Microfluidic Flow System Research Articles

Antimicrobial nisin acts against saliva derived multi-species biofilms without cytotoxicity to human oral cells.

Objectives: Nisin is a lantibiotic widely used for the preservation of food and beverages. Recently, investigators have reported that nisin may have clinical applications for treating bacterial infections. The aim of this study was to investigate the effects of ultra pure food grade Nisin ZP (>95% purity) on taxonomically diverse bacteria common to the human oral cavity and saliva derived multi-species oral biofilms, and to discern the toxicity of nisin against human cells relevant to the oral cavity.Methods: The minimum inhibitory concentrations and minimum bactericidal concentrations of taxonomically distinct oral bacteria were determined using agar and broth dilution methods. To assess the effects of nisin on biofilms, two model systems were utilized: a static and a controlled flow microfluidic system. Biofilms were inoculated with pooled human saliva and fed filter-sterilized saliva for 20–22 h at 37°C. Nisin effects on cellular apoptosis and proliferation were evaluated using acridine orange/ethidium bromide fluorescent nuclear staining and lactate dehydrogenase activity assays.Results: Nisin inhibited planktonic growth of oral bacteria at low concentrations (2.5–50 μg/ml). Nisin also retarded development of multi-species biofilms at concentrations ≥1 μg/ml. Specifically, under biofilm model conditions, nisin interfered with biofilm development and reduced biofilm biomass and thickness in a dose-dependent manner. The treatment of pre-formed biofilms with nisin resulted in dose- and time-dependent disruption of the biofilm architecture along with decreased bacterial viability. Human cells relevant to the oral cavity were unaffected by the treatment of nisin at anti-biofilm concentrations and showed no signs of apoptotic changes unless treated with much higher concentrations (>200 μg/ml).Conclusion: This work highlights the potential therapeutic value of high purity food grade nisin to inhibit the growth of oral bacteria and the development of biofilms relevant to oral diseases.

Reaction kinetics of cyclohexanone ammoximation over TS-1 catalyst in a microreactor

A continuous flow microfluidic system was developed to evaluate the reaction kinetics of cyclohexanone ammoximation over TS-1 catalyst. An ultrasonic field was introduced to avoid the sedimentation of solid catalyst. Compared with the batch reactor, the microfluidic system provides a more precise control of reaction conditions, faster reactant mixing and enhanced mass transfer. Thus, more accurate kinetic parameters can be extrapolated. The reaction order of each reactant, pre-exponential factor and activation energy were determined. A reaction kinetic model based on the mechanism through hydroxylamine route has been developed under the assumption that the reaction between hydrogen peroxide adsorbed on Ti active sites and free ammonia is the rate-determining step. The model is in good agreement with the experimental results. In addition, it has been proved that the microfluidic system with assistance of ultrasound field could be a good platform for activity evaluation of new nanocatalysts.

Continuous oxygen supply in pump-less micro-bioreactor based on microfluidics

This research aims a continuous and uniform oxygen tensions and oxygen gradients supply in microfluidic cell culture chip based micro-bioreactor without any external pumps by modifying the existing siphon based perfusion strategy using conventional tools to control constant hydrostatic pressure for constant fluid flow rate. In this study, the microfluidic based micro-bioreactor is fabricated using a polydimethylsiloxane (PDMS) replication process. The micro-bioreactor chip is composed of simple oxygen perfusion setup connecting to capillary-like branched microchannels and a cell culture and support loading area. The function of the new pump-less fluid perfusion in microfluidic cell culture chip and maintains constant oxygen supply in the cell culture area has been experimentally evaluated. And the results demonstrate that this proposed microfluidic flow system in a micro-bioreactor is proved to provide a linear distribution of oxygen throughout the cell culture area without any external and internal interference. Also, oxygen diffusion into the culture area through a microscale channel has been successfully established by the constant hydraulic pressure controlled by siphoning effect. Such flow system was used in a PDMS based microfluidic micro-bioreactor design to provide the high-throughput oxygen diffusion and perfusion, and the uniform oxygen/nutrient distribution continuously supplied for dermal fibroblast cell culture. This could be a potential and effective model to be incorporated into tissue regeneration studies, drug screening model, and in cancer tissue model studies for understanding angiogenesis, where oxygen tension and perfusion cultures play important roles.

