Described herein is an application of the copper(I)-catalyzed Huisgen [3+2] cycloaddition between azides and alkynes,
or click chemistry, for the universal two-step detection of biological macromolecules. The first step involves the
metabolic incorporation of an azide or alkyne probe into a macromolecule of interest. The second step involves the
click chemistry conjugation of the labeled macromolecule with a partner alkyne or azide-reactive fluorescent probe to
form a stable triazole ring conjugate. The fluorescently tagged molecules can be subsequently detected by a number of
different fluorescent readout platforms including flow cytometry, fluorescence imaging, and 1-D/2-D gel imaging. We
demonstrate application of this technology in two different labeling schemes. First, the labeling of newly synthesized
DNA in a novel cell proliferation assay, and second, in the labeling of specific glycoprotein subclasses for biomarker
discovery applications. In each case, azide or alkyne probes are introduced metabolically with subsequent detection
using click chemistry. Utilization of the cellular enzymatic machinery for high-fidelity target molecule labeling
combined with the superior efficiency of click chemistry detection results in highly versatile macromolecular labeling
platforms that are unmatched in sensitivity and selectivity.
The initialization of a flow field with distinct and spatially segregated scalar components represents a significant experimental difficulty. Many theoretical modeling efforts in turbulent mixing, however, seek to describe the temporal evolution of a scalar concentration field that begins with this type of idealized initial conditions experimentally. This technique uses photoactivatable (caged) fluorescence dyes dissolved in the flow medium. Caged fluorescent dyes differ from tradition dyes in that excitation and subsequent emission will not occur until a bond within the caged dye molecule is broken with an ultraviolet photon. The flow field is then tagged by activating or 'uncaging' the appropriate regions with an excimer laser. Mixing between the tagged and untagged regions is quantified by illuminating the points to be studied with an argon ion laser, and measuring the subsequent emission intensity using standard laser induced fluorescence techniques. The intensity of the emission is proportional to the concentration of the uncaged dye. High sensitivity photodetectors allow very low intensity fluctuation measurements to be made. This method is currently being used to study mixing in a turbulent pipe flow, and shows potential to be used in a large number of other flow situations.