Introduction

The diffusion processes are slow, and the inertial effects are negligible on micro-scale with low Reynolds number ^{[Angelescu2011]}. Hence microfluidics enables complex control of the intercellular microenvironment. Microfluidic experiments in conjunction with live fluorescence microscopy were designed and performed to verify and characterize the signal transmission mechanism in the developed IOD band system, and to verify the developed yeast-induced blood agglutination mechanism.

Soft-lithography

Fabricated microfluidic devices (three PDMS molds bonded to single glass slide)

Microfluidic channels were formed using PDMS soft-lithography technology, which has proven biocompatible and readily applicable to available laminar flow cabinets ^[Fikar2015]. Photomask and silicon master fabrication was outsourced. Fabrication of microfluidic devices was divided into two subsequent steps. In the first step, silicon masters were used for the PDMS molding. In the second step, PDMS molds were bonded to the glass substrates to form encapsulated microfluidic devices using air plasma technology.

PDMS molding

PDMS molding workflow

Two-part silicone elastomer Sylgard 184 was used to produce PDMS. The base part was mixed with sufficient amount of curing agent (10:1 ratio). The mixture was centrifuged to remove air bubbles introduced by the mixing. The silicon master was placed in an aluminum foil container and the mixture was poured over. The remaining air bubbles were removed from the PDMS by the sharp tip of a needle. The poured PDMS was maintained in a perfectly horizontal position to assure good planarity and was cured in an oven for 2 hours at 80°C. The PDMS edges were cut off with a sharp tool and the PDMS was peeled off of the silicon master. The PDMS mold was sliced into sections containing individual devices. Inlets and outlets were drilled carefully by biopsy punch of the appropriate diameter at the desired locations of the PDMS replica. Detailed experimental protocol is provided here.

Bonding of PDMS to the glass substrate

Bonding process workflow

Prepared PDMS replicas with imprinted microstructures were cleaned properly with a scotch tape. The PDMS and glass substrate were treated by air plasma for 2.5 minutes. The air plasma affects the PDMS backbone and forms reactive silanol functional groups (Si-OH) enabling formation of permanent irreversible covalent bond of the PDMS to the glass substrate ^[Wong2009]. In addition, the PDMS treatment with air plasma is beneficial as it avoids non-specific adsorption, decreases cell clogging, and turns the PDMS hydrophilic. The hydrophilicity of PDMS facilitates the future microchannel wetting ^[Kalio2006]. Immediately after the air plasma treatment, the glass substrate was brought into contact with the PDMS replica and placed in the oven for 5 minutes at 80°C. The bonded devices were stored at room temperature. Inlets and outlets were sealed with scotch tape to avoid contamination. Detailed experimental protocol is provided here.

Experimental setup

Experimental setup

Microfluidic experiments were conducted on a platform already established by the [http://ccy.zcu.cz/ Georgiev lab]. The laboratory is equipped with a precise microfluidic syringe system (neMESYS Low Pressure Syringe Pumps) and a microscopic station enabling fluorescence imaging and live cell microscopy (Olympus IX83, CellSens software).

Microfluidic experiments

On-chip signal transmission characterization

Example filling of microfluidic channel.

Yeast pheromone pathway activation was characterised using microfluidic technology in conjunction with live fluorescence microscopy. Fluorescent reporter proteins were connected to specific functional products to show activation of the yeast pheromone pathway. Details on the synthesis and function of the used reporter plasmids are provided by Module 1. Synthesized reporter plasmids were transformed into specific yeast strains to enable characterization of the signal transmission between yeast cells using set of orthogonal signals designed and synthesized by Module 2.

Fluorescence microscopy image showing activation gradient of MATa cells yeast pheromone pathway with pFUS1 reporter by MATa cells producing synthetic alpha factor in the microfluidics.

Level of fluorescence and its dependence on the distance from transmitting cells. Performed on Saccharomyces cerevisiae wildtype pheromone signaling molecules and receptors.

Simple microfluidic devices with T shaped microchannels were used to characterise the signal transmission between selected yeast strains. Two cell types were selected for each experiment. One cell type transmits the signal in the form of pheromone molecules. The other cell type produces the green fluorescent protein (GFP) when the pheromone level excess certain threshold, and if the pheromone molecules are complementary to the other cells receptors. Both cell types are introduced to the microfluidic device in parallel through separated inlets. Due to the laminar nature of the flow, the individual cell types remain spatially separated inside the microfluidic device for the whole experiment. The flow is stopped when the microfluidic device is filled with cells. The rest of the experiment is performed statically. Time-lapse fluorescence microscopy is used to dynamically measure levels of GFP expression in the cells that receive the signal.

Signal transmission between Wildtype MATx ↝ MATa: STE2 receptor from Sacharomyces cerevisiae, and between MATa expressing Candida parapsilosis alpha-factor; \(\Delta\)Bar ↝ MATa: STE2 receptor from Candida parapsilosis was characterised. As a negative control, signal transmission between MATa expressing "Candida parapsilosis" alpha-factor; \(\Delta\)Bar ↝ MATa: STE2 receptor from Sacharomyces cerevisiae was studied. No significant level of fluorescence was observed suggesting these signals being orthogonal. The results show that the signal transmission range is approximately 100 μm.

Yeast induced blood agglutination on-chip

Illustration of microfluidic chip used for yeast-induced blood agglutination. No sophisticated mixing topologies were required due to the presence of the 100 μm wide channel ease. The eventual cell aggregates were observed in the subsequent chamber.

Standard agglutination tests are time consuming and require specialized equipment occupying large bench space ^[Choi2009]. Special microfluidic topologies were designed and fabricated to test yeast-induced agglutination on-chip to lower the time and sample volume requirements. Prior to the experiment, the synthetic yeast cells were perfused and dissolved in plasma, which is the natural medium for the IOD band diagnostic. Blood sample and yeast culture were introduced to the microfluidic chip through separate inlets, and were brought to contact in the chip. During the experiment, the flow was reversed few times to provide sufficient time for the cells to form the eventual cell aggregates, and to decrease the required sample volume. The overall experiment duration did not exceed 60 seconds. The amount of blood necessary for the experiment was approximately 5 μl.

Functioning of location tags was proven by cell clumping consistently observed in mixed samples of yeast cells with the anti-HuA location tag and red blood cells from a type A donor. The control samples, where anti-HuA location tag display was not induced, showed negligible aggregation. The experiments also prove the compatibility of yeast cells with blood samples as no significant clumping or other abnormalities that would indicate overall concept infeasibility were observed in control samples.

Personnel

• Martin Cienciala - Microfluidic experiments
• Vaclav Pelisek - Fabrication of microfluidic devices
• Pavel Fikar - Scientific advisor

References

1. ↑ Dan E. Angelescu (2011). Highly Integrated Microfluidic Design. Artech House, Norwood.
2. ↑ Ieong Wong (2009). Surface molecular property modifications for poly(dimethylsiloxane) (pdms) based microfluidic devices. Microfluid Nanofluid, 7:291–306.
3. ↑ Johana Kuncova-Kallio (2006). Pdms and its suitability for analytical microfluidic devices. In Proceedings of the 28th IEEE EMBS Annual International Conference, New York City, USA, 9, IEEE.
4. ↑ Sung Hwan Choi (2009). Microinjection molded disposable microfluidic lab-on-a-chip for efficient detection of agglutination. Microsystem Technologies, 15(2):309-316.
5. ↑ P. Fikar (2015). SU8 microchannels for live cell dielectrophoresis improvements. In Proceedings of the Design, Test, Integration and Packaging of MEMS/MOEMS Symposium, Montpellier, FR.