NT-08 * A NOVEL HIGH-THROUGHPUT MICROFLUIDIC DEVICE DESIGNED TO ACCELERATE THE DISCOVERY OF GLIOBLASTOMA-TARGETING LIGANDS FROM OBOC LIBRARIES

Molecular targeting using ligands specific to glioblastoma (GBM) markers has shown great promise for the early detection of the pathology and directed therapy of the disease. Although conventional screening strategies of combinatorial libraries have demonstrated great potential for the discovery of novel targeting ligands, it is usually challenging and immensely costly. High-throughput screening approaches that exploit the tendency of magnetic prey beads (2-microns) coated with the target protein to specifically associate with the larger peptide-coated bait library beads (90-microns) are inexpensive, and have significantly accelerated the identification of targeting probes from one-bead-one-compound (OBOC) libraries. Using this approach, our objective is to utilize magnetic beads coated with a GBM-specific target protein to screen and capture novel GBM-binding peptides from an OBOC library. To facilitate high-throughput separation of positive hit beads from the rest of the library, we implemented a microfluidic magnetic flow system of our own design. Here, we describe a miniature microfluidic device designed to divert library beads coated with magnetic beads from one laminar flow path to another using an external magnetic force. Microfluidic channels connected to two inlets and two outlets are built using soft lithography, with dimensions of 2cm x 0.6mm x 0.2mm (L x W x H). A negative mold is created using SU-8 photoresist, and poly(dimethylsiloxane) (PDMS) is poured onto the mold to fabricate the channels. A strong neodymium magnet is placed adjacent to the channel to pull positive magnetic beads from the source flow path into the collection flow stream, allowing these beads to exit through the collection outlet. This novel on-chip technology can facilitate screening of large OBOC libraries (> 1 million beads) with great accuracy in a matter of hours, paving the way for rapid and efficient identification of targeting ligands specific to pathological targets in brain cancer and other diseases.

Abstract 3077: Label free prostate cancer cell isolation from blood by acoustic standing wave technology - acoustophoresis

Abstract There is intense search for disease biomarkers that can reliably detect lethal prostate cancer (PCa) at early stage, monitor response to treatment, detect disease recurrence, and predict survival. Enrichment, enumeration, and transcriptional profiling of rare circulating tumor cells (CTCs) can predict survival of patients with disseminated tumors, and may provide clinical benefit in predicting treatment responses of targeted therapies. We exploit ultrasound radiation forces to enable label free diversion of cancer cells from nucleated blood cells in a temperature-controlled, microfluidic continuous flow system (acoustophoresis). A third generation, standing-wave ultrasound microchip (silicon/glass) incorporated a cell pre-alignment channel, and a microchannel with a trifurcated inlet and outlet. The system is driven by compressed air (500 mBar) to mitigate and minimize continuous flow-fluctuations and provide more reproducible and reliable discrimination of tumor cells from nucleated blood cells compared to previous syringe driven setups, and allow us to process clinically relevant sample volumes of blood (5-10 mL). We fixed, spiked, and processed 50 or more PCa cells per mL in undiluted white blood cells (WBC) in the acoustic microchip. The separation system is very flexible and the ratio between cancer cell recovery and cell contamination can be tuned by changing the sample flow rates or the acoustic energy. A consistent and reproducible recovery of cancer cells above 90% with less than 0.4% contamination of WBC was provided using flow rates of 100 l/min. We also found that about 90% of the contaminating WBCs were granulocytes, whereas the vast majority of lymphocytes were reliably diverted. Our results were consistent using different PCa cell lines (DU145, LNCaP, PC3) along with the breast cancer cell line (MCF7). The isolation of cancer cells by acoustophoresis is a label free method that consistently enables high recovery of cancer cells independent of the expression of surface antigens. This allows for separation of EpCAM negative populations of CTCs, mesenchymal cells along with cancer stem cells, which can be identified with subsequent qRT-PCR analysis. In acoustophoresis, cells are separated according to size, density and compressibility (dV/dp), where size is the major factor. Using non-fixed samples, we can separate viable from dead cells, since dead cells display significantly different acoustic properties to their live counterpart. Acoustophoresis is a gentle separation method and does not affect cell viability, allowing post isolation ex vivo cultivation of CTCs. This allows functional assays to test CTCs ability to form metastasis. It is therefore a promising method for future non-invasive molecular interrogation of metastatic cancers and the characterization of CTCs will relate CTCs to disease outcome and predict for sensitivity to treatment and match patient to drug response. Citation Format: Cecilia Magnusson, Per Augustsson, Benedikta Haflidadottir, Andreas Lenshof, Yvonne Ceder, Thomas Laurell, Hans Lilja. Label free prostate cancer cell isolation from blood by acoustic standing wave technology - acoustophoresis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3077. doi:10.1158/1538-7445.AM2014-3077

