Laminar-Flow Fluid Mixer for Fast Fluorescence Kinetics Studies

• Published in: Biophysical journal 2002-11, Vol.83 (5), p.2872-2878
• Main Authors: Pabit, Suzette A., Hagen, Stephen J.
• Format: Article
• Language: English
• Subjects: Biophysics - methods; Dose-Response Relationship, Drug; Fluorescence; Kinetics; Microscopy, Video; Nucleic Acid Conformation; Nucleic Acids - chemistry; Physics; Protein Folding; Proteins; Proteins - chemistry; Sodium Iodide - pharmacology; Spectrometry, Fluorescence - methods; Time Factors; Tryptophan - chemistry; Ultraviolet Rays
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(02)75296-X

The ability to mix aqueous liquids on microsecond time scales, while consuming minimal amounts of sample and maintaining UV-visible optical access to the mixing region, is highly desirable for a range of biophysical studies of fast protein and nucleic acid interactions and folding. We have constructed a laminar coaxial jet mixer that allows the measurement of UV-excited fluorescence from nanoliter and microliter quantities of material, mixed at microsecond rates. The mixer injects a narrow cylindrical stream (radius a < 1 μm) of fluorescent sample into a larger flow of diluting buffer that moves through a capillary (100 μm i.d.) at a speed ∼20 cm/s, under laminar flow conditions ( Re ≈ 14). Construction from a fused silica capillary allows the laser excitation (at 266 nm) and detection (at 350 nm) of tryptophan fluorescence at reasonably low working concentrations, without interference from background fluorescence. Using this mixer we have measured sub-millisecond fluorescence quenching kinetics while consuming fluorescent sample at rates no greater than 6 nl/s. Consumption of the diluting buffer is also very modest (∼1–3 μl/s) in comparison with other rapid mixer designs.