A Continuous-Flow Capillary Mixing Method to Monitor Reactions on the Microsecond Time Scale

Abstract

A continuous-flow capillary mixing apparatus, based on the original design of Regenfuss et al. (Regenfuss, P., R. M. Clegg, M. J. Fulwyler, F. J. Barrantes, and T. M. Jovin. 1985. Rev. Sci. Instrum. 56:283-290), has been developed with significant advances in mixer design, detection method and data analysis. To overcome the problems associated with the free-flowing jet used for observation in the original design (instability, optical artifacts due to scattering, poor definition of the geometry), the solution emerging from the capillary is injected directly into a flow-cell joined to the tip of the outer capillary via a ground-glass joint. The reaction kinetics are followed by measuring fluorescence versus distance downstream from the mixer, using an Hg(Xe) arc lamp for excitation and a digital camera with a UV-sensitized CCD detector for detection. Test reactions involving fluorescent dyes indicate that mixing is completed within 15 micros of its initiation and that the dead time of the measurement is 45 +/- 5 micros, which represents a >30-fold improvement in time resolution over conventional stopped-flow instruments. The high sensitivity and linearity of the CCD camera have been instrumental in obtaining artifact-free kinetic data over the time window from approximately 45 micros to a few milliseconds with signal-to-noise levels comparable to those of conventional methods. The scope of the method is discussed and illustrated with an example of a protein folding reaction.

Identification and characterization of protein folding intermediates

“…As described above, the existence of folding intermediates is often proposed from indirect observations, such as the presence of sub-millisecond burst phases or roll-over effects. However, the development of instrumentation for protein folding studies, for example ultra-rapid mixing devices [32], and temperature jump relaxation techniques [33], has allowed the direct characterization of events taking place in the dead-time of conventional stopped-flow instruments. These techniques have provided new insight in the role of intermediates in protein folding [10].…”

Multi-step synthesis of nanoparticles performed on millisecond time scale in a microfluidic droplet-based system

“…16 Turbulent flow at high Re mixes reagents more quickly and limits dispersion, but requires high flow rates and increased sample consumption. 18 We have previously demonstrated millisecond time control of a single-step reaction in microfluidic channels by isolating multiple streams of reagents within plugs -aqueous droplets surrounded and transported by fluorocarbons immiscible with water. 1 We refer to the fluorocarbon mixtures used in this study as "oils".…”

Reassessing the folding of the KIX domain: Evidence for a two‐state mechanism

“…In this study, we exemplify that the saturation of the apparent rate constants at low urea may induce apparent rollover effect. In the latter case, ultrafast mixing experiments, classically introduced to monitor the accumulation of short-lived intermediates, [26][27][28][29][30] allow to infer unambiguously the existence of an intermediate, if any. By integrating stopped-flow and continuous-flow mixing experiments, in fact, we obtained for the folding kinetics of KIX a completely linear chevron plot typically diagnostic of a simple two-state model.…”

“…Continuous-flow measurements were performed using a homemade instrument of design and methodology similar to that published by Shastry and Roder, 30 as described in Refs. 31 and 32.…”