Conference Agenda

Microdroplets in Microfluidics: towards a new tool for single cell experiments

Wilhelm Huck

Radboud University, Netherlands, The

In recent years there has been an enormous interest in exploiting droplet-based microfluidic devices for performing on-chip biochemical reactions including enzyme kinetics, protein crystallization, PCR and in vitro translation and transcription. Nanoliter droplets of water in oil emulsions can be created inside microfluidic devices and can be merged, split and sorted using electric fields, while the contents can be analyzed with sensitive optical techniques. The very high throughput of these devices, up to 10,000 per second, drives research in this area to include directed evolution on-chip and single cell gene expression experiments.

In my talk, I will present some of our recent results on in vitro translation and transcription inside droplets, parallel enzymatic reactions, emulsion separation on chip, coupling of fluorescence detection with sorting and mass spectrometric analysis of droplets contents, and progress in developing tools for single cell ‘cellulomics’.

Recent publications

Simultaneous Determination of Gene Expression and Enzymatic Activity in Individual Bacterial Cells in Microdroplet Compartments

J. U. Shim, L. F. Olguin, G. Whyte, D. Scott, A. Babtie, C. Abell, W. T. S. Huck, F. Hollfelder

J. Am. Chem. Soc. 2009, 131, 15251-15256.

Coupling Microdroplet Microreactors with Mass Spectrometry: Reading the Contents of Single Droplets Online

L. M. Fidalgo, G. Whyte, B.T. Ruotolo J. L. P. Benesch, F. Stengel, C. Abell, C. V. Robinson, W. T. S. Huck

Angew. Chem. Int. 2009, 48, 3665-3668

From Microdroplets to Microfluidics: Selective Emulsion Separation in Microfluidic Devices

L. M. Fidalgo, G. Whyte, D. Bratton, C. F. Kaminski, C. Abell, W. T. S. Huck

Angew. Chem. Int. 2008, 47, 2042-2045

Microtechnologies: New Dimensions for Cytometry

Jonathan West

ISAS, Germany

Single cell analysis has been underpinned by the microscope and the flow cell cytometer (FCC). Microscopy images stationary cells to reveal spatial information with sub-cellular resolution, whereas FCC is a high throughput continuous flow process for describing the population by capturing single cell events. The two technologies have continued to evolve to the present day state-of-the-art. In parallel with these developments planar array and fluidic microtechnologies have emerged as complementary tools which can bring new qualities and quantities of information to modern cytometry. In my presentation I will illustrate the reach of microtechnologies by using examples from research in my laboratory which spans pharmaceutical and neurotoxicology screening as well as basic research concerning communication processes at the single cell level.

Patterning the adhesion microenvironment can be used for spatially standardizing the culture microenvironment [1] for reproducible and statistically powerful cell analytics. In the first case, uniform tumour spheroids were mass produced (>10000) [2] and the array dimensions were varied to modulate growth, metabolic and pathophysiological characteristics and the associated chemosensitivity traits [3]. In a second case, the neurite outgrowth assay, a classical morphological end-point measure for neurotoxicology was refined in the form of the network formation assay (NFA). Here, the array format eliminates length measurements and also assesses connectivity, a higher order predictor of functional impact than outgrowth alone [4]. The NFA was used for the rapid and reproducible assessment of the inhibitory and degenerative effects of neurotoxins, and the effects of neuroprotective agents [4,5].

Single cell arrays can also be prepared using deterministic microfluidic approaches. Using a novel valving concept where the cells naturally transform from a suspension to an adhesive state, we used this array platform for the high throughput pairing of single cells in readiness for investigating juxtacrine signalling [6]. Using similar fluidic principles a microfluidic circuit was developed for establishing compartmentalized neuronal co-cultures with one-to-one connectivity [7]. These are being used by our collaborators to investigate disease propagation following fluidically targeted chemical or pathogenic insults. Lastly, microfluidics presents the opportunity to define cellular reaction sequences with unprecedented temporal resolution. To this end we have developed a continuous flow single cell manipulation platform which resembles traditional FCC but instead is used to stimulate, incubate and capture ligand–receptor signal transduction events with millisecond resolution [8]. This opens the possibility of capturing the timing and sequence of molecular transitions occurring at the communication front between the cell and its microenvironment.