Efficient and scalable synthesis of quantum dots using hexane as the solvent in a non-microfluidic flow reactor system

Quantum dots (QDs) synthesis has been widely carried out in Schlenk line systems. However, there are two drawbacks of these systems, which have restricted their use for large scale or commercial production of QDs. One is the use of expensive and high-boiling point solvents and the other is that QDs need to be continuously produced with high production rate to reduce production costs. Nowadays, microfluidic flow systems are the most popularly used systems for the continuous production of QDs. However, their narrow channels limit the reaction volume and flow rate. In this report, a non-microfluidic flow reactor system has been developed to synthesize high quality CdSe QDs by injecting the solution containing the mixture of Cd–oleic acid (Cd–OA) and Se–tributylphosphine (Se–TBP) into near critical or supercritical hexane. The optical properties of the produced CdSe QDs can be fine-tuned to almost cover the entire visible region (450–630 nm) by changing the reaction parameters. The CdSe QDs produced with this approach are similar to those produced in Schlenk line, and can be functionalized with a ZnS shell to enhance PL efficiency. Moreover, we have also achieved a continuous synthesis of 5 g CdSe QDs with a production rate of 2.5 g h−1 to demonstrate the potential capability of mass production with this system. White light emitting diodes can be prepared by using CdSe QDs as the phosphor dye. Finally, other types of QDs, such as CdS, ZnS, and ZnSe, were also synthesized to demonstrate the generalization of the developed approach.

A simple and cost-effective molecular diagnostic system and DNA probes synthesized by light emitting diode photolithography

This work introduces a novel chip to be used in the future as a simple and cost-effective method for creating DNA arrays using light emission diode (LED) photolithography. The DNA chip platform contains 24 independent reaction sites, which allows for the testing of a corresponding amount of patients’ samples in hospital. An array of commercial UV LEDs and lens systems was combined with a microfluidic flow system to provide patterning of 24 individual reaction sites, each with 64 independent probes. Using the LED array instead of conventional laser exposure systems or micro-mirror systems significantly reduces the cost of equipment. The microfluidic system together with microfluidic flow cells drastically reduces the amount of used reagents, which is important due to the high cost of commercial reagents. The DNA synthesis efficiency was verified by fluorescence labeling and conventional hybridization.

Microfluidic conformal coating of non-spherical magnetic particles.

