Microfabricated multiple-channel glass chips were successfully interfaced to an electrospray ionization mass spectrometer (ESI-MS). The microchip device was fabricated by standard photolithographic, wet chemical etching, and thermal bonding procedures. A high voltage was applied individually from each buffer reservoir for spraying sample sequentially from each channel. With the sampling orifice of the MS grounded, it was found that a liquid flow of 100-200 nL/min was necessary to maintain a stable electrospray. The detection limit of the microchip MS experiment for myoglobin was found to be lower than 6 x 10(-8) M. Samples in 75% methanol were successfully analyzed with good sensitivity, as were aqueous samples. The parallel mutliple-channel microchip system allowed ESI-MS analysis of different samples of standard peptides and proteins in one chip.

Much attention has been focused recently on the detection and physical characterization of individual molecules. Using such methods to study the chemical properties, such as reactivity, of single molecules offers the potential to investigate how these might vary from molecule to molecule, and for individual molecules as a function of time. The complex structures of biomolecules such as enzymes make them particularly attractive targets for studying how subtle changes or differences at the molecular level might influence chemical reactivity. We have shown previously that very small (zeptomole) amounts of enzymes can be studied using a fluorescence microassay; single enzyme molecules have also been detected in oil-dispersed droplets by fluorescence microscopy. Here we report the observation of reactions of individual molecules of lactate dehydrogenase (LDH-1), which produces NADH from lactate and nicotinamide adenine dinucleotide (NAD+). When they are present at very low concentrations in a narrow capillary, each enzyme molecule produces a discrete zone of NADH; these can be manipulated electrophoretically and monitored by fluorescence spectroscopy. We find that the activity of individual electrophoretically pure enzyme molecules can vary by up to a factor of four, and that these activities remain unchanged over a two-hour period. We suggest that the origin of the activity differences may lie in the presence of several stable forms of the enzyme.

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Trace amounts of enzymes within single human erythrocytes can be quantified by a combination of on-column reaction and capillary electrophoresis. A detection limit of 1.3 x 10(-21) mol of LDH was achieved with laser-induced fluorescence by monitoring the product of the enzyme-catalyzed reaction between lactate and NAD+. Single erythrocyte analysis clearly isolates the major forms of LDH. The variation of total LDH activity in a population of cells from a single individual is large, but the relative activities of the isoenzymes LDH-1 and LDH-2 are fairly constant. This can be explained by the distribution of cell age in the population. A lower enzyme activity is indicative of senescence. The efficient separation of different LDH forms and the high detection sensitivity opens up the possibility of multiple-enzyme assays with a single mammalian cell.