Scaling of nucleic acid assays on microelectrophoresis array devices: high-dynamic range multi-gene readout from less than ten transcripts.

In this paper we describe progress in using the prodigious data-collecting ability of multilane microelectrophoresis instruments to bear on problems in scaled nucleic acid assays. We emphasize compound stacking and solid-support loading as means to concentrate <100 pg samples for direct injection. Reaction Mapping is applied to readout quantitative polymerase chain reaction gene-expression and as a way to practically overcome difficulty in interpreting amplification curves of multiplexed quantitative polymerase chain reaction at 20-50 gene/well complexity.

Reaction-mapped quantitative multiplexed polymerase chain reaction on a microfluidic device.

We have applied multiple-time-point reaction mapping to generate high-dynamic-range quantitative data from PCR multiplexes. The approach measures, then compensates, numerous PCR slope nonidealities across the multiplex without prejudice. A multilane microelectophoresis device with a novel scanning detector that reports redundantly over more than six decades in signal strength was used to collect data with multiple readings for each amplification point and with double internal calibration (lane standards and gene standards).

Solid-support sample loading for DNA sequencing.

We present a new method for simplified low-quantity DNA loading onto microelectrophoresis devices. The method is based on combined solid-phase extraction, purification, and transport of DNA reversibly bound on paramagnetic microspheres. DNA is adsorbed onto the microspheres, captured with a magnetized permalloy wire, and then directly injected as a highly focused sample plug into the separation channel.

Reversible ratiometric probe for quantitative DNA measurements.

We have designed a reversible fluorescent DNA probe that can be used to determine the concentration of single-stranded DNA in solution by a ratiometric fluorescence measurement. The probe consists of a single-stranded dual fluorescently labeled DNA molecule that adopts a stem-loop conformation in its nonhybridized state. The stem length and the length of the loop region complementary to the target were chosen to allow for reversible binding.

Coupling fiber optics to a permeation liquid membrane for heavy metal sensor development.

We present the first sensing system for metal ions based on the combination of separation/preconcentration by a permeation liquid membrane (PLM) and fluorescence detection with an optical fiber. As a model, a system for the detection of Cu(II) ions was developed. The wall of a polypropylene hollow fiber serves as support for the permeable liquid membrane.

Mechanisms of glutamate metabolic signaling in retinal glial (Müller) cells.

Retinal Müller (glial) cells metabolize glucose to lactate, which is preferentially taken up by photoreceptor neurons as fuel for their oxidative metabolism. We explored whether lactate supply to neurons is a glial function controlled by neuronal signals. For this, we used subcellular fluorescence imaging and either amperometric or optical biosensors to monitor metabolic responses simultaneously from mitochondrial and cytosolic compartments of individual Müller cells from salamander retina.