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RESEARCH PROFILES Dopamine transport at small volumes microelectrodes and sensors with micro- pipette; when the sensing portion of the As analytical instrumentation and techelectrode was placed close to the injecsecond temporal response. Other reniques have become more sensitive, tor tip, less time was needed for the sigchemists have been able to detect fewer ports have involved fabricating electronal to reach its maximum value, and chemical sensors in the bases of 600-pL and fewer molecules. However, the vice-versa. They also challenge before many found that the cyclic chemists today is to use voltammetric current these trace techniques rapidly increased after to measure phenomena dopamine was injected in microenvironments. into the vial, indicating Mark Wightman and rapid delivery and distriKevin Troyer at the bution of electroactive University of North species. Carolina–Chapel Hill To study cellular take up that challenge uptake, the researchers and, in the October 15 chose HEK-293 cells, issue of Analytical which came from an Chemistry (pp 5370– immortal line of kid5375), introduce a ney cells that had been novel technique that almanipulated to express lows them to monitor, the dopamine transwith subsecond time porter. In vivo, the resolution, the active dopamine transporter transport of dopamine protein plays a central molecules by single cells Illustration of complete microvial, placed horizontally, showing the integral refrole in clearing dopainside a picoliter vial. erence electrode (silver wire inside vial) and working electrode (carbon-fiber mine from the extraPerforming the exper- microelectrode). cellular space following iments in a 150-pL vial its secretion; many substances, such as wells to monitor secretion from single permits tight control of the extracellular cocaine and amphetamines, can inhibit cells. “We simply combined the idea of volume, and changes in the extracellular this protein and its transport of dopasmall volumes and microsensors,” says compartment can be monitored at the mine into the cell. Wightman, “something that had not single-cell level. “To watch dynamic In these uptake experiments, the processes at single cells allows one to ex- been done before.” A common problem with performing dopamine concentration in the vial deamine the degree of cell-to-cell variaanalysis at small volumes is that evapora- creased with time at a rate consistent tion,” says Wightman. with the kinetics measured in cell popution usually occurs within a matter of Wightman and Troyer fabricated lations. “We were watching, in real each 150-pL vial from a fused-silica cap- seconds and often leads to an increase time, proteins on the electrode surface in solution osmolarity. To prevent samillary and placed the vial horizontally transporting the dopamine molecules ple evaporation, the vials are capped under a microscope in a culture plate into the cell,” Wightman says. with mineral oil. Dopamine is then inof cells. The vial was filled with NaCl Wightman hopes the technique can serted into the vial, and its uptake into buffer, and a single ~15-µm-diam cell be used in the future to gain a better the cell is determined using a fast-scan was inserted using a transfer pipette. understanding of cell behavior, especialcyclic voltammeter, which measures the After a ~10-min acclimation period, the ly when measuring chemical reactions in electroactive species in the vial, says culture plate was removed, and a disksingle cells in the midst of a population Wightman. shaped, 3-µm carbon-fiber microelecof cells. “The principle illustrated by the A benefit of using smaller-volume trode was inserted into the vial with an measurements is that not only can we vials for cell analysis is how quickly the Ag/AgCl wire that served as the referdetect very small quantities of material, vial’s contents can reach uniform conence electrode. but we can measure the rate at which centrations, even without stirring. Previous reports have looked primathey undergo a transformation,” rily at static chemical composition or in- Wightman and Troyer found that the he says. a mixing time depended on the relative vestigated processes at the cell surfaces —Wilder D. Smith location of the electrode and injection with micrometer-sized carbon-fiber 566 A
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