Maria Carles

Microfabricated PCR-electrochemical device for simultaneous DNA amplification and detection

Microfabricated silicon/glass-based devices with functionalities of simultaneous polymerase chain reaction (PCR) target amplification and sequence-specific electrochemical (EC) detection have been successfully developed. The microchip-based device has a reaction chamber (volume of 8 microl) formed in a silicon substrate sealed by bonding to a glass substrate. Electrode materials such as gold and indium tin oxide (ITO) were patterned on the glass substrate and served as EC detection platforms where DNA probes were immobilized. Platinum temperature sensors and heaters were patterned on top of the silicon substrate for real-time, precise and rapid thermal cycling of the reaction chamber as well as for efficient target amplification by PCR. DNA analyses in the integrated PCR-EC microchip start with the asymmetric PCR amplification to produce single-stranded target amplicons, followed by immediate sequence-specific recognition of the PCR product as they hybridize to the probe-modified electrode. Two electrochemistry-based detection techniques including metal complex intercalators and nanogold particles are employed in the microdevice to achieve a sensitive detection of target DNA analytes. With the integrated PCR-EC microdevice, the detection of trace amounts of target DNA (as few as several hundred copies) is demonstrated. The ability to perform DNA amplification and EC sequence-specific product detection simultaneously in a single reaction chamber is a great leap towards the realization of a truly portable and integrated DNA analysis system.

Design and fabrication of an integrated microsystem for microcapillary electrophoresis

A capillary electrophoresis microsystem integrated with feed-through platinum electrodes was designed and fabricated for the separation of DNA fragments. A novel glass-to-silicon bonding technology, which allows anodic bonding of a glass wafer to a silicon wafer coated with a thick dielectric film by the inclusion of a thin intermediate amorphous silicon layer, was developed and employed to construct the microsystem. Despite the existence of a thick insulating material and non-uniform topography, robust devices without fluid leakage were obtained. Electrophoretic manipulation and separation of DNA fragments after capillary pre-treatment have been demonstrated and several operational considerations are discussed. The system performance suggests that silicon-based microsystems can be advantageous and practical for the fabrication of integrated microcapillary electrophoresis devices.

Thermal Management and Surface Passivation of a Miniaturized PCR Device for Traditional Chinese Medicine

A robust, efficient, low power and ready-to-integrate Si-based miniaturized reactor for polymerase chain reaction (PCR) has been developed. With this chip reactor, we have demonstrated DNA amplification of Traditional Chinese Medicine (TCM) named Fritillaria cirrhosa (~600 bp) as efficient as that of the conventional thermal cycler. This 8 Ill reactor has Pt heaters and temperature sensors integrated on top of a thin Si membrane capping the reaction chamber for accurate temperature control and rapid thermal cycling of the PCR reagent. A temperature accuracy of ±0.05°C together with 5°C/s heating rate and 3.5°C/s cooling rate has been achieved using digital proportional and integral (PI) control algorithm. Moreover, a power of 1.5 W is sufficient to heat up the reaction chamber to DNA melting temperature of 95°C. The low thermal budget PCR module can eventually be integrated with sample preparation, product separation and detection modules to realize a portable and battery-operated “Genechip” for bioassay as well as drug development of TCM.

DNA kinetics in microfabricated devices

The DNA kinetics in micro-capillary electrophoresis is presented. The mobility and diffusion coefficient of 14bp-DNA fragments as a function of concentration in two types of separation sieving matrices, hydroxyethylcellulose (HEC) polymer solution and agarose gel, are extracted through a series of experiments performed in microfabricated devices. In addition, the motion of a DNA plug through a miter bend and splitting a plug in a branch are quantitatively characterized. The concept of equivalent length is introduced to quantify the effect of a bend on the DNA plug motion. In a branching system, a simple kinematic relationship was discovered relating the quantity of DNA in each downstream branch to its relative channel cross-sectional area.

Plant DNA Fingerprinting and Barcoding

A brief history of taxonomy, for the most part plant oriented, is provided, which demonstrates the use of morphology early on, through the stages when different technologies became available at different times until the present use of genomic tools. Genomic authentication facilitates with greater precision than ever before the identifi cation of an organism or part thereof. In this chapter I made an attempt to stress that, in general, but more so for genomic authentication, the use of the variation inherent in taxa down to the lowest level of the hierarchy of classifi cation needs to be used to achieve a high degree of correct authentication.

Glass-Silicon Bonding Technology with Feed-Through Electrodes for Micro Capillary Electrophoresis

A new technology approach for the design, fabrication and application of a novel integrated microsystem for micro-capillary electrophoresis is presented. The technology approach, using amorphous-silicon-assisted glass-to-silicon anodic bonding, allows the integration of feed-through electrodes formed over a thick insulating layer. In spite of the thick insulating layer and non-uniform topography, hermetic and strong bonding can be achieved due to the contribution of α-silicon during the bonding. Electrophoresis experiments have been performed in the fabricated devices, and the transport of DNA fragments has been demonstrated. The generation of gas bubbles due to electrolysis, which can interfere with the electrophoresis process, and potential solutions are discussed.

Characterization of immobilized DNA probes on the surface of silicon compatible materials

In this paper, the objectives and methodologies of deoxyribonucleic acid (DNA) analysis based on micro arrays produced by microelectronic fabrication technology is reviewed. The important issue of material compatibility between DNA material and silicon based material is addressed. With a special surface treatment, DNA probes have been successfully attached (or immobilized) on the surface of silicon compatible materials (mainly silicon dioxide). Through the use of optical labels that emit optical fluorescence under the illumination of a UV light source, the number of DNA probes immobilized on the silicon dioxide surface can be quantified. The results are further analyzed with atomic force microscopy to visualize the surface conditions. The successful immobilization of DNA probes on silicon wafers is very important for the fabrication of DNA chips for DNA analysis.