Jungkyu Kim

Microfluidic sample preparation: cell lysis and nucleic acid purification.

Due to the lack of development in the area of sample preparation, few complete lab-on-a-chip systems have appeared in recent years that can deal with raw samples. Cell lysis and nucleic acid extraction systems are sufficiently complex even before adding the complexity of an analysis system. In this review, a variety of microfluidic sample preparation methods are discussed and evaluated. Microsystems for cell lysis are discussed by grouping them into categories based on their lysis mechanisms: mechanical, chemical, thermal or electrical. We classify the nucleic acid purification techniques according to the mechanism that links nucleic acids to substrates: silica-based surface affinity, electrostatic interaction, nanoporous membrane filtration, and functionalized microparticles. The techniques for microfluidic cell lysis and nucleic acid purification are compared based on the ease of microfabrication and integration, and sample flexibility. These assessments can help us determine the appropriate sample preparation technique for generating a true lab-on-a-chip.

Rapid prototyping of microfluidic systems using a PDMS/polymer tape composite

Rapid prototyping of microfluidic systems using a combination of double-sided tape and PDMS (polydimethylsiloxane) is introduced. PDMS is typically difficult to bond using adhesive tapes due to its hydrophobic nature and low surface energy. For this reason, PDMS is not compatible with the xurography method, which uses a knife plotter and various adhesive coated polymer tapes. To solve these problems, a PDMS/tape composite was developed and demonstrated in microfluidic applications. The PDMS/tape composite was created by spinning it to make a thin layer of PDMS over double-sided tape. Then the PDMS/tape composite was patterned to create channels using xurography, and bonded to a PDMS slab. After removing the backing paper from the tape, a complete microfluidic system could be created by placing the construct onto nearly any substrate; including glass, plastic or metal-coated glass/silicon substrates. The bond strength was shown to be sufficient for the pressures that occur in typical microfluidic channels used for chemical or biological analysis. This method was demonstrated in three applications: standard microfluidic channels and reactors, a microfluidic system with an integrated membrane, and an electrochemical biosensor. The PDMS/tape composite rapid prototyping technique provides a fast and cost effective fabrication method and can provide easy integration of microfluidic channels with sensors and other components without the need for a cleanroom facility.

Microfluidic approaches for gene delivery and gene therapy

Recent advances in microfluidics have created new and exciting prospects for gene delivery and therapy. The micro-scaled environment within microfluidic systems enables precise control and optimization of multiple processes and techniques used in gene transfection and the production of gene and drug transporters. Traditional non-viral gene transfection methods, such as electroporation, microinjection and optical gene transfection, are improved from the use of innovative microfluidic systems. Additionally, microfluidic systems have also made the production of many viral and non-viral vectors controlled, automated, and reproducible. In summary, the development and application of microfluidic systems are producing increased efficiency in gene delivery and promise improved gene therapy results.

Quantitative and qualitative analysis of a microfluidic DNA extraction system using a nanoporous AlOx membrane

A nanoporous aluminium oxide membrane was integrated into a microfluidic system designed to extract hgDNA (human genomic DNA) from lysed whole blood. The effectiveness of this extraction system was determined by passing known concentrations of purified hgDNA through nanoporous membranes with varying pore sizes and measuring the amount of hgDNA deposited on the membrane while also varying salt concentration in the solution. DNA extraction efficiency increased as the salt concentration increased and nanopore size decreased. Based on these results, hgDNA was extracted from whole blood while varying salt concentration, nanopore size and elution buffer to find the conditions that yield the maximum concentration of hgDNA. The optimal conditions were found to be using a low-salt lysis solution, 100 nm pores, and a cationic elution buffer. Under these conditions the combination of flow and ionic disruption were sufficient to elute the hgDNA from the membrane. The extracted hgDNA sample was analysed and evaluated using PCR (polymerase chain reaction) to determine whether the eluted sample contained PCR inhibition factors. Eluted samples from the microfluidic system were amplified without any inhibition effects. PCR using extracted samples was demonstrated for several genes of interest. This microfluidic DNA extraction system based on embedded membranes will reduce the time, space and reagents needed for DNA analysis in microfluidic systems and will prove valuable for sample preparation in lab-on-a-chip applications.

Microfluidics-assisted in vitro drug screening and carrier production

Microfluidic platforms provide several unique advantages for drug development. In the production of drug carriers, physical properties such as size and shape, and chemical properties such as drug composition and pharmacokinetic parameters, can be modified simply and effectively by tuning the flow rate and geometries. Large numbers of carriers can then be fabricated with minimal effort and with little to no batch-to-batch variation. Additionally, cell or tissue culture models in microfluidic systems can be used as in vitro drug screening tools. Compared to in vivo animal models, microfluidic drug screening platforms allow for high-throughput and reproducible screening at a significantly lower cost, and when combined with current advances in tissue engineering, are also capable of mimicking native tissues. In this review, various microfluidic platforms for drug and gene carrier fabrication are reviewed to provide guidelines for designing appropriate carriers. In vitro microfluidic drug screening platforms designed for high-throughput analysis and replication of in vivo conditions are also reviewed to highlight future directions for drug research and development.

