We show that coupled electrorotation (CER) of microscopic particles using microfabricated electrodes can be used for localized sensing and mixing. The effective use of microelectromechanical systems and micro total analysis systems requires many types of control. These include the abilityto (1) manipulate objects within microchannels by noncontact means, (2) mix fluids, and (3) sense local chemical parameters. Coupled electrorotation, in which the interactions between induced electric dipoles of adjacent particles lead to particle rotation, addresses aspects of all three challenges simultaneously. CER is a simple means of controlling the rotation of dielectric objects using homogeneous external radio frequency electric fields. CER is sensitive to several chemical and physical parameters such as the solution conductivity, pH, and viscosity. As a step toward integrating CER devices into microfluidic systems, a simple chip was designed to induce local mixing and to detect local changes in salt concentration, pH, and viscosity.

We report a novel approach to micro- and nanoparticle rotation, uniting the fine translational control afforded by optical trapping with the flexibility and simplicity of dipole-field-induced coupled electrorotation (CER). Fluorescence imaging using a microparticle photopatterning technique was combined with optical trapping to quantify both the senses and speeds of rotation for individual pairs of particles. Laser tweezers allowed controlled positioning of a pair of particles within a dipole field while simultaneously providing an axis about which the particles rotated. The particle-particle interactions inherent in CER offer several distinct advantages compared with electrorotation in multipole fields. Results from several investigations highlight the utility of this approach, including quantification of rotation in spheres as small as 750 nm in diameter, observation of rotation rates as high as 1800 rpm, fabrication of coupled electrorotational "antigears", trapping and rotation of sphere dimers, and exploitation of the registry of sphere rotation to probe the dielectric properties of immobile objects.

We describe the fabrication of nanoengineered holding pipets with concave seating surfaces and fine pressure control. These pipets were shown to exhibit exceptional stability in capturing, transporting, and releasing single cells and liposomes 1-12 microm in diameter, which opens previously inaccessible avenues of research. Three specific examples demonstrated the utility and versatility of this manipulation system. In the first, carboxyrhodamine was selectively incorporated into individual cells by electroporation, after which nearly all the medium (hundreds of microliters) surrounding the docked and tagged cells was rapidly exchanged (in seconds) and the cells were subsequently probed by laser-induced fluorescence (LIF). In the second study, a single liposome containing carboxyrhodamine was transported to a dye-free solution using a transfer pipet, docked to a holding pipet, and held firmly during physical agitation and interrogation by LIF. In the third study, pairs of liposomes were positioned between two microelectrodes, held in contact, and selectively electrofused and the resulting liposomes undocked intact.

As organisms, we are amazingly complex living laboratories. As we move, breathe, think, and eat, seemingly endless chemical reactions and interactions occur inside us. The test tubes, beakers, and flasks used to separate and selectively mix the myriad of reactants involved are cells, vesicles, and organelles. Taking the analogy further, whereas chemists typically mix chemicals milliliters or more in volume, biological systems carry out their biochemistry in containers that are femtoliters or less in volume. As researchers we assume, with good reason, that the material surfaces of our laboratory test tubes do not substantially affect the kinetics we measure. This assumption might not hold were we to shrink our containers to the femtoliter scale. At such a small scale, collision rates between reactants and their container walls become significant [1], and the inner surface, particularly in biological containers, is chemically complex. The bilayers of cells and organelles are composed of a variety of lipids. These varieties assemble into domains [2] in a process partly controlled by the transmembrane proteins in them [3]. Cellular and organellar control of chemical reactions may thus come, in part, from alterations in the composition and arrangement of the molecular species making up the bilayer membrane [4]. How do systematic alterations to the bilayer composition of a liposome alter the kinetics of reactions within the liposome interior? We may find that the potential physiological significance of lipid domains within bilayers to the kinetics of in-plane reactions [5] (i.e., for proteins and other molecules moving within the bilayer) has applications to molecules within liposomes that interact with the inner bilayer surface.

A method for cell-cell and cell-liposome fusion at the single-cell level is described. Individual cells or liposomes were first selected and manipulated either by optical trapping or by adhesion to a micromanipulator-controlled ultramicroelectrode. Spatially selective fusion of the cell-cell or cell-liposome pair was achieved by the application of a highly focused electric field through a pair of 5-micrometer o.d. carbon-fiber ultramicroelectrodes. The ability to fuse together single cells opens new possibilities in the manipulation of the genetic and cellular makeup of individual cells in a controlled manner. In the study of cellular networks, for example, the alteration of the biochemical identity of a selected cell can have a profound effect on the behavior of the entire network. Fusion of a single liposome with a target cell allows the introduction of the liposomal content into the cell interior as well as the addition of lipids and membrane proteins onto the cell surface. This cell-liposome fusion represents an approach to the manipulation of the cytoplasmic contents and surface properties of single cells. As an example, we have introduced a membrane protein (gamma-glutamyltransferase) reconstituted in liposomes into the cell plasma membrane.

A method to study single-molecule reactions confined in a biomimetic container is described. The technique combines rapid vesicle preparation, optical trapping and fluorescence confocal microscopy for performing simultaneous single-vesicle trapping and single-molecule detection experiments. The collisional environment between a single enzyme and substrate inside a vesicle is characterized by a Brownian dynamics Monte Carlo simulation.

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.

The behavior of water in;a benzene solution of C-undecylcalix[4]resorcinarene (UR) is studied by H-1 2D NMR exchange spectroscopy and by 1D NMR. The rate constants for exchange of the UR hydroxyl protons, H-a, with water over the temperature range 298-338 K are 0.7-4.6 s(-1) and those for the exchange of UR hydroxyl protons, Hb, over the temperature range 298-328 K are 1.8-8.1 s(-1). Activation energies calculated for these exchange processes are 9.0 +/- 0.5 kcal/mol and 10.2 +/- 1.0 kcal/mol, respectively. The chemical shift of dissolved water is largely dependent upon the concentration of UR. An upper limit for the critical aggregation concentration of UR is 10(-5) M as shown by H-1 NMR.