References

1. J.P. Frimat et al, Anal. Bioanal. Chem., 2009, 395(3), 601–609.

2. F. Hirschhaeuser et al, J. Biotechnol., 2010, 148, 3–15.

3. H. Hardelauf et al, Lab Chip, 2011, 11(3), 419–428.

4. J.P. Frimat et al, Lab Chip, 2010, 10, 701–709.

5. H. Hardelauf et al, Lab Chip, 2011, 11(16), 2763 – 2771.

6. J.P. Frimat et al, Lab Chip, 2011, 11(2), 231–237.

7. H. Hardelauf et al, 6th IEEE MMB Conference, Lucerne, Switzerland, 2011.

8. Y.Y. Chiang et al, 6th IEEE MMB Conference, Lucerne, Switzerland, 2011.

Sub-μm Fluidic Structures for Single Cell Trapping and Analysis of Prokaryotic Production Strains

Dietrich Kohlheyer

Forschungszentrum Juelich GmbH, Germany

This abstract describes a microfluidic chip to hydrodynamically capture single bacteria cells for long-term growth and production studies. The trapping and cultivation of single bacteria within a laminar flow, as well as fluorescence based lysine detection was successfully performed. The presented system is specially designed for parallel single cell experiments to determine growth rates and production heterogeneity. In-house developed industrial productions strains mainly based on E. coli and C. glutamicum will be used in further experiments. An important finding within this field was shown by Wang et al. in 2010. They developed a micro system having parallel growth channels to study growth behavior of E. coli [1]. The microfluidic system shown here, allows the application of chemical gradients for screening and heterogeneity studies. Furthermore, trapping of bacteria in one focal plane enables fluorescence based measurements of bacteria arrays in quasi high-throughput. Fully motorized live cell imaging microscopy and well controllable cell environment allows for long-term investigation in a time lapsed manner [2].

Using soft-lithographic methods, a disposable poly(dimethylsiloxane) (PDMS) microfluidic chip was fabricated, containing several arrays of traps for single bacteria capturing.

This work lays the foundation for future system biological and metabolic investigations such as cell heterogeneity studies or mutant screening of high content producing cells within our group and institute. It is expected to present latest results on single cell analysis within microfluidic devices.

1. Ping Wang, Lydia Robert, James Pelletier, Wei Lien Dang, Francois Taddei, Andrew Wright and Suckjoon Jun, Robust Growth of Escherichia coli. Current Biology, 2010. 20(12): p. 1099-1103.

2. Alexander Grünberger, Christopher Probst, Stephan Binder, Lothar Eggeling, Wolfgang Wiechert and Dietrich Kohlheyer, Single cell trapping and analysis of prokaryotic production strains in sub-μm fluidic structures, Proceedings of Micro TAS Conference 2011 (accepted for publication), October 2-6 2011, Seattle, USA

The Envirostat Chip - Contactless Isolation and Controlled Cultivation of Single Bacteria

Frederik Sven Ole Fritzsch¹, Katrin Rosenthal¹, Anna Kampert¹, Christian Dusny¹, Hendrik Kortmann¹, Oliver Frick², Lars Mathias Blank², Andreas Schmid^1,2

¹Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V., D-44227 Dortmund, Germany; ²Laboratory of Chemical Biotechnology, TU Dortmund University, D-44227 Dortmund, Germany

Cell to cell differences (cell heterogeneity) within a population can be analyzed by flow cytometry. Its opto based snapshots are however restricted to one point in time and hence do not allow time resolved investigations of single cells. Time resolved analysis of single cells during growth in a controlled environment enables deciphering of cellular mechanisms, such as cause and effect in regulation cascades. The analytical investigation of such mechanisms on the level of a single cell is challenging.

Rarely considered during single cell analysis, contact-free cultivation is desirable to avoid surface induced unknown changes in cell phenotype. Our recently developed Envirostat technology allows contactless and environmental controlled single cell cultivation employing negative dielectrophoresis (nDEP) for cell trapping in a flow of growth medium. The technology enables systematic perturbation experiments as carried out in many systems biology studies, but on the single cell level. Hence the experimentator is not relying on cell averages. The technology was used to analyze growth and protein secretion of single yeast. Until now, it was not possible to use contactless nDEP cultivation for the majority of bacteria because of their small cell size.

We will present a nDEP microfluidic chip that overcomes this limitation with a sophisticated microelectrode and microchannel design, developed and characterized using computational modeling. Microelectrode geometries optimized for bacteria cell dimensions and designed microfluidics enables: first, single bacteria isolation from other cells, secondly, a fluid removal of dead cells and residues of media used for population cultivation and third a precise environmental controlled nDEP trapping of a chosen cell in a microchamber for single cell cultivation. The contactless cultivated single bacterium is continuously in a flow of medium which can be used for environmental controlled perturbation experiments. Subsequent sampling of secreted molecules can be taken by an on-tube-seal microfluidic connection and for example be analyzed by mass spectroscopy. Finally, the analyzed single cell or daughter cell can be transferred in a cultivation well to retrieve a population. Importantly, the manufactured chip is biocompatible and enables isolation and cultivation of single bacteria in an environment defined by the experimentator and therefore overcomes major limitations in microfluidic based single bacteria analysis.