High-throughput screening of filamentous fungi using nanoliter-range droplet-based microfluidics.

Filamentous fungi are an extremely important source of industrial enzymes because of their capacity to secrete large quantities of proteins. Currently, functional screening of fungi is associated with low throughput and high costs, which severely limits the discovery of novel enzymatic activities and better production strains. Here, we describe a nanoliter-range droplet-based microfluidic system specially adapted for the high-throughput sceening (HTS) of large filamentous fungi libraries for secreted enzyme activities. The platform allowed (i) compartmentalization of single spores in ~10 nl droplets, (ii) germination and mycelium growth and (iii) high-throughput sorting of fungi based on enzymatic activity. A 10(4) clone UV-mutated library of Aspergillus niger was screened based on α-amylase activity in just 90 minutes. Active clones were enriched 196-fold after a single round of microfluidic HTS. The platform is a powerful tool for the development of new production strains with low cost, space and time footprint and should bring enormous benefit for improving the viability of biotechnological processes.

Membraneless glucose/O2 microfluidic biofuel cells using covalently bound enzymes.

We developed a new covalent enzyme immobilization technique compatible with lithography processes. Carbon nanotube electrodes patterned on glass slides were used to create Y-shaped membraneless glucose/O(2) microfluidic biofuel cells. An original extremophilic laccase, CotA from Bacillus subtilis, was used in the cathodic compartment of these miniaturized biofuel cells.

A novel microfluidics-based method for probing weak protein-protein interactions.

We report the use of a novel microfluidics-based method to detect weak protein-protein interactions between membrane proteins. The tight junction protein, claudin-2, synthesised in vitro using a cell-free expression system in the presence of polymer vesicles as membrane scaffolds, was used as a model membrane protein. Individual claudin-2 molecules interact weakly, although the cumulative effect of these interactions is significant. This effect results in a transient decrease of average vesicle dispersivity and reduction in transport speed of claudin-2-functionalised vesicles. Polymer vesicles functionalised with claudin-2 were perfused through a microfluidic channel and the time taken to traverse a defined distance within the channel was measured. Functionalised vesicles took 1.19 to 1.69 times longer to traverse this distance than unfunctionalised ones. Coating the channel walls with protein A and incubating the vesicles with anti-claudin-2 antibodies prior to perfusion resulted in the functionalised vesicles taking 1.75 to 2.5 times longer to traverse this distance compared to the controls. The data show that our system is able to detect weak as well as strong protein-protein interactions. This system offers researchers a portable, easily operated and customizable platform for the study of weak protein-protein interactions, particularly between membrane proteins.

DC-biased AC-electrokinetics: a conductivity gradient driven fluid flow.

This paper studies the principles of fluid flow manipulation based on DC-biased AC-electrokinetics. This method makes use of planar parallel electrodes in a microfluidic channel in contact with an electrolyte solution, with a DC biased AC electrical signal applied to the electrode pair. Due to the application of DC bias, incipient Faradaic electrolytic reactions take place resulting in an increase of the ionic content of the bulk solution. The ionic content was found to be dissimilar at the cathodic and anodic sides of the channel and a conductivity difference of approximately 10% was measured for 2 V(DC). Fluid flow is generated by the action of the DC biased AC electric signal acting on the transverse conductivity gradient generated across the microchannel. The induced flow in the form of vortex was characterized experimentally and the results substantiated theoretically. The velocity of the induced flow vortex under the employed experimental conditions was ~600 to 700 µm s(-1) which is faster than those obtained in conventional AC-electroosmosis and AC-electrothermal types of flows.

Investigation of sensing mechanism and signal amplification in carbon nanotube based microfluidic liquid-gated transistors via pulsating gate bias.

The advent of a carbon nanotube liquid-gated transistor (LGFET) for biosensing applications allows the possibility of real-time and label-free detection of biomolecular interactions. The use of an aqueous solution as dielectric, however, has traditionally restricted the operating gate bias (VG) within |VG| < 1 V, due to the electrolysis of water. Here, we propose pulsed-gating as a facile method to extend the operation window of LGFETs to |VG| > 1 V. A comparison between simulation and experimental results reveals that at voltages in excess of 1 V, the LGFET sensing mechanism has a contribution from two factors: electrostatic gating as well as capacitance modulation. Furthermore, the large IDS drop observed in the |VG| > 1 V region indicates that pulsed-gating may be readily employed as a simple method to amplify the signal in the LGFET and pushes the detection limit down to attomolar concentration levels, an order of magnitude improvement over conventionally employed DC VG biasing.

Femtomolar detection of 2,4-dichlorophenoxyacetic acid herbicides via competitive immunoassays using microfluidic based carbon nanotube liquid gated transistor.

Monitoring of environmental pollutants has become increasingly important due to concern over potential health and environmental impact inflicted by these chemicals. In this contribution, we focus on the development of an all-plastic biosensor comprising laminated single-walled carbon nanotubes as the active element and its conductance modulation in a liquid-gated field effect transistor, as the principle of transduction, for the detection of 2,4-dicholorophenoxy acetic acid (2,4-D) herbicide. The reported biosensor is capable of performing real-time label-free detection of analytes in liquid environment. This biosensor which relies on immunoassay principle for specificity is able to detect down to 500 fM levels of 2,4-D in soil samples.