Microfluidic device architecture for electrochemical patterning and detection of multiple DNA sequences.

Electrochemical biosensors pose an attractive solution for point-of-care diagnostics because they require minimal instrumentation and they are scalable and readily integrated with microelectronics. The integration of electrochemical biosensors with microscale devices has, however, proven to be challenging due to significant incompatibilities among biomolecular stability, operation conditions of electrochemical sensors, and microfabrication techniques. Toward a solution to this problem, we have demonstrated here an electrochemical array architecture that supports the following processes in situ, within a self-enclosed microfluidic device: (a) electrode cleaning and preparation, (b) electrochemical addressing, patterning, and immobilization of sensing biomolecules at selected sensor pixels, (c) sequence-specific electrochemical detection from multiple pixels, and (d) regeneration of the sensing pixels. The architecture we have developed is general, and it should be applicable to a wide range of biosensing schemes that utilize gold-thiol self-assembled monolayer chemistry. As a proof-of-principle, we demonstrate the detection and differentiation of polymerase chain reaction (PCR) amplicons diagnostic of human (H1N1) and avian (H5N1) influenza.

Selection of mammalian cells based on their cell-cycle phase using dielectrophoresis.

An effective, noninvasive means of selecting cells based on their phase within the cell cycle is an important capability for biological research. Current methods of producing synchronous cell populations, however, tend to disrupt the natural physiology of the cell or suffer from low synchronization yields. In this work, we report a microfluidic device that utilizes the dielectrophoresis phenomenon to synchronize cells by exploiting the relationship between the cell's volume and its phase in the cell cycle. The dielectrophoresis activated cell synchronizer (DACSync) device accepts an asynchronous mixture of cells at the inlet, fractionates the cell populations according to the cell-cycle phase (G(1)/S and G(2)/M), and elutes them through different outlets. The device is gentle and efficient; it utilizes electric fields that are 1-2 orders of magnitude below those used in electroporation and enriches asynchronous tumor cells in the G(1) phase to 96% in one round of sorting, in a continuous flow manner at a throughput of 2 x 10(5) cells per hour per microchannel. This work illustrates the feasibility of using laminar flow and electrokinetic forces for the efficient, noninvasive separation of living cells.

Rapid, sequence-specific detection of unpurified PCR amplicons via a reusable, electrochemical sensor.

We report an electrochemical method for the sequence-specific detection of unpurified amplification products of the gyrB gene of Salmonella typhimurium. Using an asymmetric PCR and the electrochemical E-DNA detection scheme, single-stranded amplicons were produced from as few as 90 gene copies and, without subsequent purification, rapidly identified. The detection is specific; the sensor does not respond when challenged with control oligonucleotides based on the gyrB genes of either Escherichia coli or various Shigella species. In contrast to existing sequence-specific optical- and capillary electrophoresis-based detection methods, the E-DNA sensor is fully electronic and requires neither cumbersome, expensive optics nor high voltage power supplies. Given these advantages, E-DNA sensors appear well suited for implementation in portable PCR microdevices directed at, for example, the rapid detection of pathogens.

Differential labeling of closely spaced biosensor electrodes via electrochemical lithography.

Electrochemical biosensors offer the promise of exceptional scalability and parallelizability. To achieve this promise, however, will require the development of new methods for the differential labeling of closely spaced electrodes with specific biomolecules such as DNA or proteins. Here we report a simple, highly selective method for passivating and differentially labeling closely separated gold electrodes with oligonucleotides or other biomolecules. Analogous to photolithography, where a light-sensitive resist is selectively removed to expose specific surfaces to further modification, we passivate gold electrodes with a self-assembled alkanethiol monolayer that protects them from modification. The monolayer is then electrochemically desorbed at relatively low potentials, allowing for the subsequent labeling of the now exposed array element with a specific sensing biomolecule. The observed passivation is highly efficient: using a C11-OH monolayer as the passivating agent, we do not observe any detectable cross-contamination of adjacent electrodes (95 microm separation) upon labeling with a stem-loop DNA probe. Critically, the conditions employed are sufficiently gentle that depassivation reduces the DNA load on adjacent electrodes by only approximately 1%, allowing for the sequential labeling of multiple, closely spaced electrodes. This technology paves the way for labeling multiple array elements sequentially without observable cross-contamination in a fast and controlled manner.

Integrated microsystem for dielectrophoretic cell concentration and genetic detection.

We have directly integrated a continuous-flow, electrokinetic method of bacterial cell concentration with room temperature, sequence-specific genetic detection. The system we have developed traps cells from a continuous-flow sample stream via dielectrophoresis, providing a 160-fold increase in cell concentration. PDMS microvalves then define a 100 nL volume around the trapped cells to which cell lysis buffer and genetic detection components are introduced. Direct, optical detection of Escherichia coli MC1061 cells is then accomplished via the sequence-specific hybridization of an rRNA-directed optical molecular beacon. This integrated microsystem is capable of sequence-specific genetic detection of 25 cells within 30 min.

Surface micromachined capacitive ultrasonic transducers.

The current state of novel technology, surface microfabricated ultrasonic transducers, is reported. Experiments demonstrating both air and water transmission are presented. Air-coupled longitudinal wave transmission through aluminum is demonstrated, implying a 110 dB dynamic range for transducers at 2.3 MHz in air. Water transmission experiments from 1 to 20 MHz are performed, with a measured 60 dB SNR at 3 MHz. A theoretical model is proposed that agrees well with observed transducer behavior. Most significantly, the model is used to demonstrate that microfabricated ultrasonic transducers constitute an attractive alternative to piezoelectric transducers in many applications.

Racial variation of factor VII activity and antigen levels and their correlates in healthy Chinese and Indians at low and high risk for coronary artery disease.

Plasma factor VII activity (FVIIc) is one of the independent risk factors of coronary artery disease (CAD) and is controlled by both genetic and environmental factors. South Asians including Indians have one of the highest prevalence and mortality rates from CAD while the Chinese have a much lower risk. Generally accepted risk factors cannot explain the high mortality from CAD in Indians. We examined two hundred and seventy seven Chinese (124 m, 153 f); and 216 healthy Indian (150 m, 66 f) adults for serum lipids; plasma FVIIc and FVIIag levels in order to examine racial variations of these and their correlates in these two populations. Both Indian men and women had significantly higher FVIIc levels (12% and 11%, respectively) than the Chinese even after adjustments of age, BMI and lipids (P < 0.01). In contrast, Indians had significantly lower plasma FVIIag levels than Chinese (8% and 9%, respectively in men and women; P < 0.01). Multiple linear regression analysis shows a strong correlation of FVIIc with serum triglycerides accounting for 4-8% of the total variability of FVIIc in different groups. Further, there was a stronger correlation between FVIIc and FVIIag in Indians than that in the Chinese (0.43 vs. 25) suggesting a greater activation resulting in higher FVIIc in Indians inspite of lower FVIIag levels. The higher FVIIc and stronger activation by triglycerides observed in this study partly explain the higher risk of CAD in Indians.