RESUMEN
Individual phospholipid vesicles, 1 to 5 micrometers in diameter, containing a single reagent or a complete reaction system, were immobilized with an infrared laser optical trap or by adhesion to modified borosilicate glass surfaces. Chemical transformations were initiated either by electroporation or by electrofusion, in each case through application of a short (10-microsecond), intense (20 to 50 kilovolts per centimeter) electric pulse delivered across ultramicroelectrodes. Product formation was monitored by far-field laser fluorescence microscopy. The ultrasmall characteristic of this reaction volume led to rapid diffusional mixing that permits the study of fast chemical kinetics. This technique is also well suited for the study of reaction dynamics of biological molecules within lipid-enclosed nanoenvironments that mimic cell membranes.
Asunto(s)
Bioquímica/métodos , Liposomas , Fosfatasa Alcalina/metabolismo , Calcio/metabolismo , ADN/metabolismo , Difusión , Electroquímica , Electroporación , Fluoresceínas/metabolismo , Fluorescencia , Colorantes Fluorescentes/metabolismo , Concentración de Iones de Hidrógeno , Membrana Dobles de Lípidos , Microelectrodos , Microscopía Confocal , Microscopía Fluorescente , Miniaturización , Técnicas de Placa-Clamp , FosfolípidosRESUMEN
A novel injection scheme is described in which ultrasmall samples in the attoliter (10(-18) L) and low femtoliter (10(-15) L) range, or even single molecules, are controllably introduced into a tapered capillary so that electrophoretic separation can be carried out. To match the dimensions of the capillary inlet with that of the sample, capillary tips are tapered to an inside diameter ranging from hundreds of nanometers to a few micrometers. To inject an ultrasmall sample, optical trapping is used to immobilize and manipulate the sample in order to place it inside or next to the capillary inlet. A small controlled suction results in the loading of the sample into the capillary.