Trends in Biotechnology
Volume 27, Issue 10, October 2009, Pages 572-581
Journal home page for Trends in Biotechnology

Review
Integrated microfluidic systems for high-performance genetic analysis

https://doi.org/10.1016/j.tibtech.2009.07.002Get rights and content

Driven by the ambitious goals of genome-related research, fully integrated microfluidic systems have developed rapidly to advance biomolecular and, in particular, genetic analysis. To produce a microsystem with high performance, several key elements must be strategically chosen, including device materials, temperature control, microfluidic control, and sample/product transport integration. We review several significant examples of microfluidic integration in DNA sequencing, gene expression analysis, pathogen detection, and forensic short tandem repeat typing. The advantages of high speed, increased sensitivity, and enhanced reliability enable these integrated microsystems to address bioanalytical challenges such as single-copy DNA sequencing, single-cell gene expression analysis, pathogen detection, and forensic identification of humans in formats that enable large-scale and point-of-analysis applications.

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

References (72)

  • D.A. Wheeler

    The complete genome of an individual by massively parallel DNA sequencing

    Nature

    (2008)
  • E. Check

    Human genome: Patchwork people

    Nature

    (2005)
  • H. Ellegren

    Comparative genomics and the study of evolution by natural selection

    Mol Ecol

    (2008)
  • M. Acar

    Stochastic switching as a survival strategy in fluctuating environments

    Nat Genet

    (2008)
  • P.J. Choi

    A stochastic single-molecule event triggers phenotype switching of a bacterial cell

    Science

    (2008)
  • M.A. Jobling

    Encoded evidence: DNA in forensic analysis

    Nat. Rev. Genet.

    (2004)
  • D. Ivnitski

    Nucleic acid approaches for detection and identification of biological warfare and infectious disease agents

    Biotechniques

    (2003)
  • I. Rech

    Microchips and single-photon avalanche diodes for DNA separation with high sensitivity

    Electrophoresis

    (2006)
  • J.M. Butler

    Forensic DNA typing by capillary electrophoresis using the ABI Prism 310 and 3100 genetic analyzers for STR analysis

    Electrophoresis

    (2004)
  • D.J. Harrison

    Micromachining a Miniaturized Capillary Electrophoresis-Based Chemical-Analysis System on a Chip

    Science

    (1993)
  • K. Jensen

    Chemical kinetics - Smaller, faster chemistry

    Nature

    (1998)
  • R.G. Blazej

    Microfabricated bioprocessor for integrated nanoliter-scale Sanger DNA sequencing

    Proc. Natl. Acad. Sci. U. S. A.

    (2006)
  • N.J. Panaro

    Evaluation of DNA fragment sizing and quantification by the Agilent 2100 Bioanalyzer

    Clin. Chem.

    (2000)
  • S.R. Quake et al.

    From micro- to nanofabrication with soft materials

    Science

    (2000)
  • G.S. Fiorini et al.

    Disposable microfluidic devices: fabrication, function, and application

    Biotechniques

    (2005)
  • E.T. Lagally

    Fully integrated PCR-capillary electrophoresis microsystem for DNA analysis

    Lab Chip

    (2001)
  • M.A. Burns

    Microfabricated structures for integrated DNA analysis

    Proc. Natl. Acad. Sci. U. S. A.

    (1996)
  • A.F.R. Huhmer et al.

    Noncontact infrared-mediated thermocycling for effective polymerase chain reaction amplification of DNA in nanoliter volumes

    Anal. Chem.

    (2000)
  • R. Luharuka et al.

    A bistable electromagnetically actuated rotary gate microvalve

    J Micromech Microeng

    (2008)
  • E.T. Lagally

    Integrated portable genetic analysis microsystem for pathogen/infectious disease detection

    Anal. Chem.

    (2004)
  • J.S. Marcus

    Microfluidic single-cell mRNA isolation and analysis

    Anal. Chem.

    (2006)
  • D.J. Beebe

    Functional hydrogel structures for autonomous flow control inside microfluidic channels

    Nature

    (2000)
  • C. Yu

    Flow control valves for analytical microfluidic chips without mechanical parts based on thermally responsive monolithic polymers

    Anal. Chem.

    (2003)
  • R.H. Liu

    Validation of a fully integrated microfluidic array device for influenza A subtype identification and sequencing

    Anal. Chem.

    (2006)
  • B. Zhao

    Control and applications of immiscible liquids in microchannels

    J Am Chem Soc

    (2002)
  • C.G. Koh

    Integrating polymerase chain reaction, valving, and electrophoresis in a plastic device for bacterial detection

    Anal. Chem.

    (2003)
  • Cited by (124)

    • Field-based detection of biological samples for forensic analysis: Established techniques, novel tools, and future innovations

      2018, Forensic Science International
      Citation Excerpt :

      By the 2000’s, DNA-based detection methods had advanced significantly. Lab-on-a-chip technology was a much-publicised area of research, and as such by the end of the decade, several micro total analysis systems for forensic and clinical applications had been developed [43–45], accounting for the sharp rise in publications seen during this time span. Similarly, non-DNA based methods also became more sophisticated during this time, benefiting from advancements in engineering and miniaturisation of existing technology to allow for on-site usage.

    • Centrifugal microfluidic system for a fully automated N-fold serial dilution

      2018, Sensors and Actuators, B: Chemical
      Citation Excerpt :

      In particular, accurate and precise pipetting of the solution is troublesome when the sample volume is in microliter scale or below. Microfluidics has facilitated the integration and automation of various fluidic unit operations on a miniaturized chip and it results in fast, accurate and highly efficient manipulation of fluids in microliter scale volume [1–3]. On-chip serial dilution with both linear and non-linear shape of the concentration gradient have been also demonstrated [4–9].

    View all citing articles on Scopus
    View full text