We present the conformal coating of non-spherical magnetic particles in a co-laminar flow microfluidic system. Whereas in the previous reports spherical particles had been coated with thin films that formed spheres around the particles; in this article, we show the coating of non-spherical particles with coating layers that are approximately uniform in thickness. The novelty of our work is that while liquid-liquid interfacial tension tends to minimize the surface area of interfaces-for example, to form spherical droplets that encapsulate spherical particles-in our experiments, the thin film that coats non-spherical particles has a non-minimal interfacial area. We first make bullet-shaped magnetic microparticles using a stop-flow lithography method that was previously demonstrated. We then suspend the bullet-shaped microparticles in an aqueous solution and flow the particle suspension with a co-flow of a non-aqueous mixture. A magnetic field gradient from a permanent magnet pulls the microparticles in the transverse direction to the fluid flow, until the particles reach the interface between the immiscible fluids. We observe that upon crossing the oil-water interface, the microparticles become coated by a thin film of the aqueous fluid. When we increase the two-fluid interfacial tension by reducing surfactant concentration, we observe that the particles become trapped at the interface, and we use this observation to extract an approximate magnetic susceptibility of the manufactured non-spherical microparticles. Finally, using fluorescence imaging, we confirm the uniformity of the thin film coating along the entire curved surface of the bullet-shaped particles. To the best of our knowledge, this is the first demonstration of conformal coating of non-spherical particles using microfluidics.

Selective DNA extraction with microparticles in segmented flow

Droplet-based segmented flow microfluidic systems have proven their potential for high throughput detection and quantification of analytes. However, the required sample preparations are often performed off-chip, as on-chip methods are lacking. Microparticles are especially suited for the extraction and purification of target molecules from a sample and are successfully used in other microfluidic systems and in laboratory-scale methods. The current magnetic separation methods in segmented flow microfluidics are limited in their function to purify, as only a limited part of the original droplet volume can be removed from the particles. In this paper, we report the implementation of a selective DNA extraction assay with microparticles in a segmented flow microfluidic system. The combination of magnetic separation with asymmetric droplet splitting allows the removal of 90 % or even 95 % of the original sample volume in a single separation step. It is shown that the hybridization and capture efficiency of the particles are identical for off-chip and on-chip methods. Next, the effect of the particle separation efficiency on the extraction efficiency is tested for different splitting regimes. When up to 90 % of the droplet volume is removed, nearly all particles are correctly separated and the non-separated particle fraction remains below 5 %. Only if 95 % of the original volume is removed, the unseparated fraction becomes significant (>10 %). Finally, the impact of separation at a higher splitting ratio for the repeated washing of the particles is discussed. With this novel system, more complex and relevant bioassays can be implemented completely in a droplet-based segmented flow context.

Simulation of particulate fouling at a microchannel entrance region

A computational study is reported on the dynamics of particles near the entrance of a microchannel, as used in microfluidic flow systems. The collision of particles with fins separating the microchannels in these entrance regions makes them particularly susceptible to particle fouling. The study employs a soft-sphere discrete-element method with van der Waals and electric double-layer forces between the particles and the wall. A multi-block-structured grid fit to the flow domain boundaries is used to compute the fluid velocity field using a finite-volume method. The fluid flow is interpolated onto a Cartesian grid for efficient flow field interpolation at the particle locations, and a level-set method is used to represent the flow field boundaries in the particle computation. Particles adhering to the upstream face of the fin separating two microchannels are found to form clusters, which are pulled laterally by the stagnation-point flow near the fin. Even under conditions where the adhesion is sufficiently strong that there is little or no particle rolling on the fin face, the particles are still removed from the fin due to collisions with other particles. Individual particles are removed quickly from the fin face due to collision with upstream particles, whereas particle clusters have sufficient adhesion force with the fin surface that they can withstand collision with other particles for longer periods of time before they are removed in a cascade-like process.

A Surface Enhanced Raman Scattering (SERS) microdroplet detector for trace levels of crystal violet

We report on a microfluidic platform that integrates a winding microdroplet chip and a surface-enhanced Raman scattering (SERS) detection system for trace determination of crystal violet (CV). Colloidal silver was applied to generate SERS. Compared to the continuous flow microfluidic system, the microdroplet based detection described here effectively eliminates any memory effects. Effects of flow pattern, droplet size, surfactant, and position of detection were optimized. Under optimal conditions, there is a linear correlation between signal and the concentration of CV in the 10 nM to 800 nM range, with a correlation coefficient (R2) of 0.9967. The limit of detection in water is 3.6 nM.

