Separations of 15 pM rhodamine 6G and 30 pM rhodamine B performed in a micromachined electrophoresis channel were detected by counting fluorescence bursts from individual molecules. The migration times, peak widths, and analyte concentrations were estimated from the number and the migration time distribution of the detected molecules. Concentration detection limits estimated at >99% confidence were 1.7 pM rhodamine 6G and 8.5 pM rhodamine B. The separations required <35 s and the relative migration time uncertainties were less than 2.0%. These are the lowest detection limits reported for microchip separation devices and the first example of single-chromophore molecular counting for detection of chemical separations.

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Acridine yellow dissolved in a rigid saccharide glass is proposed as a sensor material for optical thermometry. Following efficient excitation in the visible, triplet states of the dye are produced with a high quantum yield. Activated reverse intersystem crossing from the triplet to the singlet excited state, followed by delayed fluorescence, provides a temperature-dependent decay pathway that competes with phosphorescence to depopulate the triplet state. Either the triplet-state lifetime or ratio of delayed fluorescence-to-phosphorescence intensities may be used to monitor temperature. Lifetimes of >100 ms are observed at ambient temperatures which require modest instrumentation to measure and process. Since fluorescence and phosphorescence spectra are well separated, their intensity ratio can be determined using interference filters. The thermometer performance can be predicted from photophysical models for the temperature dependence of the triplet-state decay. The relative sensitivities of the triplet-state lifetime and of the ratio of delayed fluorescence-to-phosphorescence intensities to temperature over the range of -50 to 50 °C are 2.0 and 4.5%/ °C, respectively, which are ~10 times greater than typical optical thermometers. The high sensitivities to temperature change result in temperature uncertainties of less than 1 °C over this range.Recent years have seen growing applications for fiber-optic sensors in remote temperature measurements. Fiber-optic thermometers permit operation in the vicinity of electromagnetic fields and in hazardous environments. Sensor materials fashioned into compact fiber-optic probes can be used with minimal perturbation to their surroundings. In order to reduce instrument cost and complexity, the sensor material should facilitate the use of visible excitation sources and uncomplicated signal processing techniques. To maximize sensitivity of the thermometer, the optical characteristics of the sensor material should have a high relative change with temperature. To meet these goals, optical thermometers have been designed around temperature dependent-excitedstate lifetimes,1-9 shifts in fluorescence or absorption spectra,10-13 or black-body radiation spectra.14 Each approach exhibits certain (1)

Cross-correlation electrophoresis with fluorescence detection on a microchip device was used to resolve a mixture of 63 pM fluorescein and 70 pM dichlorofluorescein. The signal-to-noise ratios of analyte peaks in a correlogram derived from a 9-bit pseudorandom injection sequence were ∼17 for dichlorofluorescein and ∼6 for fluorescein. In contrast, neither peak was detected in a conventional electropherogram obtained on the microchip. Detection limits estimated from a correlogram derived from the average of 12 7-bit correlograms were 6.5 pM dichlorofluorescein and 21 pM fluorescein. These are the lowest detection limits reported for a microchip separation device using analog detection and the first example of cross-correlation electrophoresis on a monolithic device. The small injection volumes, injection reproducibility, and rapid separations enabled by microchip devices make cross-correlation multiplex injection schemes a viable approach for achieving enhanced sensitivity.

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