples stored at-80 ℃ were removed and transferred to a pre-set anaerobic incubator. Monitoring of O Using anaerobic monitors2Concentration, O2The concentration is usually less than 1 ppm. After 2h anaerobic treatment, the fecal samples were lysed and suspended with liquid YCFA medium. To prevent clogging of the microfluidic chip, a 40 μm filter screen removes large food residues and other large particle mixtures from the stool sample.
Diluted bacterial suspension OD6000.01 enters the chip as a dispersed phase, which is further encapsulated by a continuous phase, resulting in droplets. The droplets are typically collected in Eppendorf tubes filled with mineral oil, which prevents the droplets from breaking.
Before screening the strains of interest, it is first ensured that the droplets produced are stable and homogeneous. A stable droplet means that the droplet does not break up over time and a uniform droplet helps to achieve single cell encapsulation. Surfactants play an important role in stable and uniform droplet formation. The main function of using surfactants is to reduce the interfacial tension between the dispersed and continuous phases by adsorbing on the interface and preventing the droplets from coalescing with each other, thereby stabilizing the droplets in a stable emulsion state, allowing longer storage in tubes, plates or vials.
To screen for appropriate surfactants and concentrations, we used Span80 as a surfactant, compatible with living cells and preserving cell viability.
We used 2% Span80 as a prior exploration. When 2% Span80 was used as the continuous phase in mineral oil, a small amount of droplet coalescence occurred. At this time, the droplet diameter is usually around 18.24. + -. 5.54. mu.m (see FIGS. 4A and 4B).
The Span80 functions to ensure stability and uniformity of the droplets. To explore a suitable range of concentrations, we further used 5% Span80 in mineral oil, and the results showed that uniform droplets were produced with increasing Span80 concentration from 2% to 5% in mineral oil, and that the droplets produced became more uniform with increasing concentration, with 5% Span80 having an average diameter of 14.20 ± 0.26 μm and smaller and more uniform droplet diameters (see fig. 5A, 5B).
The use of mineral oil with 5% Span80 added as the continuous phase is effective in reducing the occurrence of droplet coalescence, so the choice of mineral oil with 5% Span80 is preferred. At this time, droplets of 1.41-1.59pL with a diameter of 14.20. + -. 0.27. mu.m were produced at a production rate of 5500 droplets per second, which allows isolation of the strains by encapsulating a single strain in each droplet.
The above results show that: the microfluidic device of the present invention has higher droplet throughput and produces smaller droplets, which is more advantageous for screening strains than other methods. The device allows isolation of strains from the fecal microbiota and packaging of each strain in a single droplet, followed by collection of these empty droplets and droplets with single cells for culture via Eppendorf tubes. The bacteria can maintain good activity and even proliferate in the droplets.
Since too much amount of Span80 makes the continuous phase too viscous to flow in the chip channel. We recommend the amount of Span80 to be 2% -5%.
Example 4: comparison of growth abundance of fecal bacteria on the same YCFA plate
The bacterial suspension prepared by 5% Span80 in example 3 and the collected droplets encapsulating the cells were respectively laid on YCFA plates for anaerobic culture for 24 hours, then 100 colonies were scraped for 16srRNA gene sequencing, primers were directed to the V3-V4 region, and the growth abundance of the probiotic flora strains was compared.
Total genomic DNA was extracted from the samples according to the manufacturer's protocol. DNA concentration was monitored by the Qubit dsDNA HS Assay Kit. Sequencing libraries were constructed using the MetaVX library preparation kit. Briefly, 20-30ng of DNA was used to generate amplicons covering all hypervariable regions of the bacterial 16srRNA genes V3 and V4. The forward primer comprises the sequence "CCTACGGRRBGCASCAGKVRVGAAT" (SEQ ID NO:1) the reverse primer comprises the sequence "GGACTACNVGGGTWTCTAATCC" (SEQ ID NO: 2).
25ul PCR mix was prepared with 2.5ul TransStart buffer, 2ul dNTPs, 1ul of each primer, 0.5ul TransStart Taq DNA polymerase and 20ng template DNA.