High Throughput‐Per‐Footprint Inertial Focusing

Matching the scale of microfluidic flow systems with that of microelectronic chips for realizing monolithically integrated systems still needs to be accomplished. However, this is appealing only if such re-scaling does not compromise the fluidic throughput. This is related to the fact that the cost of microelectronic circuits primarily depends on the layout footprint, while the performance of many microfluidic systems, like flow cytometers, is measured by the throughput. The simple operation of inertial particle focusing makes it a promising technique for use in such integrated flow cytometer applications, however, microfluidic footprints demonstrated so far preclude monolithic integration. Here, the scaling limits of throughput-per-footprint (TPFP) in using inertial focusing are explored by studying the interplay between theory, the effect of channel Reynolds numbers up to 1500 on focusing, the entry length for the laminar flow to develop, and pressure resistance of the microchannels. Inertial particle focusing is demonstrated with a TPFP up to 0.3 L/(min cm²) in high aspect-ratio rectangular microfluidic channels that are readily fabricated with a post-CMOS integratable process, suggesting at least a 100-fold improvement compared to previously demonstrated techniques. Not only can this be an enabling technology for realizing cost-effective monolithically integrated flow cytometry devices, but the methodology represented here can also open perspectives for miniaturization of many biomedical microfluidic applications requiring monolithic integration with microelectronics without compromising the throughput.

Microfluidic radiosynthesis and biodistribution of [18F] 2‐(5‐fluoro‐pentyl)‐2‐methyl malonic acid

Microfluidics technology has emerged as a powerful tool for the radiosynthesis of positron emission tomography (PET) and single-photon emission computed tomography radiolabeled compounds. In this work, we have exploited a continuous flow microfluidic system (Advion, Inc., USA) for the [(18) F]-fluorine radiolabeling of the malonic acid derivative, [(18) F] 2-(5-fluoro-pentyl)-2-methyl malonic acid ([(18) F]-FPMA), also known as [(18) F]-ML-10, a radiotracer proposed as a potential apoptosis PET imaging agent. The radiosynthesis was developed using a new tosylated precursor. Radiofluorination was initially optimized by manual synthesis and served as a basis to optimize reaction parameters for the microfluidic radiosynthesis. Under optimized conditions, radio-thin-layer chromatography analysis showed 79% [(18) F]-fluorine incorporation prior to hydrolysis and purification. Following hydrolysis, the [(18) F]-FPMA was purified by C18 Sep-Pak, and the final product was analyzed by radio-HPLC (high-performance liquid chromatography). This resulted in a decay-corrected 60% radiochemical yield and ≥98% radiochemical purity. Biodistribution data demonstrated rapid blood clearance with less than 2% of intact [(18) F]-FPMA radioactivity remaining in the circulation 60 min post-injection. Most organs showed low accumulation of the radiotracer, and radioactivity was predominately cleared through kidneys (95% in 1 h). Radio-HPLC analysis of plasma and urine samples showed a stable radiotracer at least up to 60 min post-injection.

An investigation into dispersion upon switching between solvents within a microfluidic system using a chemically resistant integrated optical refractive index sensor

A planar Bragg grating device has been developed that is capable of detecting changes in the refractive index of a wide range of fluids including solvents, acids and bases. The integration of this high precision refractive index sensor within a chemically resistant microfluidic flow system has enabled the investigation of diverse fluid interactions. By cycling between different solvents, both miscible and immiscible, within the microfluidic system it is shown that the previous solvent determines the nature of the refractive index profile across the transition in composition. This solvent dispersion effect is investigated with particular attention to the methanol-water transition, where transients in refractive index are observed that are an order of magnitude larger in amplitude than the difference between the bulk fluids. The potential complications of such phenomenon are discussed together with an example of a device that exploits this effect for the unambiguous composition measurement of a binary solvent system.

