Scott J. Stelick

Nucleic acid purification using microfabricated silicon structures

A microfluidic device has been designed, fabricated and tested for its ability to purify bacteriophage lambda DNA and bacterial chromosomal DNA, a necessary prerequisite for its incorporation into a biosensor. This device consists of a microfabricated channel in which silica-coated pillars were etched to increase the surface area within the channel by 300-600%, when the etch depth is varied from 20 to 50 microm. DNA was selectively bound to these pillars in the presence of the chaotropic salt guanidinium isothiocyanate, followed by washing with ethanol and elution with low-ionic strength buffer. Positive pressure was used to move solutions through the device, removing the need for centrifugation steps. The binding capacity for DNA in the device was approximately 82 ng/cm2 and on average, 10% of the bound DNA could be purified and recovered in the first 50 microl of elution buffer. Additionally, the device removed approximately 87% of the protein from a cell lysate. Nucleic acids recovered from the device were efficiently amplified by the polymerase chain reaction suggesting the utility of these components in an integrated, DNA amplification-based biosensor. The miniaturized format of this purification device, along with its excellent purification characteristics make it an ideal component for nucleic acid-based biosensors, especially those in which nucleic acid amplification is a critical step.

Hydrodynamic optical alignment for microflow cytometry

A microfabricated flow cytometer has been developed that is capable of detecting nearly all of the microparticles in an aqueous suspension. Current design allows for integrated coupling between an optical fiber-based detection system and the particle stream via hydrodynamic focusing. By adjusting the relative flow-rates at the auxiliary inputs of the focusing manifold, the particle stream can be steered out-of-plane relative to the illuminating laser, and similarly the particle stream can be squeezed or expanded. The microfabricated device was constructed in polydimethylsiloxane with cross-sectional microfluidic dimensions of 125 µm×125 µm. Using the present device and method, fluorescent microparticles in aqueous solution were counted at an absolute counting efficiency of 91±4%. The coefficient of variation of the fluorescence pulse-heights for far-red fluorescent microparticles was 15%. The device exhibited a linear response to fluorescence intensity calibration microparticles as shown by comparison with a commercial cytometer instrument.

A microchip-based DNA purification and real-time PCR biosensor for bacterial detection

A miniaturized, fully-automated, PCR (polymerase chain reaction)-based detection system has been developed for the rapid detection of bacteria. Monolithic DNA purification/real-time PCR silicon chips were fabricated and tested for their ability to purify and detect the pathogenic bacteria Salmonella typhimurium. Using silica-coated microstructures, nucleic acids could be selectively bound, washed and eluted for subsequent real-time PCR. These microstructures were integrated into a detection microchip containing two distinct regions, one for DNA purification and one for real-time PCR. Using an automated detection platform with integrated microprocessor, pumps, valves, thermocycler and fluorescence detection modules, microchips were used to purify and detect bacterial DNA by real-time PCR amplification using SYBR Green fluorescent dye. As few as 2/spl times/10/sup 3/ S. typhimurium cells could be detected using this system with an average time for detection of 45 min. Detection was augmented by on-chip melting curve analysis capable of differentiating between positive and false-positive results.

PCR-based detection of Bacillus anthracis using an integrated microfluidic platform

A highly-integrated PCR-based detection system has been developed for the rapid identification of pathogenic bacteria. Nanofabricated fluidic cartridges were used to carry out SYBR Green-based fluorogenic PCR assays for the detection of Bacillus anthracis which incorporated the chromosomal BA813 locus as the target for amplification. Real-time PCR assay conditions and operating parameters were optimised to increase detection sensitivity. Optimisation of the system resulted in the detection of as few as 40 B. anthracis colony forming units (CFU) with an average time to detection of 60 min, inclusive of DNA purification and PCR amplification, and a dynamic range of 40 to 400,000 CFU. Real-time fluorescence curves were analysed using a simplified mathematical method to determine threshold cycle (Ct) values with comparable results to a statistically-based analysis algorithm. These results support the utility of the system for rapid, sensitive detection of B. anthracis as well as potential for quantitative determination of target cell concentration.

NiColoy: a versatile electroforming process for biosensor fabrication by embossing

High strength, hard, electroformed stampers are formed from a silicon master using Nickel-Cobalt electrodeposited alloy. They are found to replicate structures from sub-micron dimensions like optical gratings and to hundreds of microns like micro-fluidic channels without distortion. The channels and optical elements constitute building blocks of many biosensors. Electroform is a negative replica of the silicon master and a low cost reproduction of the master is obtained by embossing it onto plastic substrates. We find that damage free reproduction in the plastic substrate is achieved when the aspect ratio of the structures is less than 3.