Rapid mixing of two fluids has remained a challenge within the microfluidics community. Current techniques that rely on diffusion as a mixing mechanism require mixing times up to several minutes and mixing lengths on the order of meters — making it difficult to analyze rapid biochemical reactions. Recently, a group of researchers from the Universities of California, Irvine, and San Diego introduced a laser-induced technique that allows microsecond micron-scale mixing of two fluids.Time-resolved photography depicts the mixing process from left to right: laser-induced plasma formation produces a bubble (a) that expands within a 200-μm channel (b) on the microfluidic device. After the first split, jet formation toward the channel walls causes another split (c) and allows the fluids to mix (d). The first of three studies conducted by the group utilized time-resolved photography to visualize the mixing dynamics. Two parallel streams of water and an 8-mg/ml solution of green dye were injected into the microfluidic device, which was placed on the stage of a Zeiss inverted microscope. Pulses 6 ns long from a 532-nm Q-switched Nd:YAG Spectra-Physics laser were introduced into the rear aperture of the microscope objective, producing a localized nanoliter of mixed fluid.Plasma-induced bubbleThe individual laser pulses induced plasma formation, which generated a 240-μm-long bubble within the channel in only 4.4 μs. Its rapid collapse caused the bubble to split along the axis parallel to the channel walls and then, again, perpendicularly, forming four smaller bubbles. The jet formation associated with the second bubble collapse destabilized the interface between the two fluids, and it allowed them to mix. Whereas this expansion and collapse occurred in ∼50 μs, the mixing dynamics continued for as long as 15 ms, with the fluid interface fully restored after 50 ms. The researchers imaged these mixing dynamics using a Roper Scientific intensified CCD camera attached to the microscope.With this optical setup, Nd:YAG laser pulses are introduced to a microfluidic device and cause localized mixing of two fluids.Replacing the green dye with 10 μM of fluorescein and bringing the two fluids together at a Y-junction on the microfluidic device, the group used fluorescence detection to examine the dynamics ∼7 mm downstream from the laser-induced mixing site. The researchers monitored the presence of fluorescent molecules with the output of a continuous-wave, 488-nm argon-ion laser, which was coupled through a single-mode fiber optic and reflected into the rear aperture.An optical setup comprising a Hamamatsu photomultiplier tube, a long-pass filter to block argon-ion laser light and a 50-μm-diameter pinhole for confocal detection was mounted on a translation stage above the microfluidic device. This allowed positioning of both the detection system at a defined distance from the Nd:YAG laser pulse site and the argon-ion beam focal volume at desired locations within the channel. A volume of mixed fluid was found to occupy the width of the channel and was carried downstream with the main flow.Chemical reactionFinally, the researchers demonstrated the ability of the technique to initiate a biochemical reaction and imaged the process with fluorescence videomicroscopy. Two non-fluorescent reactants, Amplex Red and hydrogen peroxide, were mixed in the presence of horseradish peroxidase to produce fluorescent resorufin.The inverted microscope setup equipped with a 100-W mercury lamp enabled the researchers to obtain bright-field and fluorescence images of the reaction, which they recorded with a Panasonic digital video camera.The technique holds several advantages over current methods: it does not require a customized microfluidic device design, it provides a localized region of mixed fluid that can be produced at any optically accessible location within a microfluidic chip, and the mixing has the potential to be rapidly switched to create individual boluses of mixed fluid or continuous mixing. The group is currently investigating the dependence of the fluid mixing dynamics on the duration, energy, wavelength and repetition rate of the laser pulse.Analytical Chemistry, published online May 18, 2007.