We have demonstrated the ability to perform real-time homogeneous, sequence specific detection of PCR products in silicon microstructures. Optimal design/ processing result in equivalent performance (yield and specificity) for high surface-to-volume silicon structures as compared to larger volume reactions in polypropylene tubes. Amplifications in volumes as small as 0.5 microl and thermal cycling times reduced as much as 5-fold from that of conventional systems have been demonstrated for the microstructures.

No abstract

Capillary electrophoresis (CE) continues to grow rapidly as an analytical technique in a wide range of application areas (1-5). Typically, CE separations are performed in fusedsilica capillaries with internal diameters of 25-100 pm. High heat dissipation efficiencies in the narrow columns allow separations to be performed at high field strengths (200-500 V/cm), resulting in short analysis times and peak efficiencies of up to millions of theoretical plates.

An experimental investigation of the distance dependence of long-range electron transfer in zinc/ruthenium-modified myoglobins has been performed. The modified proteins were prepared by substitution of zinc mesoporphyrin IX diacid (ZnP) for the heme in each of four previously characterized pentaammineruthenium(II1) (a5Ru; a = NH,) derivatives of sperm whale myoglobin (Mb): a5Ru(His-48)Mb, aSRu(His-12)Mb, a5Ru(His-l 16)Mb, a5Ru(His-81)Mb. Electron transfer from the ZnP triplet excited state (,ZnP*) to Ru3+, 3ZnP*-Ru3t -+ ZnP+-Ru2+ (AEo -0.8 V) was measured by time-resolved transient absorption spectroscopy: rate constants (kf) are 7.0 X lo4 (His-48), 1.0 X IO2 (His-12), 8.9 X 10' (His-1 16), and 8.5 X IO' (His-81) s-' a t 25 OC. Activation enthalpies calculated from the temperature dependences of the electron-transfer rates over the range 5-40 O C are 1.7 i 1.6 (His-48), 4.7 i 0.9 (His-12), 5.4 i 0.4 (His-116), and 5.6 i 2.5 (His-81) kcal mol-'. Electron-transfer distances (d = closest ZnP edge to a5Ru(His) edge; angstroms) were calculated to fall in the following ranges: . The rate-distance equation is kf = 7.8 X lo8exp [-0.9l(d - it is generally assumed t h a t t h e rates of these transfers are determined to a large extent by the donor-acceptor separation and t h e nature of the intervening medi~m,l'-~' very few experiments have addressed these points in a systematic manner.'^^.''One approach t o t h e study of long-range electron transfer is to attach a redox-active complex t o a specific surface site of a structurally characterized heme or blue copper protein, thereby producing a two-site molecule with a fixed donor-acceptor distance. T h e redox-active complex t h a t has been employed successfully in several previous experiments is a5Ru2+/3+ (a = NH,), which covalently bonds to surface histidines.8-12 This complex can be attached by t h e reaction of a5Ru(OH,)2t with native protein under mild conditions, and t h e ruthenated protein can be purified by ion-exchange chromatography.In order t o probe distance effects on long-range electron transfer, we have replaced the heme in four ruthenated myoglobins (a5Ru(His-48)Mb; a5Ru(His-1 2)Mb; a5Ru(His-l 16)Mb; a5Ru-(His-81)Mb: Mb = sperm whale myoglobin)'O by zinc mesoporphyrin IX diacid (ZnP). The ZnP excited triplet state (3ZnP*) is a much more powerful electron donor t h a n a reduced heme (hE0(3ZnP*-Ru3+ -ZnP+-Ru2+) -0.8 V)," thereby allowing four different electron-transfer distances to be examined in a single protein molecule. Experimental SectionMaterials and Apparatus. Distilled water, filtered through a Barnstead Nanopure water purification system (No. 2794, specific resistance >18 Ma-cm), was used in the preparation of all aqueous solutions. 2-Butanone (MCB) was stored over aluminum oxide (Woelm neutral, Waters Associates) at 4 OC to prevent the accumulation of peroxides. All other reagents were used as received.Carboxymethylcellulose cation-exchange resin, CM-52 (Whatman, preswollen, microgranular), was equilibrated as indicated by the manu...

Abstract-Polymerase chain reaction (PCR) using micromachined structures promises improved temperature uniformity and cycling time together with decreased reagent and sample volumes. Thermal design of these structures will benefit from measurements of the temperature distribution in the reacting liquid. We report measurements of temperature uniformity and time constant in a microfabricated 18-vessel array using encapsulated liquid crystals suspended in the liquid. Separate sets of crystals are used to image temporal and spatial temperature variations near the processing thresholds of 55 C and 95 C with a resolution of 0.1 C. While the thermometry technique developed here is particularly useful for characterizing microfabricated PCR systems, it can also aid with the thermal design of a broad variety of microfluidic devices. [330]