In this paper, we demonstrate immobilization and stretching of single lambda-phage DNA molecules within microfluidic systems using ac fields. We present a novel "thiol-on-gold"-based immobilization technique for fixing one specific end (3' end) of a DNA molecule onto a gold electrode. A polymer-enhanced medium (approximately 3.75 wt % linear polyacrylamide in Tris-HCl) is used to obtain fully stretched configurations (21 microm) of fluorescently stained lambda-DNA molecules. We also present an optimized microelectrode design with pointed electrodes and an electrode spacing of 20 microm for stretching DNA molecules with an ac field (1 MHz, 3 x 10(5) V/m). Finally, using these techniques, we immobilize a single DNA molecule at one electrode edge, stretch the molecule, and fix the other end at an adjacent electrode edge, forming a bridge between two electrodes within a microfabricated device.
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.
In this paper we describe the design, construction and operation of a micropump that delivers continuous, ultra-low flow velocities at ∼100 µm s −1. The pumping concept is based on the commonly observed phenomenon of transpiration in plant leaves. A liquid meniscus is pinned inside a microchannel by selective hydrophobic patterning and the evaporation rate of the liquid at the meniscus is controlled. The controlled evaporative flux results in a regulated flow of the liquid from a reservoir to the meniscus. Using this technique, precise flow control (5 nl min −1) has been achieved in several channel geometries for extended periods of time (∼2 h). Various factors affecting the performance of the pump were studied and theoretical predictions along with experimental results are presented. Such a micropump could find applications in emerging biological assays such as single-molecule studies of DNA and cell adhesion analyses.
A short focused pulse of light was used to selectively cut lambda-phage DNA molecules at specific restriction sites. Lambda DNA (48.5 kbp) was stretched and placed in a solution containing a restriction enzyme (Sma 1), caged magnesium ions (using a DM-Nitrophen complex), and a chelating agent (EDTA). When a pulse of UV light was directed at a particular location on the stretched DNA molecule, magnesium ions were released into solution. A series of binding reactions then occur in which the enzyme and the chelating agent compete for free Mg2+ ions. Since Sma 1 functions only in the presence of Mg2+, as is true of most endonucleases, the site(s) in the vicinity of the pulse (typically approximately 6 microm) were cut while other sites (three total for this DNA/enzyme pair) were not. The ratio of the concentration of the chelating agent to that of the magnesium ions was used to control the radius of this reaction zone with higher ratios leading to smaller, localized reaction areas. This optically based reaction mechanism could be useful to understand single molecule enzymatic kinetics, and when coupled with other DNA analysis techniques, this could be used to construct complex genotyping and sequencing devices that would analyze parts of single DNA molecules.
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