How to perform microfluidic single spore encapsulation?

Introduction to microfluidic single spore encapsulation

This single spore encapsulation application note will show you how highly monodispersed droplets can be easily generated to encapsulate single spore of fungus using a microfluidic droplet generation system. For more insight into single spore encapsulation, please watch Sehrish Iftikhar‘s webinar presenting single spore encapsulation for phytopathogenic fungi or the review about fungus identification in microfluidics. A protocol on how to perform antifungal screening on chip using droplet based microfluidics is also available.

In this work, several experimental conditions have been tested to achieve the encapsulation of single spore of fungi within highly monodispersed droplets.

This technique can be adapted to encapsulate different type of fungus, with different spore size (ex: Alternaria alternata, A. tenuissima, Curvularia spp. Fusarium spp. etc.) or even cells.

We will first start by preparing a spore suspension solution at a precise concentration according to the Poisson Law distribution. Then microdroplets of this solution will be generated using a microfluidic system.

There are various advantages to single spore encapsulation, including protection against coarse external environment, physical and chemical isolation of spores, less chances of contamination against foreign organisms, enhanced isolation and fast and efficient reagent mixing.

To discover more tips and tricks about droplets generation and manipulation, please check our droplet Userguide!

Setup for single spore encapsulation

Microfluidic single spore encapsulation: list of components

Microfluidic single spore encapsulation: Materials

Hardware:

• OB1 flow controller with at least two channels 0/2000 mbar
• 2x flow sensors MFS3
• Kit starter pack Luer Lock + 1/32 tubings + 1/32 sleeves + 23G needles
• 2x 50 mL Falcon reservoirs
• Microfluidic chip (hydrophobic channels)
• Optical microscope for observation
• Fast camera to register droplets (optional)

Chemicals:

• 2% surfactant in HFE 7500 fluorinated oil (Emulseo)
• Tween 20 (Sigma Aldrich)

Design of the PDMS chip

PDMS droplet generation chip for single spore encapsulation: droplets are produced by flow-focusing of the aqueous stream with two streams of fluorinated oil containing surfactant (for more details please refer to White Paper: Droplet based microfluidics.)

Microfluidic single spore encapsulation: a quick start guide

Solutions preparation

Step 1: Prepare the spore suspension of fungus with required concentration using Poisson distribution. Pour 50 mL of 0.1% Tween 20 in 7-8 days old sporulating culture of fungus Alternaria alternata and scratch the surface with a glass rod to release and suspend the spores in water. Filter the spore suspension using nylon filtration tissue NITEX, mesh opening 50 µm (for A. alternata).

Tips from the expert:

Preparation of spore suspension:
Use a 7 days old culture of fungus to prepare the spore suspension.
Always filter the spore suspension to get rid of any mycelium and to avoid clogging of the channels.
Use mesh pore size depending on the size of the spore.
Use 0.1% tween to avoid clumping of spores together.

Poisson Law Distribution:
The encapsulation rate of single element inside each droplet can be estimated by using the Poisson law distribution (Collins et al, 2015) where the mean number of elements in the volume of each droplets is λ = [0.05;0.1].

Microdroplet generation

Standard droplet preparation procedure, here with spore suspension instead of water.

Step 2: Connect your OB1 pressure controller to an external pressure supply using pneumatic tubing, and to a computer using an USB cable. For detailed instructions on OB1 pressure controller setup, please read OB1 user guide.

Step 3: Fill your microfluidic tanks with dispersed (spore suspension) and continuous phase (2% surfactant diluted in HFE 7500 fluorinated oil (Emulseo).

Step 4: Plug microfluidic tanks to the OB1 pressure controller outlet. The Elveflow Reservoirs connection instructions are covered by a specific guide (see Elveflow Microfluidic Reservoirs Assembly Instructions).

Step 5: Turn on the OB1 by pressing the power switch on the front panel of the instrument.

Step 6: Launch the Elveflow software. The Elveflow Smart Interface’s main features and options are covered by the Smart Interface’s guide. Please refer to those guides for a detailed description.

Step 7: Press Add Instrument \ Choose OB1 \ set as MK3+, set pressure channels if needed, give name for the instrument and press OK to save changes. Your OB1 now should be in the list of recognized devices.

Step 8: Calibrate the OB1 calibration for the first use. Please refer to the OB1 user guide.

Step 9: Use the supplied 1/32” OD tubing to connect microfluidic tanks with the chip.

Tips from the expert:

Tubing connection to the chip:
For easier insertion of tubing into the PDMS chip, it is recommended to cut the tube at a slight angle, then use a flat (with ridges) tweezer to insert the tubing into PDMS. However, at high pressures, it is better to cut the tubing flat to make it hold better. At insertion, push the tubing until reaching the glass, then slightly pull back so as not to clog the channels with the tubing.

Step 10: Set pressures (and other parameters if needed) and start pumping liquids into the chip. Wait until air escapes from the chip and both liquids are flowing.
Change pressure of spore suspension channel to start generating monodispersed droplets with single spore. Their size and frequency will depend on the pressure, flow rate and viscosity of the liquids used. See droplet size/pressure diagram in the “Results” chapter.

Tips from the expert:

Chip priming:
To begin with, run liquids into tubes until the liquid starts to drip. Only then, connect them to the chip, starting from the continuous phase (in this case – oil).
Once the oil tubing is connected, apply a pressure of around 100 mbar and wait until the chip is filled with oil.
Only then plug the spore suspension channel (500 mbar could be used to flow in the tube, then after connecting, go back to 100 mbar).
To begin with droplet generation, start by putting both channels at 100 mbar.

Step 11: Collect the spore encapsulated droplets in the collection vial.
Here, the droplets were collected in collection tubes and imaged at 40x magnification. Each droplet forms an independent reaction vessel.

Tips from the expert:

Make sure that the output tube from the chip plunges in the liquid of the collection vial to avoid dripping.

Using the Elveflow® smart interface to generate controlled size droplets:

RESULTS

Single spore of Alternaria alternata encapsulated in droplet (100%)

DROPLET SIZE vs PRESSURE DIAGRAM