A PCR reactor with an integrated alumina membrane for nucleic acid isolation

Recently, there has been a growing interest in point-of-care devices capable of detecting nucleic acids (NA) in clinical and environmental samples. Nucleic acid detection requires, however, various sample preparation steps that complicate device operation. An attractive remedy is to integrate many, if not all, sample preparation operations and nucleic acid amplification into a single reaction chamber. A microfluidic chip that integrates, in a single chamber, polymerase chain reaction (PCR) amplification with solid-phase extraction of nucleic acids using a nanoporous, aluminium oxide membrane (AOM) is described. Samples suspected of containing target bacteria and/or viruses are mixed with lysis agents and a chaotropic salt and loaded into a plastic chip housing a nanoporous, aluminium oxide membrane. The nucleic acids in the lysate bind to the membrane. The membrane is then washed, the chamber is filled with the PCR reaction reagents, and the chambers temperature is cycled to amplify the captured nucleic acids and produce detectable products. Both DNA and RNA (with reverse-transcription) isolation and amplification are demonstrated. Due to the dry membranes high resistance to liquid flow, a specialized flow control system was devised to facilitate sample introduction and membrane washing.

Universal Microfluidic Automaton for Autonomous Sample Processing: Application to the Mars Organic Analyzer

A fully integrated multilayer microfluidic chemical analyzer for automated sample processing and labeling, as well as analysis using capillary zone electrophoresis is developed and characterized. Using lifting gate microfluidic control valve technology, a microfluidic automaton consisting of a two-dimensional microvalve cellular array is fabricated with soft lithography in a format that enables facile integration with a microfluidic capillary electrophoresis device. The programmable sample processor performs precise mixing, metering, and routing operations that can be combined to achieve automation of complex and diverse assay protocols. Sample labeling protocols for amino acid, aldehyde/ketone and carboxylic acid analysis are performed automatically followed by automated transfer and analysis by the integrated microfluidic capillary electrophoresis chip. Equivalent performance to off-chip sample processing is demonstrated for each compound class; the automated analysis resulted in a limit of detection of ~16 nM for amino acids. Our microfluidic automaton provides a fully automated, portable microfluidic analysis system capable of autonomous analysis of diverse compound classes in challenging environments.

Magnetic Relaxation Detector for Microbead Labels

A compact and robust magnetic label detector for biomedical assays is implemented in 0.18-μ m CMOS. Detection relies on the magnetic relaxation signature of a microbead label for improved tolerance to environmental variations and relaxed dynamic range requirement, eliminating the need for baseline calibration and reference sensors. The device includes embedded electromagnets to eliminate external magnets and reduce power dissipation. Correlated double sampling combined with offset servo loops and magnetic field modulation, suppresses the detector offset to sub-μ T. Single 4.5-μ m magnetic beads are detected in 16 ms with a probability of error <; 0.1%.

Microvalve Enabled Digital Microfluidic Systems for High-Performance Biochemical and Genetic Analysis

Microfluidic devices offer unparalleled capability for digital microfluidic automation of sample processing and complex assay protocols in medical diagnostic and research applications. In our own work, monolithic membrane valves have enabled the creation of two platforms that precisely manipulate discrete, nanoliter-scale volumes of sample. The digital microfluidic automaton uses two-dimensional microvalve arrays to combinatorially process nanoliter-scale sample volumes. This programmable system enables rapid integration of diverse assay protocols using a universal processing architecture. Microfluidic emulsion generator array (MEGA) devices integrate actively controlled three microvalve pumps to enable on-demand generation of uniform droplets for statistical encapsulation of microbeads and cells. A MEGA device containing 96 channels confers the capability of generating up to 3.4 × 106-nL volume droplets per hour for ultrahigh-throughput detection of rare mutations in a vast background of normal genotypes. These novel digital microfluidic platforms offer significant enhancements in throughput, sensitivity, and programmability for automated sample processing and analysis.

Integration of programmable microfluidics and on-chip fluorescence detection for biosensing applications

We describe the integration of an actively controlled programmable microfluidic sample processor with on-chip optical fluorescence detection to create a single, hybrid sensor system. An array of lifting gate microvalves (automaton) is fabricated with soft lithography, which is reconfigurably joined to a liquid-core, anti-resonant reflecting optical waveguide (ARROW) silicon chip fabricated with conventional microfabrication. In the automaton, various sample handling steps such as mixing, transporting, splitting, isolating, and storing are achieved rapidly and precisely to detect viral nucleic acid targets, while the optofluidic chip provides single particle detection sensitivity using integrated optics. Specifically, an assay for detection of viral nucleic acid targets is implemented. Labeled target nucleic acids are first captured and isolated on magnetic microbeads in the automaton, followed by optical detection of single beads on the ARROW chip. The combination of automated microfluidic sample preparation and highly sensitive optical detection opens possibilities for portable instruments for point-of-use analysis of minute, low concentration biological samples.