Antifungal screening on chip

Introduction to antifungal screening

Pressure-driven flow controlled droplet-based microfluidics and single spore encapsulation offer the key for a breakthrough in antifungal screening and fungicide discovery. 

This application note focuses on an application of droplet-based microfluidics through a platform for single-spore analysis and high-throughput antifungal screening.

Fungicide discovery requires screening of a large number of candidates for evaluating their antifungal efficacy, cytotoxicity and other possible effects. Those experiments result in high experimental costs and require complex in vitro and in vivo studies until the development of commercial products, thus creating a major bottleneck to the advancement of antifungal discoveries and new screening technologies.
Indeed, many pathogens develop resistance against fungicides, leading to the need for sensitivity screening of fungicides to assess their efficiency [1-2]. The conventional techniques for this type of screening, like water agar and 96-well plate assays,  are time consuming, laborious, based on bulk methods and prevent single spore level analysis. Considering the importance of standard high throughput screening (HTS) methods for fungicide sensitivity testing, a microfluidic platform is a powerful tool for large-scale, automated antifungal screening and sensitivity studies.

Advantages of microfluidics of antifungal screening

Droplet-based microfluidics are an increasingly interesting alternative to conventional microtiter plate approaches for high-throughput screening (HTS) of fungi against various antifungal agents. Droplet-based antifungal screening provides various advantages:

• Easy handling 
• Reproducibility
• Less time consuming
• Downsizing a bench-top laboratory to a chip
• Low reagent consumption 
• High-throughput (HT) analysis
• Reduced analysis time
• Monitoring with high spatial and temporal resolution
• Observing the dynamic behaviour of many spores
• Spore-to-spore variation in a heterogeneous fungal population
• Protection against harsh external environments
• Physical and chemical isolation of the spore
• Reduced chances of contamination against foreign organisms

Principle of antifungal screening using droplet based microfluidics

The droplet-based microfluidic platform introduced in this application note consists of: 

1. Encapsulation at the single-spore level of filamentous fungi Alternaria alternata into droplets, 
2. Trapping and storage of droplets inside the microfluidic chip,
3. Readout over time by optical analysis.

To generate and trap monodisperse droplets, a polycarbonate (PC) microfluidic chip (fluidic 719 from microfluidic ChipShop, Germany) is used. This microfluidic chip allows the co-encapsulation of the spores with the reagent mix by a flow focusing junction. The OB1 pressure controller with flow sensors, tubings and connectors are used to reliably generate monodisperse droplets (CV < 3%) in a high-throughput manner.

Figure 1: Picture of the microfluidic platform including (from left to right): an OB1 flow controller with three channels pressurizing three 15 mL tubes, carrying the different solutions through the three flow sensors to the Fluidic 719 microfluidic chip.

Antifungal screening setup diagram

Materials

Hardware

1. OB1 flow controller with at least three 0–2000 mbar channels
2. 3 x Flow sensor MFS 3D 2.4–80 µL/min
3. Kit starter pack Luer Lock + 1/32”OD tubing with sleeves
4. 3 x 50 mL Falcon reservoirs
5. Microfluidic chip (Fluidic 719 with 1 pack of mini luer connectors and 1 pack of mini luer plugs from microfluidic ChipShop)
6. Microscope for observation
7. High-speed camera for imaging (optional)

Chemicals

1. HFE-7500 + 1 % FluoSurf surfactant (Emulseo, France)
2. Aquapel (Autoserv, Germany) for the hydrophobic treatment
3. Tween 20 (Sigma Aldrich)
4. Medium (Potato dextrose broth, Dutscher)
5. Spore suspension (Alternaria alternata)
6. Kunshi/tezuma fungicide (Belchim Crop Protection France S.A.,)

Design of the antifungal screening chip

Fluidic 719: Droplet generation and storage chips

Figure 2: Overview of the Fluidic 719 microfluidic chip (a) picture of the microfluidic chip, (b) droplet generation using three inlets ((1) spore suspension inlet, (2) fungicide inlet and (3) oil inlet), (c-f) droplets trapped inside the storage positions from 500 to 50 µm scale

Antifungal screening on chip quick start guide

Instrument connection

• Connect your OB1 pressure controller to an external pressure supply using pneumatic tubing, and to a computer using a USB cable. For detailed instructions on OB1 pressure controller setup, please read the OB1 user guide.
• Connect the flow sensors to the OB1. For more details, refer to the MFS user guide.
• Turn on the OB1 by pressing the power switch.
• Launch the Elveflow software. The Elveflow Smart Interface’s main features and options are covered in the ESI user guide. Please refer to the guide for a detailed description.
• Press Add instrument \ choose OB1 \ set as MK3+, set pressure channels if needed, give a name to the instrument and press OK to save changes. Your OB1 should now be on the list of recognized devices.
• OB1 calibration is required for the first use. Please refer to the OB1 user guide.
• Add the flow sensors: press Add sensor \ select flow sensor \ analog or digital (choose the working range of flow rate for the sensor if you have an analog one), give a name to the sensor, select to which device and channel the sensor is connected and press OK to save the changes. Your flow sensor should be on the list of recognized devices. For details refer to MFS user guide.
• Open the OB1 Window.

