Lab on a Chip Blog

In a recent paper in Lab on a Chip, which featured as the Cover Article of Issue 22, Vol 13, researchers from the Ecole Polytechnique, Palaiseau, France describe a micro-droplet based method for measuring reaction kinetics using tiny (20 nL) sample volumes.

When working with precious reagents such as enzymes, minimizing sample volume is of the utmost importance.  Droplet-based microfluidic methods can greatly reduce sample volumes, but still waste considerable amounts of sample in fluid handling processes (e.g. pushing the sample through a channel).  A team led by Marten Vos and Charles Baroud has utilized a unique “rails and anchors” method¹ of forming and controlling the movement of droplets to observe the kinetics of a chemical reaction using only 20 nL of each reagent solution.

In this scheme, the flow chamber geometry is carefully designed so that when an aqueous solution is injected into the oil-filled chamber, droplets of a reproducible size are released and constrained between the top and bottom surfaces of the chamber.  Then, widening grooves (“rails”) in the bottom of the chamber guide the droplets via surface tension to “anchors” in the center.  (See figure a and b.)  In this way, no additional solution is wasted pushing droplets around.

Droplet-based scheme for observing reaction kinetics (Fig 2 from paper)

The authors used the “rails and anchors” to place two droplets, each containing a different reagent, in proximity, and then used an IR laser pulse to break the surface tension between them (figure c). The interface between the two solutions (in this case, DCPIP and ascorbic acid) can clearly be seen in figure d below.  By tracking the front of the color change that moves across the droplet as the reaction proceeds (figure e), it is possible to calculate the reaction rate (accounting also for the diffusion rates of the reactants).

The researchers also demonstrated the potential of multiplexing their technique by monitoring six different reactions in parallel droplets. They were able to obtain results in good agreement with a commercial stopped-flow spectrometer with greatly reduced cost and time.

Read this HOT article in Lab on a Chip today!

Parallel Measurements of Reaction Kinetics using Ultralow Volumes, Etienne Fradet, Paul Abbyad, Marten H. Vos, and Charles N. Baroud, DOI: 0.1039/c3lc50768h

References:

1. P. Abbyad et al., Lab on a Chip 2011, 11, 813-821.

These HOT articles were recommended by our referees and are free to access for 4 weeks*

One-step microfluidic generation of pre-hatching embryo-like core–shell microcapsules for miniaturized 3D culture of pluripotent stem cells
Pranay Agarwal, Shuting Zhao, Peter Bielecki, Wei Rao, Jung Kyu Choi, Yi Zhao, Jianhua Yu, Wujie Zhang and Xiaoming He  
Lab Chip, 2013,13, 4525-4533
DOI: 10.1039/C3LC50678A

A microfluidic approach to synthesizing high-performance microfibers with tunable anhydrous proton conductivity
Mohammad Mahdi Hasani-Sadrabadi, Jules J. VanDersarl, Erfan Dashtimoghadam, Ghasem Bahlakeh, Fatemeh Sadat Majedi, Nassir Mokarram, Arnaud Bertsch, Karl I. Jacob and Philippe Renaud  
Lab Chip, 2013,13, 4549-4553
DOI: 10.1039/C3LC50862E

The microfluidic Kelvin water dropper
Álvaro G. Marín, Wim van Hoeve, Pablo García-Sánchez, Lingling Shui, Yanbo Xie, Marco A. Fontelos,  Jan C. T. Eijkel, Albert van den Berg and Detlef Lohs  
Lab Chip, 2013,13, 4503-4506
DOI: 10.1039/C3LC50832C

A cell rolling cytometer reveals the correlation between mesenchymal stem cell dynamic adhesion and differentiation state
Sungyoung Choi, Oren Levy, Mónica B. Coelho, Joaquim M. S. Cabral, Jeffrey M. Karp and Rohit Karnik  
Lab Chip, 2013, Advance Article
DOI: 10.1039/C3LC50923K

Ultrahigh-throughput sorting of microfluidic drops with flow cytometry
Shaun W. Lim and Adam R. Abate  
Lab Chip, 2013,13, 4563-4572
DOI: 10.1039/C3LC50736J

A programmable and reconfigurable microfluidic chip
Raphael Renaudot, Vincent Agache, Yves Fouillet, Guillaume Laffite, Emilie Bisceglia, Laurent Jalabert, Momoko Kumemura,   Dominique Collard and Hiroyuki Fujita  
Lab Chip, 2013,13, 4517-4524
DOI: 10.1039/C3LC50850A

The term ‘microfluidics’ immediately invokes the image of elegant, tiny devices with a small footprint in which fluids are manipulated with ease. It is an image that is most definitely correct. Still, every researcher in the field of microfluidics is aware of ‘the dirty little secret’ of these devices. Namely, that they often require bulky pumps and lots of tubing to be operated.

The requirement for macroscopic equipment to control microfluidic devices is a serious problem. It hinders the development of complex and integrated microfluidic circuits and it prevents the integration of the devices in small portable equipment.

Many organisms can direct their movements according to their chemical environment. In bacteria, this chemical-directed migration (i.e. chemotaxis) is enabled by receptor-mediated signaling to the bacterial flagellar motors.¹ Equipped with this remarkable sensory-motor system, bacteria can measure chemical gradients and swim towards nutritious substances (positive chemotaxis) or flee from cytotoxic chemicals (negative chemotaxis).

