Trends in Biotechnology
ReviewIntegrated microfluidic systems for high-performance genetic analysis
Introduction
With the completion of the reference human genome sequence and additional individual sequences 1, 2, 3, even more ambitious goals for future genome-related research are being planned. Exploring the implications of genome variation for speciation, evolution, and disease 4, 5, studying gene expression and regulation at the single-cell level 6, 7, as well as improving forensic and clinical genetic analysis 8, 9, are now on the horizon. Technologies that will enable these advances of genetic analysis must be fast and inexpensive, have high sensitivity, and provide flexible and robust platforms.
Automated genetic analysis has advanced significantly in the past decade through the application of robotics, but several intrinsic drawbacks are increasingly evident. First, the liquid-handling limits of robotic analytical techniques are usually in the microliter scale, which not only consumes expensive reagents, but also leads to inevitable sample dilution. For example, to analyze a single gene in a cell, we conventionally put it into a working volume of 10 μL, which results in extreme dilution down to <10–18 M. This is problematic because the most sensitive systems for DNA detection typically require concentrations in the femtomolar to picomolar range [10]. Second, during conventional genetic analysis, samples are transferred between multiple instruments, which can cause further sample dilution and loss. For instance, in DNA capillary electrophoresis (CE) analysis, the loaded sample is typically 1–2 μL, but only ∼2 nL of this sample volume is effectively injected into the capillary for separation and detection [11]. Third, contamination issues become prominent when dealing with low-copy-number or single-cell samples because contaminants can overwhelm the real target signals. Current analytical processes which have multiple open sample transfer steps make contamination inevitable [12]. Paradoxically, the final analytical systems for genetic analysis typically do not require a large amount of sample. For example, only 106–107 molecules are sufficient for CE detection. While robotics provides the macro integration of analytical processes which can address some of the problems mentioned above, a fully integrated and automatic system that operates on the nanoliter scale would enable improved performance.
Section snippets
Integrated Microfluidic Systems
Micro-total analysis systems have the potential to overcome all problems mentioned above due to their capability of integrating multiple analytical steps into a single microdevice at the pL–nL volume scale using microfabrication technology. The advantages provided by such a “laboratory-on-a-chip” system are recognized as high-speed, high-throughput, low reagent consumption, and reduction of instrument size 13, 14. Moreover, the limited diffusion distances and concentrated reagents achieved by
Development of Integrated Microdevices
To develop fully integrated microsystems for gene expression and genetic analysis, four elements critically impact process integration: i) device material, ii) heaters and temperature sensors for thermal cycling of reactions, iii) microvalves for partitioning analytical steps, and iv) sample/product transport between analytical steps. The choices made in each of these areas determine the challenges and successes achieved in the integrated analytical system.
Applications of Integrated Microfluidic Devices
The development of fully integrated microfluidic devices is advancing rapidly. This development has led to significant achievements in the areas of DNA sequencing, gene expression analysis, pathogen detection, and forensic STR typing, which are discussed in detail below.
Conclusions and Prospects
Over the past two decades, microfluidic devices for genetic and gene expression analysis have advanced rapidly. Most of the analytical steps have been successfully translated into chip formats where they demonstrated at least ten-times better performance over conventional counterparts. However, thus far, microfluidic systems are primarily utilized by the academic research community. We believe fully integrated microfluidic systems that contain all the necessary analytical components and provide
Acknowledgements
We thank Robert G. Blazej, Palani Kumaresan, and Samantha A. Cronier for providing figures of the MINDS bioprocessor. We also thank Richard Novak, Samantha A. Cronier, Yong Zeng, Amy Twite and Erik C. Jensen for valuable discussions. This work was supported by grant number 2007-DN-BX-K142 awarded by the National Institute of Justice, Office of Justice Programs, US Department of Justice. The opinions in this document are those of the authors and do not necessarily represent the official position
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