Light‐Governed Capillary Flow in Microfluidic Systems

Light-based flow systems for point-of-care devices are of interest because, in principle, sunlight could be used to operate them, potentially allowing for high functionality with minimal device complexity and expense. A light-operated method to drive flow using poly(N-isopropylacrylamide), a 'smart' polymer that changes wettability as a function of temperature, is introduced. It is grafted onto a carbon black-polydimethylsiloxane surface, which converts light into a thermal pattern that valves flow at user-defined locations. Flow rates are demonstrated ranging from 4 μL min(-1) at 25 °C to 0.1 μL min(-1) at 40 °C. The valving dynamics are also characterised, and a response time of less than 4 s is shown. Light-operated flow could provide the simple architecture and advanced functionality needed in low-resource point-of-care devices.

Droplet microfluidics based microseparation systems

Lab on a chip (LOC) technology is a promising miniaturization approach. The feature that it significantly reduced sample consumption makes great sense in analytical and bioanalytical chemistry. Since the start of LOC technology, much attention has been focused on continuous flow microfluidic systems. At the turn of the century, droplet microfluidics, which was also termed segmented flow microfluidics, was introduced. Droplet microfluidics employs two immiscible phases to form discrete droplets, which are ideal vessels with confined volume, restricted dispersion, limited cross-contamination, and high surface area. Due to these unique features, droplet microfluidics proves to be a versatile tool in microscale sample handling. This article reviews the utility of droplet microfluidics in microanalytical systems with an emphasize on separation science, including sample encapsulation at ultra-small volume, compartmentalization of separation bands, isolation of droplet contents, and related detection techniques.

Tailoring Surface-Enhanced Raman Scattering Effect Using Microfluidics

Surface-enhanced Raman scattering (SERS)-based microfluidic platforms are investigated to optimize the detection of biomolecules using silver nanoparticles. The method is established using pefloxacin (an antibacterial agent) as a probe. We first monitor the continuous SERS signal of a 10–5 M pefloxacin solution diffusing continuously into a silver nanoparticle stream across the shared interface separating the two-phase, segmented microfluidic laminar flow system. Diffusion of the pefloxacin adsorbate begins immediately after the two streams merge, generating silver aggregates and producing a huge enhancement of the Raman bands of pefloxacin. We show that the evolution of the SERS signal presents a diffusion-like behavior, whose dynamics can be easily monitored as the signal measured in the flow-wise direction is proportional to the reaction time. Finally, we demonstrate that it is possible to optimize SERS intensity spectra by adding a controlled amounts of chloride ions through a side channel to control ...

Imaging Techniques for Quantifying Passive Diffusion Across Lipid Bilayer Membranes

Passive diffusion of small-molecule species across the cell membranes is an essential step in drug delivery since molecules that can easily permeate the plasma membrane have high oral bioavailability. There are several techniques for characterizing the passive transmembrane transport of small molecules; however, there are many instances in which these techniques produce widely varying results. For instance, literature values for the permeabilities of low-molecular-weight carboxylic acids across phosphatidylcholine membranes span three orders of magnitude.We have developed a technique for precisely quantifying lipid membrane permeability to small molecules based on imaging of the concentration field surrounding giant unilamellar lipid vesicle (GUV) membranes. High-speed spinning disk confocal microscopy can be used to image dynamic changes in the concentration field and precisely measure permeabilities as fast as 0.2 cm/s. This technique can be used to measure the transport of both dye-containing species and species that result in a change of fluorescence intensity of a dye molecule. For instance, by using a pH-sensitive fluorophore, the transport of non-fluorescent carboxylic acid molecules can by observed.This technique can be combined with direct imaging of the membrane to simultaneously measure distinct transport rates across different phase-separated regions of a lipid vesicle. We have also imaged transport across GUVs with compositionally asymmetric membranes. These GUVs are formed in a multiphase microfluidic flow system in which the composition of each leaflet of the membrane can be controlled independently. We have measured proton and carboxylic acid transport across membranes with charge asymmetry. Early results show that the permeability of membranes with charge asymmetry depends on the direction in which small molecules and proton species are moving with respect to the membrane.

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1 In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented. Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.