Preparation method of single-cell liquid drops
Polyacrylamide-encapsulated single-cell droplets were prepared by adding specific chemical agents and emulsified oil systems to the fecal cell suspension, which solved the problem of low cell loss and activity in traditional methods, and achieved efficient and economical preparation of fecal cell suspension and high-throughput experimental support.
Patent Information
- Application Number
- CN202510167517.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-16
- Publication Date
- 2025-05-09
AI Technical Summary
The traditional method of preparing fecal cell suspension has problems such as large cell losses, low cell activity, and high impurity content. The microbial DNA in fecal samples is prone to degradation during preservation and transportation, which affects subsequent experimental analysis.
Using a single-cell droplet preparation method, an aqueous suspension was prepared by adding a specific chemical agent to a suspension of aqueous fecal bacteria with a cell concentration of 460,000 per microliter, and evenly dispersed in the oil through a specific emulsified oil system to form single-cell droplets wrapped in polyacrylamide.
It realizes efficient and economical preparation of fecal cell suspension, ensures the accuracy and high throughput of experiments, provides a solution of applicability and flexibility, and provides support for the template preparation of single-cell fusion gene technology.
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Figure CN119955898A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microbial detection. Background Art
[0002] In biomedical research and clinical diagnosis, stool samples have broad application prospects as a non-invasive source of biomarkers. However, the cell composition in stool samples is complex and easily affected by environmental factors, which leads to decreased cell activity and stability, thus affecting subsequent experimental results and diagnostic accuracy.
[0003] Traditional methods for preparing fecal cell suspensions usually involve simple mechanical disruption and centrifugation steps, but this method often suffers from problems such as large cell loss, low cell activity, and high impurity content. In addition, the microbial DNA in fecal samples is also prone to degradation during storage and transportation, further affecting subsequent experimental analysis. Summary of the invention
[0004] In view of this, the present invention discloses a method for preparing a single cell droplet, comprising the following steps: adding a 10% mass concentration of ammonium persulfate solution, a 9.4% mass concentration of acrylamide solution and a 0.25% mass concentration of N,N'-bis(acryloyl)cystamine acrylamide solution to 30 µL of an aqueous fecal bacterial suspension with a cell concentration of 460,000 per microliter to prepare an aqueous suspension; adding the aqueous suspension dropwise at a uniform speed to a mixed emulsified oil, and vortexing to obtain an oil-in-water system; the mixed emulsified oil comprises 45 mL of Span 80, 4 mL of Tween 80, 0.5 mL of polyethylene glycol monooctylphenyl ether and 950.5 mL of mineral oil; adding 25 μL tetramethylethylenediamine was added, vortexed and allowed to stand to obtain an oil-in-water single-cell system; water-saturated diethyl ether was added to the oil-in-water single-cell system and immediately inverted to mix to form a visible precipitate, the upper liquid of the precipitate was removed, and the oil layer was removed after repeated washing with pure water, and then the water layer was removed to obtain a single-cell droplet wrapped with polyacrylamide.
[0005] Furthermore, the preparation method of the aqueous fecal bacterial suspension includes the following steps: diluting a fecal sample with 25% Ringer's solution to obtain an initial fecal suspension; grinding and filtering the initial fecal suspension to obtain a fecal filtrate; treating the fecal filtrate with 25% Ringer's solution, and filtering through a 40 μm cell filter to obtain a fecal secondary filtrate; ultrasonicating the fecal secondary filtrate for 45 seconds and then vortexing for 10 seconds, repeating 9 cycles to obtain an aqueous fecal bacterial suspension.
[0006] Furthermore, the 25% Ringer's solution includes the following ingredients: 2.250 g sodium chloride, 0.105 g potassium chloride, 0.120 g calcium chloride and 0.050 g sodium bicarbonate.
[0007] Compared with the prior art, the present invention has the following beneficial effects: using the method provided by the present invention, in particular, preparing an oil-in-water emulsified system with the aid of a specific emulsified oil, the emulsified oil is used as a continuous phase, and the aqueous suspension is used as a dispersed phase, so that the suspension is evenly dispersed in the oil in the form of tiny droplets. Therefore, the present method can not only ensure accuracy, but also achieve high throughput, and is an economical and efficient method for preparing a fecal cell suspension, providing an applicable and flexible solution for the preparation of templates for single-cell fusion gene technology. BRIEF DESCRIPTION OF THE DRAWINGS
[0008] Figure 1 It is a mixed solution of aqueous suspension and emulsified oil.
[0009] Figure 2 It is an oil-in-water single-cell system wrapped by polyacrylamide.
[0010] Figure 3 It is a single-cell droplet encapsulated by polyacrylamide. DETAILED DESCRIPTION
[0011] Example
[0012] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail and completely in conjunction with specific embodiments below, and the advantages and features of the present invention will become clearer as the description progresses. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.