PCR was performed according to the following procedure: denaturation at 94 ℃ for 3min, annealing at 95 ℃ for 5s, annealing at 57 ℃ for 90s, extension at 72 ℃ for 10s, and final extension at 72 ℃ for 5min for 24 cycles. The indexed adapters were added to the ends of the amplicons by limited cycle PCR. Finally, the library was purified using magnetic beads. The concentration was measured by a microplate reader, and the fragment size was measured by 1.5% agarose gel electrophoresis, which was expected to be about 600 bp. Sequencing was performed on the Illumina Miseq/Novaseq platform.
The results are shown in FIG. 6:
we compared the abundance of all bacteria including lactococcus, streptococcus, pseudomonas, enterococcus, lactobacillus, bacillus, proteus, escherichia, etc., with or without droplet encapsulation, and found that the present droplet encapsulation method resulted in increased abundance of bacteria in lactococcus, streptococcus, escherichia, lactobacillus, and enterococcus (see fig. 6A).
Comparing probiotics (including lactobacillus and bifidobacterium), the method disclosed by the invention has the advantages that the probiotics obtained after the liquid drop encapsulation and the probiotics obtained after the non-liquid drop encapsulation have significant difference, the method disclosed by the invention is greatly enriched in the probiotics, and the relative abundance can reach 27.8% (see fig. 6B).
Meanwhile, we also compared the Chao1 index for indicating the abundance size of flora and the Shannon index for indicating the diversity size of flora, and the results show that the bacteria obtained after the liquid drop encapsulation of the invention are higher than the bacteria without encapsulation (see fig. 6C and 6D) in both the abundance of bacteria (the Chao1 index reaches 10) and the diversity of bacteria (the Shannon index reaches 1.91).
According to the list of probiotics released by the ministry of health in china that are available for food, the probiotic flora is mainly from the genera bifidobacterium and lactobacillus. Clearly, it is prudent to select strains from both genera to screen for potential probiotics. However, some intestinal bacteria are difficult to culture by conventional culture methods due to low abundance, slow growth rate, interspecies competition, etc. Using our approach, the results show an increased population abundance and diversity as measured by the Chao1 index and shannon index, compared to fecal bacteria cultured on the same plate without droplet encapsulation. In addition, we compared the effect of the two methods on the abundance of such species, and the results show that our strategy can significantly improve the abundance of this probiotic group required by the ministry of health in china. Importantly, the liquid drop can isolate interspecies competition, and the nutrient medium in the liquid drop can enable bacteria to propagate, so that the biomass of the bacteria is increased, the possibility of culturing strains is increased, and the invention also proves that the target flora can be obtained in a greater abundance after the single-cell liquid drop is packaged.
Example 5: screening and characterization of all the strains obtained
Colonies on the conditioned medium plate CMPs were picked into 20mL of medium and anaerobically cultured at 37 ℃ for 12 hours. Total DNA was extracted using the TIANAmp Bacteria DNA Kit. Extraction was performed according to the instructions of TIANAmp Bacteria DNA Kit. PCR using the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') (SEQ ID NO:3) and 1492R (5'-GGTTACCTTGTTACGACTT-3') (SEQ ID NO:4) covered almost the full-length 16SrRNA gene.
PCR reactions were performed in 50. mu.L volumes, each containing 2. mu.L of 10. mu.M forward and reverse primers, 25. mu.L of 2 × PhantaMax buffer, 17. mu.L of ddH2O, 1. mu.l of Phanta Max Super-Fidelity DNA polymerase, 1. mu.l of NTPMix and 2. mu.l of LDNA as templates.
The thermal cycling was performed as follows: 41 cycles (95 ℃, 15 seconds; 55 ℃, 15 seconds; 72 ℃, 60 seconds), initial denaturation at 95 ℃ for 3 minutes, and final extension at 72 ℃ for 5 minutes. Equal amounts of PCR products were mixed according to the concentration of the PCR products. Then, the PCR product is purified and sequenced to obtain the gene sequence. Finally, the gene sequences were submitted to NCBI to identify the strains. Sequence annotation and database searches for sequence similarity were performed using the BLAST tool available online. Typically, the 16 srna gene nucleotide sequences of these strains have 99% to 100% sequence similarity to the reference strain in NCBI GenBank, considered as different strains of the same species (Janda & Abbott, 2007).
As a control, the abundance of obesity-associated bacteria (obesity group) was selected by the method described in reference 1.
