Andrew J. de Mello

Development of quantitative cell-based enzyme assays in microdroplets.

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.

Precise temperature control in microfluidic devices using Joule heating of ionic liquids

Microfluidic devices for spatially localised heating of microchannel environments were designed, fabricated and tested. The devices are simple to implement, do not require complex manufacturing steps and enable intra-channel temperature control to within +/-0.2 degrees C. Ionic liquids held in co-running channels are Joule heated with an a.c. current. The nature of the devices means that the internal temperature can be directly assessed in a facile manner.

Rapid formation of amides via carbonylative coupling reactions using a microfluidic device

Carbonylative cross-coupling reactions of arylhalides to form secondary amides were rapidly carried out on a glass-fabricated microchip--the first time a microstructured device has been used to perform a gas-liquid carbonylation reaction.

Microchip-based synthesis and analysis: Control of multicomponent reaction products and intermediates

A miniaturised-SYNthesis and Total Analysis System (mu SYNTAS) was used for the solution-phase synthesis and on-line analysis (TOF-MS) of Ugi multicomponent reaction (MCR) products. This approach provides an unusually high degree of control of the MCR and delivers detailed, novel information on reaction intermediates in real-time. Specifically, the Ugi 4 component condensation (4CC) involving the reaction of an amine, acid, aldehyde and isocyanide species was performed at room temperature in a controllable fashion. Furthermore, observation of the nitrilium intermediate, cyclohexyl(2-piperidin-1-ylethylidyne)ammonium chloride, is presented for the first time.

On-chip generation and reaction of unstable intermediates-monolithic nanoreactors for diazonium chemistry: azo dyes.

Monolithic nanoreactors for the safe and expedient continuous synthesis of products requiring unstable intermediates were fabricated and tested by the synthesis of azo dyes under hydrodynamic pumping regimes.

Sub-microliter electrochemiluminescence detector : A model for small volume analysis systems

A small volume, electrochemical cell for the generation and detection of electrochemiluminescence from tris(2,2′-bipyridyl) ruthenium(II) has been fabricated. The flowcell is a poly(methyl methacrylate) (PMMA)–acetate–PMMA sandwich construct, containing two platinum, thin-film electrodes. Operation of the microchip establishes sub-microliter detection of tris(2,2′-bipyridyl) ruthenium(II) electrochemiluminescence in continuous flow. Initial experiments demonstrate a detection limit of 5 × 10–13M at an effective cell volume of 100 nl. This corresponds to the detection of only 30 000 molecules.

A polydimethylsiloxane/glass capillary electrophoresis microchip for the analysis of biogenic amines using indirect fluorescence detection

A polydimethylsiloxane‐glass capillary microchip is fabricated for the rapid analysis of a mixture of common biogenic amines using indirect fluorescence detection. Using a running buffer of phosphate and 2‐propanol, and Rhodamine 110 as a background fluorophore, both co‐ionic and counter‐ionic systems are explored. Studies demonstrate the separation and analysis of cations using indirect fluorescence detection for the first time in a chip‐based system. Resulting electrophoretic separations are achieved within a few tens of seconds with detection limits of approximately 6 ν M. The reduced sample handling and rapid separations afforded by the coupling of indirect fluorescence detection with chip‐based capillary electrophoresis provide a highly efficient method for the analysis and detection of molecules not possessing a chromophore or fluorophore. Furthermore, limits of detection are on a par with reported chip‐based protocols that incorporate precolumn derivatisation with fluorescence detection. The current device circumvents lengthy sample preparation stages and therefore provides an attractive alternative technique for the analysis biogenic amines.

Rapid prototyping of microfluidic devices for integrating with FT-IR spectroscopic imaging.

A versatile approach for the rapid prototyping of microfluidic devices suitable for use with FT-IR spectroscopic imaging is introduced. Device manufacture is based on the direct printing of paraffin onto the surface of an infrared transparent substrate, followed by encapsulation. Key features of this approach are low running costs, rapid production times, simplicity of design modifications and suitability for integration with FT-IR spectroscopic measurements. In the current experiments, the minimum width of channel walls was found to be approximately 120 mum and approximately 200 when a 25 mum and 12 mum spacer is used, respectively. Water and poly(ethylene glycol) are used as model fluids in a laminar flow regime, and are imaged in both transmission and attenuated total reflection (ATR) modes. It is established that adoption of transmission mode measurements yields superior sensitivity whilst the ATR mode is more suitable for quantitative analysis using strong spectral absorption bands. Results indicate that devices manufactured using this approach are suitable for use with in situ FT-IR spectroscopic imaging.

Hydrodynamic focusing in microstructures: Improved detection efficiencies in subfemtoliter probe volumes

We present a method for improving detection efficiencies in single molecule confocal fluorescence spectroscopy with subfemtoliter probe volumes within microfluidic channels. Our approach is based on hydrodynamically focusing an analyte stream within a microfluidic channel down to a width on the same order of magnitude as that of the confocal probe volume. Experiments are performed in which fluorescently labeled polystyrene microspheres (930 nm diameter) are motivated through a microchannel and passed through a focused laser beam at a variety of flow rates (0.1−11 μL∕min). Hydrodynamic focusing of the analyte stream is achieved by introduction of two sheath flow streams that flank the central analyte stream. Through variation of the relative flow rates in each input stream the analyte stream can be controlled with micron resolution. A maximum hydrodynamic focusing width of 3 μm was achieved within a 50 μm wide microfluidic channel; hence, a larger proportion of molecules traveling through the microfluidic ...

Thermal optimisation of the Reimer–Tiemann reaction using thermochromic liquid crystals on a microfluidic reactor

Microreactors incorporating thin film resistive heating elements for continuous flow organic synthesis are presented. Internal thermal conditions were monitored in real time using reflectance spectra of temperature sensitive thermochromic liquid crystals (TLC) in a collateral microfluidic network. To demonstrate the precise temperature control provided by this method, the thermal optimisation of the Reimer-Tiemann formylation of beta-naphthol was performed under hydrodynamic pumping regimes.