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Nine orders of magnitude dynamic range: picomolar to millimolar concentration measurement in capillary electrophoresis with laser induced fluorescence detection employing cascaded avalanche photodiode photon counters.

ABSTRACT: The dynamic range of capillary electrophoresis analysis is ultimately limited by molecular shot noise at low concentrations and by concentration-induced band broadening at high concentrations. We report a system that approaches these fundamental limits. A laser-induced fluorescence detector is reported that employs a cascade of four fiber-optic beam splitters connected in series to generate a primary signal and four attenuated signals, each monitored by a single-photon counting avalanche photodiode. Appropriate scaling of the signals from the five photodiodes produces a linear optical calibration curve for 5-carboxyl-tetramethylrhodamine from the concentration detection limit of 1 pM to the upper limit of 1 mM. Mass detection limits are 120 yoctomoles (70 molecules) injected into the instrument. The very-wide dynamic range instrument was used to study the metabolic products of the fluorescently labeled glycosphingolipid tetramethylrhodamine labeled GM1 (GM1-TMR) produced by single cells isolated from the rat cerebellum.

SUBMITTER: Dada OO

PROVIDER: S-EPMC3072807 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Nine orders of magnitude dynamic range: picomolar to millimolar concentration measurement in capillary electrophoresis with laser induced fluorescence detection employing cascaded avalanche photodiode photon counters.

Dada Oluwatosin O OO Essaka David C DC Hindsgaul Ole O Palcic Monica M MM Prendergast Jillian J Schnaar Ronald L RL Dovichi Norman J NJ

Analytical chemistry 20110316 7

The dynamic range of capillary electrophoresis analysis is ultimately limited by molecular shot noise at low concentrations and by concentration-induced band broadening at high concentrations. We report a system that approaches these fundamental limits. A laser-induced fluorescence detector is reported that employs a cascade of four fiber-optic beam splitters connected in series to generate a primary signal and four attenuated signals, each monitored by a single-photon counting avalanche photodi ...[more]

PMID: 21410138

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Project description:A beta camera has been developed that allows planar imaging of the spatial and temporal distribution of beta particles using a 14 × 14 mm(2) position sensitive avalanche photodiode (PSAPD). This camera system, which we call Betabox, can be directly coupled to microfluidic chips designed for cell incubation or other biological applications. Betabox allows for imaging the cellular uptake of molecular imaging probes labeled with charged particle emitters such as (18)F inside these chips. In this work, we investigate the quantitative imaging capabilities of Betabox for (18)F beta particles, in terms of background rate, efficiency, spatial resolution, and count rate. Measurements of background and spatial resolution are considered both at room temperature (21 °C ± 1 °C) and at an elevated operating temperature (37 °C ± 1 °C), as is often required for biological assays. The background rate measured with a 4 keV energy cutoff is below 2 cph mm(-2) at both 21 and 37 °C. The absolute efficiency of Betabox for the detection of (18)F positron sources in contact with a PSAPD with the surface passivated from ambient light and damage is 46% ± 1%. The lower detection limit is estimated using the Rose Criterion to be 0.2 cps mm(-2) for 1 min acquisitions and a 62 × 62 µm(2) pixel size. The upper detection limit is approximately 21 000 cps. The spatial resolution at both 21 and 37 °C ranges from 0.4 mm FWHM at the center of the field of view (FOV), and degrades to 1 mm at a distance of 5 mm away from center yielding a useful FOV of approximately 10 × 10 mm(2). We also investigate the effects on spatial resolution and sensitivity that result from the use of a polymer based microfluidic chip. For these studies we place varying layers of low-density polyethylene (LDPE) between the detector and the source and find that the spatial resolution degrades by ?180 µm for every 100 µm of LDPE film. Sensitivity is reduced by half with the inclusion of ?200 µm of additional LDPE film. Lastly, we demonstrate the practical utilization of Betabox, with an imaging test of its linearity, when coupled to a polydimethylsiloxane microfluidic chip designed for cell based assays.

Dataset Information

Nine orders of magnitude dynamic range: picomolar to millimolar concentration measurement in capillary electrophoresis with laser induced fluorescence detection employing cascaded avalanche photodiode photon counters.

Publications

Nine orders of magnitude dynamic range: picomolar to millimolar concentration measurement in capillary electrophoresis with laser induced fluorescence detection employing cascaded avalanche photodiode photon counters.

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