The results are shown in fig. 7 and table 2:
TABLE 2 sequencing analysis results of 16SrRNA genes of representative strains
Similarity is used to describe the degree of similarity of a query sequence to a target sequence. The higher the similarity, the higher the degree of matching (Pearson, 2013).
After EBPB conditioned medium is utilized, eleven strains (Bacillus cereus, Bacillus naersonii, Bacillus umbellifer, Bacillus circulans, enterococcus and Bacillus) of four genera (Lactobacillus cereus, Bacillus naersonii, Bacillus umbellifer, Bacillus circulans, enterococcus faecalis, Lactobacillus murinus, Lactobacillus enteric, Lactobacillus vaginalis, Lactobacillus reuteri, Lactobacillus johnsonii and Bifidobacterium pseudocatenulatum) with high abundance are obtained. Wherein, the abundances of lactobacillus, bifidobacterium, enterococcus and bacillus can reach 42%, 1%, 33% and 10% respectively, eleven strains comprise bacillus cereus, bacillus naersonii, bacillus umbellifer, bacillus circulans, enterococcus faecalis, lactobacillus murinus, lactobacillus entericus, lactobacillus vaginalis, lactobacillus reuteri, lactobacillus johnsonii and bifidobacterium pseudocatenulatum, and the abundances of the lactobacillus can reach 3%, 27%, 1%, 2%, 10%, 26%, 2%, 6%, 4%, 4% and 1% respectively (see fig. 7A and B).
Compared with the Lactobacillus, Bifidobacterium, Bacillus and enterococcus screened by the method in the literature 1 (obesity group), the Lactobacillus and Bifidobacterium, especially the Bifidobacterium screened by the present invention were significantly improved (see FIG. 7C).
After the droplet microfluidic platform was constructed and completed, we attempted to use the platform to screen potential probiotics that could be anti-obesity. The butyric acid yield of the engineered butyric acid producing bacteria (EBPB) constructed previously reaches 1.5g/L, and the weight gain of mice induced by high fat diet can be effectively reduced. Furthermore, metagenomic results indicate that EBPB alters the composition of the gut microbiota, promoting the abundance of some bacterial species. Obesity is associated with the intestinal microbiota. EBPB is a dry prognosis, a strain with a partial increase in the intestinal tract can be a candidate for an anti-obesity strain. However, isolation, culture and screening of this group of bacteria is a prerequisite for the detection of anti-obesity function and therapeutic potential. Eleven species belonging to four genera were obtained using our platform, from the genera lactobacillus, bifidobacterium, enterococcus and bacillus. Of course, lactobacillus and bifidobacterium are of greater interest.
Example 6: screening potential anti-obesity strains by using platform and functional analysis
As above, 11 species belonging to 4 genera were obtained using our platform, including lactobacillus, bifidobacterium, enterococcus and bacillus.
Interestingly, in previous studies, EBPB increased the abundance of lactobacilli and bifidobacteria in vivo experiments. Thus, strains from the genera lactobacillus and bifidobacterium, including lactobacillus murinus, lactobacillus enteric, lactobacillus vaginalis, lactobacillus reuteri, lactobacillus johnsonii and bifidobacterium pseudocatenulatum, are of interest.
Not all strains obtained from CMPs using the present microfluidic device may be used as alternatives to anti-obesity probiotics. As shown in fig. 8, the results of metagenome of abundance changes of the four genera (enterococcus, bacillus, bifidobacterium, lactobacillus) examined above. The grey bands indicate increased abundance and the white bands indicate no increase, specifically EBPB can increase the abundance of lactobacilli and bifidobacteria in previous experiments (figure 8), therefore we focused on these species and further reviewed the literature on probiotics and obesity and found that lactobacillus reuteri, lactobacillus johnsonii and bifidobacterium pseudocatenulatum were used to reduce obesity (table 3).
