Mark A. Burns

Microfabricated devices for genetic diagnostics

This paper presents a review of microfabricated devices for genetic diagnostics. Genetic diagnostics are powerful technology drivers and excellent candidate applications for miniaturization technologies because the demand for inexpensive genetic information is essentially unlimited, and the cost and time for the diagnostic decreases with sample volume. Genetic information is stored in long DNA molecules in solution. This information is processed and extracted using a series of enzymatic and other chemical reactions well known in molecular biology. Processing of DNA molecules in the microscale hence requires the implementation of microfluidic devices capable of handling, mixing, thermal cycling, separating, and detecting nano- and picoliter liquid samples. This paper discusses some of the fundamental macroscale protocols used for genetic analyses and how these processes scale down to microscopic volumes. The construction and performance of microfluidic devices of DNA amplification, separation, hybridization, and detection are discussed, showing that so far, no fundamental impediments exist for genetic diagnostics based on microelectromechanical systems. Some of the unresolved storage and packaging issues and future challenges for the practical implementation of these devices are also presented.

Mathematical modeling of drop mixing in a slit-type microchannel

Fast solute mixing can be achieved in a microchannel by rapid unidirectional displacement of a discrete liquid drop. The recirculation streamlines created within the liquid during the drop’s motion cause the solute to interlayer across the channel depth, provided the interlayer diffusion of the solute is small. Uniform interlayering appears when the drop is displaced by more than three drop lengths in a slit-type microchannel, thereby reducing the solute diffusion distances to a fraction of the channel depth. By fabricating the microchannel to a depth of less than 50 µm even large molecules with a low diffusivity ( D< 10 −8 cm 2 s −1 ) can be mixed in seconds. The above strategy is shown by modeling the mixing of solutes present in a drop moving in a slit-type microchannel.

Nanoliter viscometer for analyzing blood plasma and other liquid samples

We have developed a microfabricated nanoliter capillary viscometer that quickly, easily, and inexpensively measures the viscosity of liquids. The measurement of viscosity is based on capillary pressure-driven flow inside microfluidic channels (depth approximately 30 microm and width approximately 300 microm). Accurate and precise viscosity measurements can be made in less than 100 s while using only 600 nL of liquid sample. The silicon-glass hybrid device (18 mm by 15 mm) contains on-chip components that measure the driving capillary pressure difference and the relevant geometrical parameters; these components make the nanoliter viscometer completely self-calibrating, robust, and easy to use. Several different microfabricated viscometers were tested using solutions with viscosities ranging from 1 to 5 cP, a range relevant to biological fluids (urine, blood, blood plasma, etc.). Blood plasma samples collected from patients with the symptoms of hyperviscosity syndrome were tested on the nanoliter capillary viscometer to an accuracy of 3%. Such self-calibrating nanoliter viscometers may have widespread applications in chemical, biological, and medical laboratories as well as in personal health care.

Microdroplet-enabled highly parallel co-cultivation of microbial communities.

Microbial interactions in natural microbiota are, in many cases, crucial for the sustenance of the communities, but the precise nature of these interactions remain largely unknown because of the inherent complexity and difficulties in laboratory cultivation. Conventional pure culture-oriented cultivation does not account for these interactions mediated by small molecules, which severely limits its utility in cultivating and studying “unculturable” microorganisms from synergistic communities. In this study, we developed a simple microfluidic device for highly parallel co-cultivation of symbiotic microbial communities and demonstrated its effectiveness in discovering synergistic interactions among microbes. Using aqueous micro-droplets dispersed in a continuous oil phase, the device could readily encapsulate and co-cultivate subsets of a community. A large number of droplets, up to ∼1,400 in a 10 mm×5 mm chamber, were generated with a frequency of 500 droplets/sec. A synthetic model system consisting of cross-feeding E. coli mutants was used to mimic compositions of symbionts and other microbes in natural microbial communities. Our device was able to detect a pair-wise symbiotic relationship when one partner accounted for as low as 1% of the total population or each symbiont was about 3% of the artificial community.

Electrophoresis in microfabricated devices using photopolymerized polyacrylamide gels and electrode-defined sample injection

Microfabrication techniques have become increasingly popular in the development of the next generation of DNA analysis systems. While significant progress has been reported by many researchers, complete microfabricated integrated DNA analysis devices are still in the earliest stages of development. Most miniaturized analysis systems have incorporated noncross‐linked polymer solutions as the separation medium of choice and the operation of these systems necessitates the use of high electric fields and long separation lengths. In this paper, we present two techniques that may help alleviate this problem and accelerate the development of the so‐called ‘lab‐on‐a‐chip’ systems. We present the use of photodefinable polyacrylamide gels as a sieving medium for DNA electrophoresis. These gels offer the significant advantages of faster curing times, locally controlled gel interface, and simpler handling over chemically polymerized gels. We also introduce an electrode‐defined sample compaction and injection technique. This technique helps achieve sample compaction without migration into the gel and offers significant control over the size and application of the sample plug. The use of these technologies for double‐stranded DNA separations in microfabricated separation systems is demonstrated.

