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It is not uncommon for students to view laboratory instruments as black boxes. Unfortunately, this can often result in poor experimental results and interpretation. To tackle this issue, a laboratory course was designed to enable students not only to critically think about operating principles of the instrument but also to improve interpretation skills. Students were required to build their own visible spectrometer using interlocking building bricks with simple optical elements and a Raspberry Pi computer. Experiments were then conducted to explore the instrumental capabilities while, at the same time, using Python programming to plot data, perform linear least-squares fitting, and calculate errors. Instrument response and spectral measurements were followed by kinetic studies, enabling the students to tackle a "real" problem by extracting rate constants. The main learning outcomes were that the students would gain a better understanding of instrumental components and at the same time learn valuable analytical techniques such as calibration, determination of the limits of linearity, and dynamic range. These outcomes were achieved by applying a problem based learning approach.

The use of dielectrophoretic forces is crucially tied to the knowledge of Joule heating within a fluid, since the use of planar microelectrodes creates a temperature gradient within which the particle of interest is manipulated. Mapping temperature with sufficient spatial resolution within a dielectrophoretic trap is recognized to be of high importance. Herein, we demonstrate local temperature measurements in the vicinity of a trapped micrometersize particle using confocal fluorescence spectroscopy. Such measurements are shown to provide a novel calibration tool for screening temperature-mediated processes with high resolution. 10.1021/ac101557g

Microdroplets generated in microfluidic channels hold great promise for use as substrates in high-throughput chemical and biological analysis. These water-in-oil compartments can serve as isolated reaction vessels, and since they can be generated at rates in excess of 1 kHz, thousands of assays can be carded out quickly and reproducibly. Nevertheless, sampling the large amount of information generated from these platforms still remains a significant challenge. For example, considering the high droplet generation rates and velocities, reproducibility and micrometer resolution are challenging requirements that must he fulfilled. Herein we combine confocal fluorescence lifetime imaging microscopy with a statistical implementation that permits the analysis of mixing phenomena within microdroplets with a temporal resolution of 1 [micro]s. Importantly, such exquisite resolution is only possible as a result of the large number of droplets sampled and their high structural reproducibility. 10.1021/ac100055g

In this paper we describe a universal mechanism for merging multiple aqueous microdroplets within a flowing stream consisting of an oil carrier phase. Our approach involves the use of both a pillar array acting as a passive merging element, as well as built-in electrodes acting as an active merging element. The pillar array enables slowing down and trapping of the droplets via the drainage of the oil phase. This brings adjacent droplets into close proximity. At this point, an electric field applied to the electrodes breaks up the thin oil film surrounding the droplets resulting in merging.

Rapid kinetic measurements are important in understanding chemical interactions especially for biological molecules. Herein, we present a droplet-based microfluidic platform to study streptavidin--biotin binding kinetics with millisecond time resolution. With integration of a confocal fluorescence detection system, individual droplets can be monitored and characterized online to extract kinetic information. Using this approach, binding kinetics between streptavidin and biotin were observed via fluorescence resonance energy transfer. The binding rate constant of streptavidin and biotin was found to be in a range of 3.0 x [10.sup.6] - 4.5 x [10.sup.7] [M.sup.-1] [s.sup.-1].

We describe the development of an enzyme assay inside picoliter microdroplets. The enzyme alkaline phosphatase is expressed in Escherichia coli cells and presented in the periplasm. Droplets act as discrete reactors which retain and localize any reaction product. The catalytic turnover of the substrate is measured in individual droplets by monitoring the fluorescence at several time points within the device and exhibits kinetic behavior similar to that observed in bulk solution. Studies on wild type and a mutant enzyme successfully demonstrated the feasibility of using microfluidic droplets to provide time-resolved kinetic measurements.

The online characterization and detection of individual droplets at high speeds, low analyte concentrations, and perfect detection efficiencies is a significant challenge underpinning the application of microfluidic droplet reactors to high-throughput chemistry and biology. Herein, we describe the integration of confocal fluorescence spectroscopy as a high-efficiency detection method for droplet-based microfluidics. Issues such as surface contamination, rapid mixing, and rapid detection, as well as low detections limits have been addressed with the approach described when compared to conventional laminar flowbased fluidics. Using such a system, droplet size, droplet shape, droplet formation frequencies, and droplet compositions can be measured accurately and precisely at kilohertz frequencies. Taking advantage of this approach, we demonstrate a high-throughput biological assay based on fluorescence resonance energy transfer (FRET). By attaching a FRET donor (Alexa Fluor 488) to streptavidin and labeling a FRET acceptor (Alexa Fluor 647) on one DNA strand and biotin on the complementary strand, donor and acceptor molecules are brought in proximity due to streptavidin-biotin binding, resulting in FRET. Fluorescence bursts of the donor and acceptor from each droplet can be monitored simultaneously using separate avalanche photodiode detectors operating in single photon counting mode. Binding assays were investigated and compared between fixed streptavidin and DNA concentrations. Binding curves fit perfectly to Hill-Waud models, and the binding ratio between streptavidin and biotin was evaluated and found to be in agreement with the biotin binding sites on streptavidin. FRET efficiency for this FRET pair was also investigated from the binding results. Efficiency results show that this detection system can precisely measure FRET even at low FRET efficiencies.