TABLE 3 study of the related functions of Lactobacillus reuteri, Lactobacillus johnsonii and Bifidobacterium pseudocatenulatum
It can be seen that our strategy helps to improve the efficiency of screening anti-obesity probiotics. We believe that the supernatant of EBPB in CMPs exerts a screening pressure on fecal bacteria and is enriched for lactobacillus reuteri, lactobacillus johnsonii and bifidobacterium pseudocatenulatum, which may be selected as candidates for anti-obesity probiotics. And further verifies the anti-obesity mechanism function with reference to fig. 9. EBPB intervention increased the abundance of certain species in the mouse gut, which could be a potential anti-obesity strain. However, the isolation, culture and screening of this fraction of bacteria does allow us to test the function and therapeutic potential of microorganisms. Thus, the platform was created and applied to obtain lactobacillus reuteri, lactobacillus johnsonii and bifidobacterium pseudocatenulatum, suggesting that these isolates may interact with EBPB.
The results show that our method can provide guidance for screening for bacteria in the gut microbiota that can interact with engineered probiotics.
Example 7: the platform is used for separating bacteria from other samples
Firstly, separating and enriching bacteria in a milk sample:
10ml milk samples were taken, centrifuged at 6000rpm for 5 minutes, the supernatant was removed, resuspended in PBS buffer and the OD diluted600To a value of 0.01. With the concentration of the continuous phase span80 being 3%, referring to other embodiments 3-4, the single bacterium is wrapped and separated by the droplet microfluidic chip provided by the invention, the collected droplets wrapped with the single bacterium are coated or streaked on a BHI medium plate for 24h constant temperature culture, and finally sequencing and identification are carried out to obtain three strains of lactococcus lactis, enterococcus faecalis and Bacillus belgii, wherein the abundances can reach 15%, 20% and 20% respectively.
II, separating and enriching bacteria in the environmental sample:
placing crushed stone in a centrifuge tube, adding PBS buffer solution for shaking, taking supernatant and diluting OD600Value to 0.03, continuous phaseThe span80 concentration is 4%, other refer to the embodiment 3-4, the single bacterium encapsulation and separation are realized by the droplet microfluidic chip of the invention, the collected droplet coated with the single bacterium is coated or streaked on a BHI culture medium flat plate for 24h constant temperature culture, and finally sequencing and identification are carried out to obtain two strains of high-abundance bacillus altitudinis and bacillus megaterium, and the abundance can reach 20% and 30% respectively.
Therefore, the microfluidic device or the system provided by the invention can be suitable for separating and enriching bacteria of various samples, and can further enrich target flora by combining with a conditioned medium.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention can be made, and the same should be considered as the disclosure of the present invention as long as the idea of the present invention is not violated.
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Claims (10)
1. The microfluidic device is characterized by comprising a microfluidic chip, wherein the microfluidic chip comprises two inlets, an outlet and a plurality of channels, the two inlets are respectively a dispersed phase inlet and a continuous phase inlet, and the channels comprise rectangular continuous phase liquid inlet channels and linear dispersed phase liquid inlet channels.
2. A microfluidic system comprising the microfluidic device of claim 1, and a continuous phase.
3. The microfluidic system of claim 2, wherein the continuous phase comprises mineral oil and Span 80.
4. Microfluidic system according to any of claims 2 to 3, wherein the system comprises a culture unit for bacteria and/or an identification unit for bacteria.
5. Use of a microfluidic device according to claim 1 or a microfluidic system according to any of claims 2 to 4 for the isolation of bacteria.
6. A method for separating bacteria, comprising separating bacteria using the microfluidic device of claim 1 or the microfluidic system of any one of claims 2-4.
7. The separation method according to claim 6, characterized in that it comprises the steps of: 1) the bacterial suspension enters through a dispersed phase inlet; 2) the continuous phase enters through a continuous phase inlet, and 3) a droplet of a continuous phase packaging dispersed phase is formed in the microfluidic chip; 4) collecting the individual droplets, preferably, said step 1) comprises adjusting the OD of the bacterial suspension600The value is such that the bacteria are encapsulated by single cells into single cell droplets.
8. The isolation method according to any one of claims 6 to 7, wherein the bacterial sample in the bacterial suspension in step 1) is derived from a tissue sample of an organism or an environmental sample.
9. The separation method according to any one of claims 6 to 8, wherein the separation method comprises step 5): and (3) bacterial culture: culturing the droplet, and/or, step 6) identifying the bacteria.
10. A bacterial flora produced by the isolation method of any of claims 6-9, preferably enriched in bacteria comprising one or more of the genera lactobacillus, bifidobacterium, enterococcus and bacillus.
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