Lindsay A. Legendre

A fully integrated microfluidic genetic analysis system with sample-in–answer-out capability

We describe a microfluidic genetic analysis system that represents a previously undescribed integrated microfluidic device capable of accepting whole blood as a crude biological sample with the endpoint generation of a genetic profile. Upon loading the sample, the glass microfluidic genetic analysis system device carries out on-chip DNA purification and PCR-based amplification, followed by separation and detection in a manner that allows for microliter samples to be screened for infectious pathogens with sample-in–answer-out results in <30 min. A single syringe pump delivers sample/reagents to the chip for nucleic acid purification from a biological sample. Elastomeric membrane valving isolates each distinct functional region of the device and, together with resistive flow, directs purified DNA and PCR reagents from the extraction domain into a 550-nl chamber for rapid target sequence PCR amplification. Repeated pressure-based injections of nanoliter aliquots of amplicon (along with the DNA sizing standard) allow electrophoretic separation and detection to provide DNA fragment size information. The presence of Bacillus anthracis (anthrax) in 750 nl of whole blood from living asymptomatic infected mice and of Bordetella pertussis in 1 μl of nasal aspirate from a patient suspected of having whooping cough are confirmed by the resultant genetic profile.

Purification of Nucleic Acids in Microfluidic Devices

The functionality of micropillars, microposts, silica beads, silica particles, sol−gels, and porous monoliths provides a framework for sample preparation and analysis for an integrated microfluidic system.

An integrated microfluidic device for DNA purification and PCR amplification of STR fragments

This work presents the integration of DNA extraction from complex samples and PCR amplification of STR fragments in a valveless, glass microdevice, using commercially available kits and instrumentation. DNA extraction was performed using a microchannel packed with a silica solid phase and a standard syringe pump as a single pressure source driving the extraction process, followed by integrated, online microchip amplification of STR fragments in a total volume of 1.2 microL. Reported characteristics important to this work include the capacity of the device for purification of DNA from a complex biological sample (whole blood) and the timing of DNA elution from the silica solid phase for successful downstream PCR amplification by placement the microdevice into a conventional thermocycler. Potential application of this microdevice to forensic genetic analysis was demonstrated through the preliminary extraction of DNA from semen, followed by an integrated, multiplexed, on-chip amplification that yielded detectable STR amplicons. By utilizing conventional laboratory equipment, the device presented exploits the benefits of microfluidic systems without complex control systems.

Toward a Simplified Microfluidic Device for Ultra-fast Genetic Analysis with Sample-In/Answer-Out Capability: Application to T-Cell Lymphoma Diagnosis

If microfluidic devices capable of rapid genetic analysis are to affect clinical diagnostics, they ultimately must be capable of carrying out more than ultra-rapid electrophoretic separations. The last half decade has seen a groundswell of activity in defining miniaturized DNA sample preparation methodologies that can be integrated with chip-based electrophoretic separations. Successfull integration of PCR-based DNA amplification and solid-phase DNA sets the stage for integrated microminiaturized analytical systems with sample in-answer out capabilities. Here we provide a brief review of the state of the art on the microfluidic integration of sample preparation processes with discussion of several systems with highly integrated capabilities, including one capable of detection of infectious agents present in complex biofluids in less than 30 min. This overview is used as a launch point to discuss the design and functionality of similar devices capable of accepting a whole blood or fine-needle aspirate sample, purifying the DNA, amplifying target sequences of the T-cell receptor-γ gene, and eletrophoretically resolving the products for detection of a signature consistent with monoclonality. We describe the details of the early experimental success in defining the individual chip-based processes required for an integrated T-cell lymphoma chip, with a vision to a device that provide sample in-answer out capabilities for diagnosing certain blood cancers in roughly 1 h.

Rapid DNA Amplification in Glass Microdevices

The polymerase chain reaction (PCR) for amplification of DNA has become a very useful tool in scientific research and analytical laboratories, yet conventional techniques are time-consuming, and the reagents are expensive. Miniaturization of this technique has the potential to drastically reduce amplification time and reagent consumption while simultaneously improving the efficiency of the reaction. Increasing the surface area-to-volume ratio using microfluidic reaction chambers allows homogeneous solution temperatures to be achieved much more rapidly than in conventional heating blocks. Employing infrared radiation to selectively heat the reaction solution can additionally reduce the time and energy needed for thermocycling; the reaction container is not heated and can even serve as a heat sink for enhancement of cooling. Microchip systems also provide the potential for fabrication of structures for additional processing steps directly in line with the PCR chamber. Not only can amplification be integrated with product separation and analysis, but sample preparation steps can also be incorporated prior to amplification. The ultimate goal is a miniature total-analysis-system with seamlessly coupled sample-in/answer-out capabilities that consumes very low volumes of reagents and drastically reduces the time for analysis. This chapter will focus on the materials and methods involved in simple straight-channel microchip PCR on glass substrates using non-contact thermocycling.