Bacterial chemotaxis is critical for various processes including biofilm formation, biomediation, and diseases pathogenesis.² For example, at the onset of various digestive diseases, bacteria rely on chemotaxis to find a suitable colonization site in the human gastrointestinal tract. Thus, there is great interest in studying the rates and molecular mechanisms in these chemotactic processes.

In the past 10 years, microfluidic gradient-generators have improved the way scientists study bacterial chemotaxis;³ however, these studies have not addressed gas gradients (e.g. O₂ and CO₂), which are known to be important in bacterial motility.⁴ Microfluidics devices have enabled the generation of well-controlled chemical gradients and the measurement of bacterial response at high spatiotemporal resolution. Gradients are typically formed by flowing a sample liquid (e.g. aspartate in buffer) and a reference liquid (e.g. buffer) into the device.

A team of researchers at the RIKEN Advanced Science Institute, Japan and Hanyang University, Korea carried out the world’s first microfluidic study of bacterial chemotaxis in gas gradients.⁵ The microfluidic device (see picture) comprises an isolated chamber (micro-aquarium) for bacterial culture and two bypass channels for sample and reference inputs. In this design, the sample molecules in the channel permeate through the PDMS walls (150 µm thick) and diffuse into the liquid of the micro-aquarium, facilitating the generation of a stable, shear-free gradient. The bacterial response can be monitored optically and quantified via image processing.

Using this device, the research team studied the chemotactic behaviour of Euglena gracilis microbial cells in the presence of CO₂ gradients. These studies revealed that the chemotaxis of Euglena cells is rapid and is dependent on CO₂ concentration. In particular, negative chemotaxis was observed at high concentrations (>15%) and positive chemotaxis was observed at low concentrations (<15%)— the maximum positive chemotaxis was observed at ~5% CO₂, corresponding to the most favourable condition for photosynthesis. Interestingly, the team observed evidence of chemotactic “adaption” when they consecutively switched the sample and reference inputs.

The microfluidic device is also compatible with small molecule liquid samples such as ethanol, H₂O₂, and culture media. By continuously perfusing culture medium in the bypass channels, the viability of the Euglena cells in the micro-aquarium can be maintained for more than 2 months.

In summary, the research team created a microfluidic gradient-generator that is versatile and straightforward to use. The device is useful for bacterial chemotaxis studies in chemical gradients formed from gas or liquid samples. Furthermore, the device may potentially be useful for other applications including drug screening, toxicity assays, and environmental monitoring.

1. H. C. Berg, Annual Review of Biochemistry, 2003, 72, 19-54.
2. T. Ahmed, T. S. Shimizu and R. Stocker, Integrative Biology, 2010, 2, 604-629.
3. J. Wu, X. Wu and F. Lin, Lab on a Chip, 2013, 13, 2484-2499.
4. C. Douarche, A. Buguin, H. Salman and A. Libchaber, Physical Review Letters, 2009, 102, 198101.
5. K. Ozasa, J. Lee, S. Song, M. Hara and M. Maeda, Gas/liquid sensing via chemotaxis of Euglena cells confined in an isolated micro-aquarium, Lab on a Chip, 2013, 13, 4033-4039

Researchers in Munich have adapted their molecular force assay technique to a microfluidic system, allowing the strength of protein-DNA interactions to be measured in a chip format.

The measurement of intermolecular forces is a challenging and important task. Two well-known methods, atomic force microscopy and optical trapping, are capable of probing these forces¹. However, these instrumentation-intensive techniques are not suitable for every application. A team from the Ludwig-Maximilians-Universität, Germany, headed by Hermann Gaub, a leader in the field of force spectroscopy, has now developed a technique, termed molecular force assay (MFA), to probe the strength of DNA hybridization and the effects of DNA-binding proteins².

Schematic of the molecular force assay (adapted from Figure 1 of paper)

In MFA, a surface is functionalized with a carefully designed DNA construct, as seen in the left panel of the figure. This surface is brought into contact with another surface which is coated with neutravidin (center). The neutravidin binds the biotin at the end of the DNA construct, so when the two surfaces are separated, the DNA construct rips apart (dehybridization, right). The breakage can happen in one of two ways—if the hybridization of the “probe strand” is stronger than that of the “reference strand”, the reference strand will dehybridize, leaving behind a Cy3 tag. If the hybridization of the reference strand is stronger, the probe strand will dehybridize, leaving both a Cy3 tag and a Cy5 tag behind. Via the resulting fluorescence, it is possible to compare the hybridization strength of the probe strand to that of the known reference. DNA binding proteins which modify the hybridization strength of the probe strand can then be introduced.

In the original formulation of MFA, the surfaces were brought into contact using a piezoelectric device; however, in a recent paper in Lab on a Chip, the researchers have translated their assay into a microfluidic format. A microfluidic chip was used in which the lower layer contains sample wells and the upper layer contains control valves. By pressurizing button valves above each well, the top and bottom surfaces can be brought into contact and then separated. The authors demonstrated the effect of EcoRI binding on probe strand hybridization strength as a proof-of-concept, “paving the way for studies of currently unknown protein-DNA interactions, including those of transcription factors.”

Read this HOT article in Lab on a Chip today!

Protein–DNA force assay in a microfluidic format, Marcus Otten, Philip Wolf, and Hermann E. Gaub. DOI: 10.1039/C3LC50830G

References:

1. K. C. Neuman and A. Nagy, Nature Methods, 2008, 5, 491-505.

2. P. M. D. Severin et al., Lab on a Chip, 2011, 11, 856-862.