Advances in on-chip photodetection for applications in miniaturized genetic analysis systems

Microfabrication techniques have become increasingly popular in the development of next generation DNA analysis devices. Improved on-chip fluorescence detection systems may have applications in developing portable hand-held instruments for point-of-care diagnostics. Miniaturization of fluorescence detection involves construction of ultra-sensitive photodetectors that can be integrated onto a fluidic platform combined with the appropriate optical emission filters. We have previously demonstrated integration PIN photodiodes onto a microfabricated electrophoresis channel for separation and detection of DNA fragments. In this work, we present an improved detector structure that uses a PINN+ photodiode with an on-chip interference filter and a robust liquid barrier layer. This new design yields high sensitivity (detection limit of 0.9 ng µl−1 of DNA), low-noise (S/N ~ 100/1) and enhanced quantum efficiencies (>80%) over the entire visible spectrum. Applications of these photodiodes in various areas of DNA analysis such as microreactions (PCR), separations (electrophoresis) and microfluidics (drop sensing) are presented.

Microfluidic Pneumatic Logic Circuits and Digital Pneumatic Microprocessors for Integrated Microfluidic Systems

We have developed pneumatic logic circuits and microprocessors built with microfluidic channels and valves in polydimethylsiloxane (PDMS). The pneumatic logic circuits perform various combinational and sequential logic calculations with binary pneumatic signals (atmosphere and vacuum), producing cascadable outputs based on Boolean operations. A complex microprocessor is constructed from combinations of various logic circuits and receives pneumatically encoded serial commands at a single input line. The device then decodes the temporal command sequence by spatial parallelization, computes necessary logic calculations between parallelized command bits, stores command information for signal transportation and maintenance, and finally executes the command for the target devices. Thus, such pneumatic microprocessors will function as a universal on-chip control platform to perform complex parallel operations for large-scale integrated microfluidic devices. To demonstrate the working principles, we have built 2-bit, 3-bit, 4-bit, and 8-bit microprocessors to control various target devices for applications such as four color dye mixing, and multiplexed channel fluidic control. By significantly reducing the need for external controllers, the digital pneumatic microprocessor can be used as a universal on-chip platform to autonomously manipulate microfluids in a high throughput manner.

Microfluidic flow control using selective hydrophobic patterning

We have developed a method to pattern self assembled monolayer films of n-octadecyltrichlorosilane on silicon and glass substrates using a simple lift-off procedure. By defining hydrophobic regions at definite locations in microchannels and using an external pressure source, we can split off precise nanoliter volume liquid drops and control the motion of those drops through the microchannels. We have also constructed an on-chip pressure source for drop splitting and motion by heating air trapped in a micromachined chamber. Both techniques can produce and move drops on the order of 50 nl.

Asynchronous magnetic bead rotation (AMBR) biosensor in microfluidic droplets for rapid bacterial growth and susceptibility measurements

Inappropriate antibiotic use is a major factor contributing to the emergence and spread of antimicrobial resistance. The long turnaround time (over 24 hours) required for clinical antimicrobial susceptibility testing (AST) often results in patients being prescribed empiric therapies, which may be inadequate, inappropriate, or overly broad-spectrum. A reduction in the AST time may enable more appropriate therapies to be prescribed earlier. Here we report on a new diagnostic asynchronous magnetic bead rotation (AMBR) biosensor droplet microfluidic platform that enables single cell and small cell population growth measurements for applications aimed at rapid AST. We demonstrate the ability to rapidly measure bacterial growth, susceptibility, and the minimum inhibitory concentration (MIC) of a small uropathogenic Escherichia coli population that was confined in microfluidic droplets and exposed to concentrations above and below the MIC of gentamicin. Growth was observed below the MIC, and no growth was observed above the MIC. A 52% change in the sensor signal (i.e. rotational period) was observed within 15 minutes, thus allowing AST measurements to be performed potentially within minutes.

Push-pull perfusion sampling with segmented flow for high temporal and spatial resolution in vivo chemical monitoring.

Low-flow push-pull perfusion is a sampling method that yields better spatial resolution than competitive methods like microdialysis. Because of the low flow rates used (50 nL/min), it is challenging to use this technique at high temporal resolution which requires methods of collecting, manipulating, and analyzing nanoliter samples. High temporal resolution also requires control of Taylor dispersion during sampling. To meet these challenges, push-pull perfusion was coupled with segmented flow to achieve in vivo sampling at 7 s temporal resolution at 50 nL/min flow rates. By further miniaturizing the probe inlet, sampling with 200 ms resolution at 30 nL/min (pull only) was demonstrated in vitro. Using this method, L-glutamate was monitored in the striatum of anesthetized rats. Up to 500 samples of 6 nL each were collected at 7 s intervals, segmented by an immiscible oil and stored in a capillary tube. The samples were assayed offline for L-glutamate at a rate of 15 samples/min by pumping them into a reagent addition tee fabricated from Teflon where reagents were added for a fluorescent enzyme assay. Fluorescence of the resulting plugs was monitored downstream. Microinjection of 70 mM potassium in physiological buffered saline evoked l-glutamate concentration transients that had an average maxima of 4.5 ± 1.1 μM (n = 6 animals, 3-4 injections each) and rise times of 22 ± 2 s. These results demonstrate that low-flow push-pull perfusion with segmented flow can be used for high temporal resolution chemical monitoring and in complex biological environments.