The completion of the human genome draft has taken several years and is only the beginning of a period in which large amounts of DNA and RNA sequence information will be required from many individuals and species. Conventional sequencing technology has limitations in cost, speed, and sensitivity, with the result that the demand for sequence information far outstrips current capacity. There have been several proposals to address these issues by developing the ability to sequence single DNA molecules, but none have been experimentally demonstrated. Here we report the use of DNA polymerase to obtain sequence information from single DNA molecules by using fluorescence microscopy. We monitored repeated incorporation of fluorescently labeled nucleotides into individual DNA strands with single base resolution, allowing the determination of sequence fingerprints up to 5 bp in length. These experiments show that one can study the activity of DNA polymerase at the single molecule level with single base resolution and a high degree of parallelization, thus providing the foundation for a practical single molecule sequencing technology.T he Sanger method of DNA sequencing (1) and subsequent developments in automation (2) and computation (3) revolutionized the world of biological sciences and eventually led to the sequencing of the consensus human genome (4, 5). The successes of this and other genome projects have only whetted the appetite of the scientific community, and many applications of DNA sequencing have been proposed that will require cheaper, faster, or more sensitive sequencing technology than conventional methods currently provide. After the determination of the consensus human genome, there is a desire to sequence many individual human genomes to provide highresolution genotypes that can be used to determine the complex relationships among disease, pharmaceutical efficacy, and genetic variability (6-8). Similarly, aggressive technological innovation is required for the field of comparative genomics to reach its full potential (4). Finally, mRNA sequencing is valuable to determine exon splicing patterns (9) and as a tool to discover gene function from context-specific expression data (10).There have been many proposals to develop new sequencing technologies based on single molecule measurements, generally either by observing the interaction of particular proteins with DNA (6, 11-13) or by using ultra high-resolution scanned probe microscopy (14). Although none of these methods has been demonstrated experimentally, they are interesting because they promise high sensitivity, low cost, and in some cases a high degree of parallelization (15). Unlike conventional technology, their speed and read length would not be inherently limited by the resolving power of electrophoretic separation. Single molecule sensitivity might permit direct sequencing of mRNA from rare cell populations or perhaps even individual cells.A major obstacle in the development of single molecule sequencing schemes is that DNA has an extraordinarily ...

Here we describe the development of a high-throughput multi-antigen microfluidic fluorescence immunoassay system. A 100-chamber polydimethylsiloxane (PDMS) chip performs up to 5 tests for each of 10 samples. In this particular study system, the specificity of detection was demonstrated, and calibration curves were produced for C-reactive protein (CRP), prostate-specific antigen (PSA), ferritin, and vascular endothelial growth factor (VEGF). The measurements show sensitivity at and below clinically normal levels (with a signal-to-noise ratio >8 at as low as 10 pM antigen concentration). The chip uses 100 nL per sample for all tests. The developed system is an important step toward derivative immunoassay applications in scientific research and "point-of-care" testing in medicine.

We report on a fundamental technological advance for multilayer polydimethylsiloxane (PDMS) microfluidics. Vertical passages (vias), connecting channels located in different layers, are fabricated monolithically, in parallel, by simple and easy means. The resulting 3D connectivity greatly expands the potential complexity of microfluidic architecture. We apply the vias to printing nested bioarrays and building autoregulatory devices. A current source is demonstrated, while a diode and a rectifier are derived; all are building blocks for analog circuitry in Newtonian fluids. We also describe microfluidic septa and their applications. Vias lay the foundation for a new generation of microfluidic devices.ver the decade of its existence, polydimethylsiloxane (PDMS) microfluidics has progressed from the plain microchannel (1) through pneumatic valves and pumps (2, 3) to an impressive set of specialized components organized by the thousands in multilayer large-scale-integration chips (4). These devices have become the hydraulic elastomeric embodiment of Richard Feynman's dreams of infinitesimal machines (5, 6). The now established technology (7) has found successful applications in protein crystallization (8), DNA sequencing (9), nanoliter PCR (10), cell sorting and cytometry (11), nucleic acids extraction and purification (12), immunoassays (13,14), cell studies (15-18), and chemical synthesis (19), while also serving as the fluid-handling component in emerging integrated microelectromechanical devices (MEMS) (20).The energetic pursuit of applications, however, has resulted in a premature attention shift away from fundamental microfluidics. Here we report on a fundamental technological advance that allows a simple and easy access to a large increase in the architectural complexity of microfluidic devices, as well as opens new possibilities for technical developments and consequent applications. We dubbed the previously undescribed device ''via,'' in reference to its analog in modern semiconductor electronics.Vias are vertical micropassages that connect channels fabricated in different layers of the same PDMS multilayer chip. The functional result is 3D channels that lift the restrictions of the traditional architecture wherein channels could not leave their layer and two channels within the same layer could not cross without mixing. These restrictions did not prevent the emergence of expansive architectures (4), because the particular applications were shrewdly chosen to involve large-scale parallelization of simple identical operations, thereby requiring few controls and maximizing device density. However, as the field moves to functionally complex heterogeneous devices integrated on the same chip, laying out the respective circuitry would inevitably necessitate convenient, simple, and reliable vertical connectivity just as it did in the semiconductor industry. Microfluidic vias provide that 3D connectivity, lift the above architectural restrictions, and contribute morphological and functional capabilities.The pursuit...

We have developed the first fully integrated microfluidic system for DNA sequencing-by-synthesis. Using this chip and fluorescence detection, we have reliably sequenced up to 4 consecutive bps. The described sequencer can be integrated with other microfluidic components on the same chip to produce true lab-on-a-chip technology. The surface chemistry that was designed to anchor the DNA to elastomeric microchannels is useful in a broad range of studies and applications.

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A systematic experimental study and theoretical modeling of the device physics of polydimethylsiloxane "pushdown" microfluidic valves are presented. The phase space is charted by 1587 dimension combinations and encompasses 45-295 m lateral dimensions, 16-39 m membrane thickness, and 1 -28 psi closing pressure. Three linear models are developed and tested against the empirical data, and then combined into a fourth-power-polynomial superposition. The experimentally validated final model offers a useful quantitative prediction for a valve's properties as a function of its dimensions. Typical valves ͑80-150 m width͒ are shown to behave like thin springs.

This review article discusses PDMS (polydimethylsiloxane) microfluidic devices and their biological applications. First, the already developed devices are classified from the viewpoints of underlying technology within a common logical framework comprising single-layer, multilayer, and integrated devices, as well as surface chemistry modifications of PDMS. Combinatorial techniques are applied to re-derive existing devices within this framework. Next, the relevant scales of both microfluidics and biology are compared, obtaining the promise and limitations of PDMS microfluidics. Finally, the body of work is reclassified in terms of addressed biological applications and compared to the standard methods in cellular and molecular biology, to offer insights for future devices and applications.

Herein we report on reliable reproducible quantification of protein analytes in human serum by fluorescence sandwich immunoassays in disposable PDMS microfluidic chips. The system requires 1000 times less sample than typical clinical blood tests and is specifically shown to measure ferritin down to 250 pM in human serum. The in-built calibration method of spiking the serum with known concentrations of commercially available antigen avoids common sources of error and improves the reliability of the test results. The reported microfluidic system is an important new tool for fundamental scientific research, offering sensitive immunoassay measurements in small but complex biosamples. The system is also a further step towards comprehensive affordable ''pointof-care'' biomedical diagnostics.