[0013] The method for preparing fecal cell suspension comprises the following specific steps:
[0014] (1) Dilute the stool sample to obtain the initial stool suspension:
[0015] The stool sample was diluted at a ratio of 1:10 (volume ratio), that is, 0.200 g of fresh stool sample was accurately weighed into a 10 ml sterile centrifuge tube and diluted to 2 ml with 25% Ringer's solution (Table 1). The stool was fully soaked and evenly soaked, and impurities such as undigested food residues could be removed without resistance using a 25-gauge needle. The stool was stirred evenly until there were no obvious large particles, and the initial stool suspension was obtained.
[0016] Table 1 Preparation of 25% Ringer's solution (1 / 4 Ringer)
[0017]
[0018] (2) Grind and filter the initial fecal suspension to obtain fecal filtrate:
[0019] The insoluble fecal particles were carefully ground into a paste using a 1 ml syringe plunger as a grinding rod, and the initial fecal suspension was preliminarily filtered through a 70 μm cell filter. The filtrate was collected in a sterile centrifuge tube to obtain the fecal filtrate.
[0020] If the filter is clogged, remove the residue as necessary to ensure that the filtered suspension is free of any impurities.
[0021] (3) After treating the fecal filtrate with 25% Ringer's solution, filter it through a 40 μm cell filter to obtain the fecal secondary filtrate:
[0022] The fecal filtrate was diluted to a volume of 5 ml with 25% Ringer's solution, and then the fecal filtrate was circulated and filtered using a 1 ml syringe. The obtained filtrate was collected in a sterile centrifuge tube. This process was repeated 10 times. The obtained filtrate was filtered through a 40 μm cell filter to obtain a secondary fecal filtrate and the obtained filtrate was collected in a sterile centrifuge tube.
[0023] (4) The fecal secondary filtrate was sonicated for 45 seconds and then vortexed for 10 seconds, and the process was repeated 9 times to obtain an aqueous fecal bacterial suspension:
[0024] The ultrasonic power of the ultrasonic oscillator was set to 360 watts, the ultrasonic frequency was set to 50 kHz, and the ultrasonic temperature was set to 20°C. The fecal secondary filtrate was ultrasonicated and taken out after 45 seconds each time. The fecal suspension was vortexed at the maximum vortex speed of the vortex oscillator for 10 seconds each time. The above ultrasonic and vortexing process was repeated for 9 cycles. After treatment, an aqueous fecal bacterial suspension was obtained, and the acoustic energy density of the fecal suspension was about 16 kilojoules per liter. At this point, the aqueous fecal bacterial suspension was prepared.
[0025] (5) Quantification of cells in aqueous fecal bacterial suspensions:
[0026] The 16S rRNA gene (target fragment of 193 bp) was quantified by SYBR GREEN real-time fluorescent quantitative polymerase chain amplification reaction to count the number of cells in aqueous fecal bacterial suspensions.
[0027] The reaction system of fluorescence quantitative PCR was 20 μL, including 1× SuperReal PreMix Plus (with SYBRGreen I) (TIANGEN BIOTECH CO., LTD), 2 μL aqueous fecal bacterial suspension and 0.3 μM forward and reverse primers (Table 2, 341F and 534R).
[0028] The reaction procedure adopted a two-step method: pre-denaturation at 95 °C for 15 min (1×), denaturation at 95 °C for 10 sec, annealing at 50 °C for 30 sec (40×), and melting curve analysis (50 °C - 95 °C, increasing by 0.5 °C every 5 sec).
[0029] The standard curve was obtained by amplifying the standard plasmid by 10-fold gradient dilution. 2 It should be greater than 0.99, and the amplification efficiency should be in the range of 90% - 110%. Three replicates should be set for each group of standards and samples, and a negative control experiment should be performed at the same time.
[0030] After the reaction was completed, the corresponding copy number of the 16S rRNA gene was obtained as the number of cells in the aqueous fecal bacterial suspension: 460,000 per microliter.
[0031] Table 2 Fluorescence quantitative PCR primers (16S rRNA gene)
[0032]
[0033] (6) Preparation of aqueous suspension:
[0034] 30 µL of an aqueous fecal bacterial suspension with a cell concentration of 460,000 cells per microliter was taken as the base solution of the aqueous suspension. On this basis, 25 µL of a 10% ammonium persulfate solution and 200 µL of an acrylamide solution containing a 9.4% acrylamide solution and a 0.25% N,N'-bis(acryloyl)cystamine solution were added to prepare an aqueous suspension (a total of 255 µL).
[0035] (7) Preparation of water-in-oil system:
[0036] Prepare Span-Tween-polyethylene glycol monooctylphenyl ether mixed emulsified oil (Table 3).
[0037] Take a sterile 2 mL round-bottom microcentrifuge tube and add 600 µL of the mixed emulsified oil. On this basis, add the aqueous suspension dropwise into the mixed emulsified oil at a uniform speed to complete the water-oil mixing.
