The exploitation of microdroplets produced within microfluidic environments has recently emerged as a new and exciting technological platform for applications within the chemical and biological sciences. Interest in microfluidic systems has been stimulated by a range of fundamental features that accompany system miniaturization. Such features include the ability to process and handle small volumes of fluid, improved analytical performance when compared to macroscale analogues, reduced instrumental footprints, low unit cost, facile integration of functional components and the exploitation of atypical fluid dynamics to control molecules in both time and space. Moreover, microfluidic systems that generate and utilize a stream of sub-nanolitre droplets dispersed within an immiscible continuous phase have the added advantage of allowing ultra-high throughput experimentation and being able to mimic conditions similar to that of a single cell (in terms of volume, pH, and salt concentration) thereby compartmentalizing biological and chemical reactions. This review provides an overview of methods for generating, controlling and manipulating droplets. Furthermore, we discuss key fields of use in which such systems may make a significant impact, with particular emphasis on novel applications in the biological and physical sciences.

We demonstrate that single cells can be controllably compartmentalized within aqueous microdroplets; using such an approach we perform high-throughput screening by detecting the expression of a fluorescent protein in individual cells with simultaneous measurement of droplet size and cell occupancy.

Even though they were introduced less than a decade ago, electrochemical paper‐based devices (ePADs) have attracted widespread attention because of their inherent advantages in many applications. ePADs combine the advantages of microfluidic paper‐based devices (low cost, ease of use, equipment free pumping, etc.) for sample handling and processing with the advantages of sensitive and selective detection provided by electrochemistry. As a result, ePADs provide simplicity, portability, reproducibility, low cost and high selectivity and sensitivity for analytical measurements in a variety of applications ranging from clinical diagnostics to environmental sensing. Herein, recent advances in ePAD development and application are reviewed, focusing on electrode fabrication techniques and examples of applications specially focused on environmental monitoring, biological applications and clinical assays. Finally, a summary and prospective directions for ePAD research are also provided.

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 (6)-4.5 x 10 (7) M (-1) s (-1).

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 dropletbased 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.In recent years, microfluidic systems have been successfully used to generate multiphase flows in a variety of formats. Of particular note are those that exploit flow instabilities between immiscible fluids to generate suspended droplets. 1 In simple terms, droplets can be made to spontaneously form when laminar streams of aqueous reagents are injected into an immiscible carrier fluid. The formed droplets define picoliter volumes, and because each droplet is isolated from both channel surfaces and other droplets, each acts as an individual reaction vessel. 2 Importantly, variation of the cross-sectional dimensions of the microfluidic system and the relative flow rates of the input streams allows for exquisite control over droplet size, volume fraction in the continuous phase, frequency of droplet production, and relative concentrations of reagents contained within each droplet. Several recent studies have exploited the formation of droplets in microfluidic systems to perform a variety of analytical processes. 2 For example, droplet-based microfluidic systems have been used to perform enzyme assays, 3 small-molecule synthesis, 4-6 protein crystallization, 7-9 nanopa...

Salmonella causes over a million foodborne illnesses per year in the United States resulting in more hospitalizations and deaths than any other foodborne bacterial pathogen. To help prevent outbreaks, a rapid, portable, sensitive, and reliable method for onsite detection of bacteria that can be used in different sample matrices would be beneficial. Herein, we present a colorimetric paper-based analytical device (PAD) combined with immunomagnetic separation (IMS) for detecting Salmonella typhimurium. IMS anti-Salmonella coated magnetic beads were applied to capture and separate bacteria from the sample matrix and preconcentrate it into small volumes before testing on paper. To directly detect S. typhimurium after IMS, a sandwich immunoassay was implemented into the procedure with β-galactosidase (β-gal) as the detection enzyme. Using the antibody/enzyme complex, we performed a colorimetric assay with chlorophenol red-β-d-galactopyranoside (CPRG) for bacteria quantification. The method was confirmed to be highly specific to S. typhimurium without interference from other pathogenic bacteria like Escherichia coli. Using this system, the limit of detection of S. typhimurium was found to be 10 CFU mL in culturing solution without any pre-enrichment. In addition, distance-based detection where the concentration is read as the length of colored band formed on the reaction was also demonstrated. This assay had a detection limit of 10 CFU mL for S. typhimurium, providing an instrument-free quantitative analysis alternative to spot tests, which require image analysis. Finally, the proposed platform was applied for detection of S. typhimurium in inoculated Starling bird fecal samples and whole milk with detection limits of 10 CFU g and 10 CFU mL, respectively, and this is the first published paper-based detection method for S. typhimurium in bird feces and whole milk.

Droplet microfluidics constitutes a diverse and practical tool set that enables chemical and biological experiments to be performed at high speed and with enhanced efficiency when compared to conventional instrumentation.

Every little drop: The KD values of angiogenin (ANG) interactions as shown by FRET analysis of thousands of pL‐sized droplets agree with data from bulk‐fluorescence polarization measurements. Importantly, the use of fluorophores does not affect the activity of ANG or the binding of anti‐ANG antibodies to ANG. Such an experimental platform could be applied to the high‐throughput analysis of protein–protein interactions.magnified image