Lab on a Chip Blog

Researchers at Virginia Tech create an elegant device to perform DNA amplification starting from whole cells by taking advantage of diffusivity differences in PCR components.

Diffusion can be friend or foe in the microscale regime, depending on the application. For active mixing, relying on diffusion can lengthen reaction time and thereby decrease reaction efficiency. But for separating reaction products, low ratios of convection to diffusion (Péclet number) enable control over elements based on their diffusivity^[1]. Professors Luke Achenie and Chang Lu from the chemical engineering department at Virginia Tech took advantage of this diffusion-enabled control to combine cell lysis and PCR reactions in ‘one pot’ with temporal separation of how components add to the chamber due to diffusivity differences. Separation of cell lysis and DNA amplification steps in PCR is important as many traditional chemical reagents for cell lysis inhibit polymerases used in PCR and Phusion polymerases tolerant to surfactant lysis reagents are incompatible with downstream SYBR green dyes.

The device consists of a single reaction chamber connected on both sides to two separate loading chambers. A hydration line ensures minimal evaporation during the PCR cycle in the main chamber. The loading chambers are opened in sequence to let molecules into the reaction chamber via two-layer control valves. The substantial difference in reagent diffusivity in the lysis and amplification processes allow diffusion gradients to drive molecules from new solutions contacting the reaction chamber and replace reagents from previous steps without disturbing the DNA of interest. Taq polymerase and proteins are two orders of magnitude larger in diffusivity than typical (50 kb) DNA fragments, while primers, dNTPs, and lysis buffers are three orders smaller. Relying solely on diffusion to deliver reagents to the main chamber increases the time of the reaction, but this can be addressed by elevating the temperature or increasing concentration of starting reagents in the loading chambers.

The authors showed the functionality of their device with purified human genomic DNA as well as single cells. This work opens up new capabilities to perform multi-step preparation and amplification assays for DNA in a single chamber starting directly from few cells to a single cell.

Download the full article today – for free*

Diffusion-based microfluidic PCR for “one-pot” analysis of cells

Sai Ma, Despina Nelie Loufakis, Zhenning Cao, Yiwen Chang, Luke E Achenie and Chang Lu
DOI:10.1039/C4LC00498A

References: [1] T. M. Squires and S. R. Quake, Reviews of Modern Physics, 2005, 77, 977.

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About the Webwriter

Sasha is a PhD student in bioengineering working with Professor Beth Pruitt’s Microsystems lab at Stanford University. Her research focuses on evaluating relationships between cell geometry, intracellular structure, and force generation (contractility) in heart muscle cells. Outside the lab, Sasha enjoys hiking, kickboxing, and interactive science outreach.

In a recent paper in Lab on a Chip, a group of British researchers reported a ‘lab-in-a-briefcase’ for detection and quantification of the prostate cancer biomarker PSA in human serum and whole blood. Their lab-in-a-briefcase is a small container with a set of coated plastic capillaries, a pre-loaded microwell plate with reagents and a film scanner.

The researchers stress that their system is cheap and easy to handle, which would make it very useful for performing diagnostics in low resource areas. In addition, their lab-in-a-briefcase demonstrates the potential for point-of-care tests for prostate cancer, which would allow easy screening by non-experts in a non-clinical setting.

The concept of a lab-in-a-briefcase may have more far-reaching implications, though. Most lab-on-a-chip assays and microfluidic systems are usually developed in the context of interdisciplinary research collaborations. One research department may develop a new system, while another department has the – often unstable – samples that are used to demonstrate proof-of-concept. This complexity means that projects can quickly become logistic nightmares.

Multi-site collaborations make the portability and the standardized format that are found in the lab-in-a-briefcase and related technologies very important. It doesn’t matter if the application domain of a project is physics, biochemistry or biology. Developing a portable, standardized set-up with good documentation, automated analysis and easy read-out can contribute greatly to the success of a multi-disciplinary microfluidic engineering project, because it promotes collaboration and a wider application of the technology early on.

All of this means that the lab-in-a-briefcase is not just a niche product that is only useful for cheap point-of-care diagnostics in low resource areas. It is a design concept that anyone in the realm of microfluidic engineering needs to understand. Perhaps the concept is also applicable to the project you’re currently working on?

Go check it out for yourselves – you can download this paper fro free* for a limited time only!