[0038] The water-oil mixed solution was vortexed by a vortex oscillator, and the vortex speed was set to 3000 rpm and the time was set to 30 seconds. The vortexing process can produce about 500 million droplets (calculated based on the average droplet diameter of 10 µm), achieve complete mixing of the aqueous suspension and emulsified oil in the water-oil mixed solution, and fully and evenly disperse the oil droplets and water droplets, completing the preparation of the oil-in-water system based on a single cell.
[0039] Table 3 Preparation of Span-Tween-Polyethylene glycol monooctylphenyl ether mixed emulsified oil
[0040]
[0041] (8) Preparation of oil-in-water single cell system:
[0042] 25 μL of tetramethylethylenediamine was added to the oil-in-water system, and the mixture was vortexed evenly using a vortex oscillator, with the vortex speed set to 3000 rpm and the time set to 30 seconds. The emulsion was then left to stand for 90 minutes, and acrylamide was polymerized to form polyacrylamide, thereby obtaining an oil-in-water single cell system wrapped by polyacrylamide.
[0043] (9) Preparation of polyacrylamide-encapsulated single-cell droplets:
[0044] Add 800 µL of water-saturated diethyl ether to the 2 mL centrifuge tube of the oil-in-water single cell system, and immediately invert the mixed emulsion and diethyl ether to form a visible precipitate. Carefully aspirate the oil and ether mixture around the precipitate with a pipette, add 1 mL of ultrapure water (nuclease-free), and invert the centrifuge tube to mix. Transfer all the liquid to a new 2 mL centrifuge tube, set the speed to 12000 xg, and centrifuge for 30 s. After centrifugation, an upper oil layer, a middle water-oil mixed layer, and a transparent polyacrylamide single cell system are formed.
[0045] Use a pipette to remove the upper oil layer, add 1 mL of ultrapure water and mix by inversion, then set the speed to 12000 xg and centrifuge for 30 seconds. The process of transferring the oil layer and adding water to wash needs to be repeated until no oil layer is formed (about 5 times).
[0046] When no oil layer is formed in the last water washing, the water layer is sucked out with a pipette and discarded. At this time, the separation of oil in the oil-in-water system is completed, and single-cell droplets wrapped with polyacrylamide are obtained.
[0047] Compared with the prior art, the present invention has the following beneficial effects: using the method provided by the present invention, in particular, preparing an oil-in-water emulsified system with the aid of a specific emulsified oil, the emulsified oil is used as a continuous phase, and the aqueous suspension is used as a dispersed phase, so that the suspension is evenly dispersed in the oil in the form of tiny droplets. Therefore, the present method can not only ensure accuracy, but also achieve high throughput, and is an economical and efficient method for preparing a fecal cell suspension, providing an applicable and flexible solution for the preparation of templates for single-cell fusion gene technology.
[0048] Finally, a person skilled in the art should understand that the above examples are only specific embodiments of the present invention, and are not intended to indicate that the scope of the present disclosure (including the claims) is limited to the preparation of a stool sample cell suspension. Therefore, any omission, modification, equivalent substitution, improvement, etc. of the method for constructing a cell suspension within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for preparing a single cell droplet, characterized in that: The steps include: To 30 µL of an aqueous fecal bacterial suspension having a cell concentration of 460,000 cells per microliter, a 10% mass concentration ammonium persulfate solution, a 9.4% mass concentration acrylamide solution, and a 0.25% mass concentration N,N'-bis(acryloyl)cystamine acrylamide solution were added to prepare an aqueous suspension; The aqueous suspension was added dropwise and at a uniform speed to the mixed emulsified oil, and the mixed emulsified oil was vortexed to obtain a water-in-oil system; the mixed emulsified oil included 45 mL of Span 80, 4 mL of Tween 80, 0.5 mL of polyethylene glycol monooctylphenyl ether and 950.5 mL of mineral oil; Add 25 μL of tetramethylethylenediamine to the oil-in-water system, vortex and let stand to obtain the oil-in-water single-cell system; After adding water-saturated diethyl ether to the oil-in-water single-cell system, the mixture is immediately inverted to form a visible precipitate. The upper liquid of the precipitate is removed, and the oil layer is removed after repeated washing with pure water. The water layer is then removed to obtain a single-cell droplet wrapped in polyacrylamide.
2. The method for preparing a single cell droplet according to claim 1, characterized in that: The method for preparing the aqueous fecal bacterial suspension comprises the following steps: The stool sample was diluted with 25% Ringer's solution to obtain the initial stool suspension; Grinding and filtering the initial fecal suspension to obtain a fecal filtrate; After treating the fecal filtrate with 25% Ringer's solution, it was filtered through a 40 μm cell filter to obtain a fecal secondary filtrate; The fecal secondary filtrate was sonicated for 45 seconds and then vortexed for 10 seconds, and this was repeated for 9 cycles to obtain an aqueous fecal bacterial suspension.
3. The method for preparing a single cell droplet according to claim 2, characterized in that: The 25% Ringer's solution includes the following components: 2.250 g sodium chloride, 0.105 g potassium chloride, 0.120 g calcium chloride and 0.050 g sodium bicarbonate.