[Show abstract][Hide abstract] ABSTRACT: We report a novel approach for determining the enzymatic activity within a single suspended cell. Using a steady-state microfluidic delivery device, and timed exposure to the pore-forming agent digitonin, we controlled the plasma membrane permeation of individual NG108-15 cells. Mildly permeabilized cells (~100 pores) were exposed to a series of concentrations of FDP, a fluorogenic alkaline phosphatase substrate, with and without levamisole, an alkaline phosphatase inhibitor. We generated quantitative estimates for intracellular enzyme activity, and were able to construct both dose-response and dose-inhibition curves, at the single-cell level, resulting in an apparent Km of 15.3 μM ± 1.02 (mean ± SEM, n=16), and Ki of 0.59 mM ± 0.07 (mean ± SEM, n=14). Enzymatic activity could be monitored just 40 s after permeabilization, and 5 point dose-inhibition curves could be obtained within 150 s. This rapid approach offers a new methodology for characterizing enzyme activity within single cells.
[Show abstract][Hide abstract] ABSTRACT: Methods that can control and vary the solution environment around single cells are abundant. In contrast, methods that offer direct access to the intracellular proteome and genome in single cells with the control, flexibility, and convenience given by microfluidic methods are both scarce and in great demand. Here, we present such a method based on using a microfluidic device mounted on a programmable scanning stage and cells on-chip permeabilized by the pore-forming glycoside digitonin. We characterized the on-chip digitonin poration, as well as the solution exchange within cells. Intracellular solution exchange times vary with the dose of exposure to digitonin from less than a second to tens of seconds. Also, the degree of permeabilization obtained for cells treated with the same dose varies considerably, especially for low doses of digitonin exposure and low permeabilities. With the use of the presented setup, the degree of permeabilization can be measured during the permeabilization process, which allows for "on-line" optimization of the digitonin exposure time. Using this calibrated permeabilization method, we demonstrate the generation of intracellular oscillations, intracellular gradients, and the delivery of substrate to initiate enzymatic reactions in situ. This method holds the potential to screen and titrate intracellular receptors or enzymes or to generate intracellular oscillations, useful in the study of signaling pathways and oscillation decoding among other applications.
[Show abstract][Hide abstract] ABSTRACT: We present a concept for the post production modification of commercially available microfluidic devices to incorporate local temperature control, thus allowing for the exact alignment of heating structures with the existing features, e.g. wells, channels or valves, of a system. Specifically, we demonstrate the application of programmable local heating, controlled by computerized PI regulation, to a rapid solution exchanger. Characterisation of the system to show that both uniform temperature distributions and temperature gradients can be established, and to confirm that the solution exchange properties are undisturbed by heating, was achieved using in situ thermometry and amperometry.
Lab on a Chip 04/2008; 8(3):480-3. · 5.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We introduce a system for temperature control of a commercial microfluidic superfusion device that, in combination with patch-clamp, enables rapid acquisition of dose-response data at different temperatures. We obtained dose-response curves for the GABAA receptor, a ligand-gated ion channel, for two different agonists at temperatures between 25 and 40 degrees C. For GABA, the dose-response curves shifted toward higher EC50 values as the temperature increased, whereas for beta-alanine, the EC50 values were constant. This shows that temperature is an important factor for obtaining accurate estimations of EC50 values and also that such temperature effects can be ligand-specific. Using the EC50 values, we estimated the enthalpy of dissociation between the ligand and the receptor. Furthermore, the technology introduced here is generally applicable to all patch-clamp studies where temperature control is desirable, e.g., studies of kinetics and thermodynamics, drug screening, compliant ADME/Tox testing, and in studies of temperature-gated ion channels.
[Show abstract][Hide abstract] ABSTRACT: We present an open-volume microfluidic system capable of on-line modification of a patterned laminar flow by using programmable inlet valves. Each separate solution environment in the flow pattern can be independently exchanged between different preloaded input solutions where each exchange requires 20 s. The number of flow patterns that can be generated by one device is N(n), where N represents the number of valve inlets and n the number of microchannels in the microfluidic system. Furthermore, the system can be operated as a combinatorial mixer, in which mixture of the different input solutions can be obtained independently in each channel. Since the flow patterns are generated in an open volume, they are accessible to many different detection methods and types of probes, e.g., microelectrodes, cells, or cell fragments. This technology offers the possibility to adjust the flow pattern composition in response to an output from a probe. This is the first step toward creating an automated feedback device controlled by, for example, biological cells.
[Show abstract][Hide abstract] ABSTRACT: Algorithms and methods were developed to synthesize complex chemical waveforms in open volumes by using a scanning-probe microfluidic platform. Time-dependent variations and oscillations of one or several chemical species around the scanning probe, such as formation of sine waves, damped oscillations, and generation of more complex patterns, are demonstrated. Furthermore, we show that intricate bursting and chaotic calcium oscillations found in biological microdomains can be reproduced and that a biological cell can be used as a probe to study receptor functionalities as a function of exposure to time-dependent variations of receptor activators and inhibitors. Thus, the method allows for studies of biologically important oscillatory reactions. More generally, the system allows for detailed studies of complex time-varying chemical and physical phenomena in solution or at solution/surface interfaces.
Proceedings of the National Academy of Sciences 07/2005; 102(23):8097-102. · 9.81 Impact Factor