Solution preparation

• For the spore suspension solution: pour 50 mL of potato dextrose broth (with 0.1% Tween 20 to avoid clumping of spores together) 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 the media.
• Filter the spore suspension using nylon filtration tissue NITEX, mesh opening 50 µm (Dutscher, France) (for A. alternata).

TIP: The encapsulation rate of a single element inside each droplet can be estimated by using the Poisson law distribution where the mean number of elements in the volume of each droplet is λ = [0.05; 0.1].

• For the fungicide solution: the commercially available fungicide, kunshi/tezuma (Composition: 375 g.kg^-1 fluazinam + 250 g.kg^-1 cymoxanil) (Belchim Crop Protection France S.A.,) is used in its commercial form to prepare a stock solution (200 μg.mL^-1) in dimethylsulfoxide (DMSO) (1%). The initial concentration of kunshi (antifungal agent) is adjusted to get the desired concentrations 6, 4, 2, 1, 0.5, 0.25, and 0.015 μg.μL^-1 in the droplets.

TIP: Use double concentration of fungicide for the initial solution as the final concentration of fungicide will be half of the initial concentration used. For example, if you want to have a final concentration of 5 μg μL^-1 in the droplets, you will use 10 μg μL^-1 fungicide concentration.

• Prepare the oil solution reservoir by adding a volume of HFE-7500 + 1% FluoSurf surfactant and connect the supplied 1/32” OD tubing and the 4 mm OD coil tubing to the tank. For more details, refer to the video “connector for the OB1”.
• Repeat Step 5 for the spore suspension and fungicide solutions for this experiment.
• Treat the microfluidic chip with an hydrophobic treatment, like aquapel (Autoserv, Germany).

TIP: Aquapel is a solution that crystallises when in contact with air. The best treatment is to flush the chip with: (i) argon, (ii) aquapel (iii) argon and (iv) HFE-7500 oil.

Antifungal screening

• For flow measurement, connect the flow sensors between the microfluidic reservoirs and the chip. For more details, refer to the “MFS user guide”.
• Plug microfluidic tanks to the corresponding OB1 pressure controller outlet. For more details, refer to “Elveflow microfluidic reservoirs assembly”.
• Set a low pressure (50 mbar for example) of the oil solution until the solution starts dripping out of the tubing and then connect the tubing to the corresponding inlet. Fill the microfluidic chip with oil.

TIP: For secure insertion of tubing into the chip, it is recommended to gently press the luer connector to avoid any leaking. Do not press with force, otherwise it can damage the connectors and the microfluidic chip.

• Repeat Step 3 for the spore suspension and the fungicide solutions.
• Apply a pressure of 210 mbar for the oil solution and a pressure of 200 mbar for the spore suspension and fungicide solutions to start the generation of 100 µm droplets (important to keep the ratio 1:1 between spore suspension and fungicide solutions to ensure a 50-50 ration within the droplets).

TIP: The size of 100 µm droplets has been determined as optimal for the encapsulation of Alternaria alternata in the previous application note. For more details, refer to the application note “How to perform microfluidic single spore encapsulation?”.

• The droplets are trapped in the storage position while the experiment is ongoing.

Figure 3: Droplets confined in micron size storage positions within the Fluidic 719 microfluidic chip. Three out of five are encapsulated with one single spore of Alternaria alternata.

Results

The antifungal efficacy of kunshi (antifungal agent) was tested against spore germination of Alternaria alternata via the encapsulation of single spores into droplets with a specific concentration of the antifungal agent. A total of seven different concentrations of kunshi ranging from 0.015 µg µL^-1 to 6 µg µL^-1 were analysed.

The 6 µg µL^-1 concentration showed the maximum germination reduction rate (98.619%) after 4h of incubation which was significantly higher than the control (P < 0.05). The least reduction of spore germination (6.905%) was obtained at the concentration of 0.015 µg µL^-1 and was significantly higher than the control (P < 0.05). Also, the reduction rate between two time points for each concentration was not significantly different from each other (P < 0.05).

Figure 4: In vitro antifungal activity of kunshi (antifungal agent) against A. alternata in droplet based assay. The spores of A. alternata after treatment with fungicide at 4 h and 24 h incubation (a) Control (4h), (b) Control (24h), (c) 0.015 µg µL^-1 (4h), (d) 0.015 µg µL^-1 (24h), (e) 6 µg µL^-1 (4h), (f) 6 µg µL^-1 (24h), (g) Percentage inhibition of kunshi against A. alternata. The bars with the same letters are not significantly different (P < 0.05; Statistix 8.1). Error bars represent ± standard error of means (n = 2 for each concentration with n=1 representing 100 droplets).

To validate the results from the platform for antifungal screening, similar experiments were performed using water agar and 96-well plate assay (conventional methods in the literature for antifungal screening). The results obtained by water agar and 96-well plate screening assays were correlated to the results obtained by the droplet-based screening assay (article in submission).

Two videos are available to show the single spore encapsulation in monodisperse droplets (CV < 3%) and the trapping of those droplets in the fluidic 719 microfluidic chip.

References

Acknowledgements

Application note written by Sehrish IFTIKHAR – This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 843162 (project MAHT-FunSST).