Bioluminescence resonance energy transfer biosensor for measuring activity of a protease secreted by Pseudomonas fluorescens growing in milk.

Bacterial proteases are sporadic contributors to milk spoilage, reducing the quality of ultra-heat treated (UHT) milk and other dairy products. Current methods for measuring bacterial protease activity in milk are insensitive and too slow to be used in routine testing in dairy processing plants. We have designed a novel bioluminescence resonance energy transfer (BRET)-based biosensor to measure the activity of proteases secreted by bacteria in milk.

Resonance Energy Transfer-Based Biosensors for Point-of-Need Diagnosis-Progress and Perspectives.

The demand for point-of-need (PON) diagnostics for clinical and other applications is continuing to grow. Much of this demand is currently serviced by biosensors, which combine a bioanalytical sensing element with a transducing device that reports results to the user. Ideally, such devices are easy to use and do not require special skills of the end user.

Development and characterisation of a compact device for rapid real-time-on-chip detection of thrombin activity in human serum using bioluminescence resonance energy transfer (BRET).

Bioluminescence resonance energy transfer (BRET) is a sensitive optical detection method that can monitor changes in the relative orientation and the physical proximity of molecules in real-time. Since the light is generated internally by a bioluminescent protein, BRET does not rely on an external light source. The use of BRET simultaneously simplifies the hardware required for sensing and offers improved detection limits and sensitivity for applications targeting point-of-care bio-sensing.

A Miniature Gas Sampling Interface with Open Microfluidic Channels: Characterization of Gas-to-Liquid Extraction Efficiency of Volatile Organic Compounds.

Chemosensory protein based olfactory biosensors are expected to play a significant role in next-generation volatile organic compound (VOC) detection systems due to their ultra-high sensitivity and selectivity. As these biosensors can perform most efficiently in aqueous environments, the detection systems need to incorporate a gas sampling interface for gas-to-liquid extraction. This interface should extract the VOCs from the gas phase with high efficiency and transfer them into the liquid containing biosensors to enable subsequent detection.

Highly Sensitive and Selective Biosensor for a Disaccharide Based on an AraC-Like Transcriptional Regulator Transduced with Bioluminescence Resonance Energy Transfer.

Sensitive and selective quantification of individual sugars in complex media is technically challenging and usually requires HPLC separation. Accurate measurement without the need for separation would be highly desirable. The measurement of trace levels of lactose in lactose-reduced milk exemplifies the problem, with the added challenge that trace lactose must be measured in the presence of ≈140 mM glucose and galactose, the products of lactase digestion of lactose.

Diurnal variation in expired breath volatiles in malaria-infected and healthy volunteers.

We previously showed that thioether levels in the exhaled breath volatiles of volunteers undergoing controlled human malaria infection (CHMI) with P. falciparum increase as infection progresses. In this study, we show that thioethers have diurnal cyclical increasing patterns and their levels are significantly higher in P.

Molecular basis for the blue bioluminescence of the Australian glow-worm Arachnocampa richardsae (Diptera: Keroplatidae).

Bioluminescence is the emission of visible light by living organisms. Here we describe the isolation and characterisation of a cDNA encoding a MW ≈ 59,000 Da luciferase from the Australian glow-worm, Arachnocampa richardsae. The enzyme is a member of the acyl-CoA ligase superfamily and produces blue light on addition of D-luciferin.

Classifying continuous, real-time e-nose sensor data using a bio-inspired spiking network modelled on the insect olfactory system.

In many application domains, conventional e-noses are frequently outperformed in both speed and accuracy by their biological counterparts. Exploring potential bio-inspired improvements, we note a number of neuronal network models have demonstrated some success in classifying static datasets by abstracting the insect olfactory system. However, these designs remain largely unproven in practical settings, where sensor data is real-time, continuous, potentially noisy, lacks a precise onset signal and accurate classification requires the inclusion of temporal aspects into the feature set.

De Novo Engineering of Solid-State Metalloproteins Using Recombinant Coiled-Coil Silk.

To achieve the sophisticated chemistry required for life, nature uses metal containing proteins (metalloproteins). However, despite intensive research efforts, very few of these metalloproteins have been exploited for biotechnological applications. One major limiting factor is the poor stability of these proteins when they are removed from their cellular environment.

Analysis of Breath Specimens for Biomarkers of Plasmodium falciparum Infection.

Currently, the majority of diagnoses of malaria rely on a combination of the patient's clinical presentation and the visualization of parasites on a stained blood film. Breath offers an attractive alternative to blood as the basis for simple, noninvasive diagnosis of infectious diseases. In this study, breath samples were collected from individuals during controlled malaria to determine whether specific malaria-associated volatiles could be detected in breath.

Sub-nanomolar detection of thrombin activity on a microfluidic chip.

Bioluminescence resonance energy transfer (BRET) is a form of Förster resonance energy transfer. BRET has been shown to support lower limits of detection than fluorescence resonance energy transfer (FRET) but, unlike FRET, has not been widely implemented on microfluidic devices for bioanalytical sensing. We recently reported a microscope-based microfluidic system for BRET-based biosensing, using a hybrid, high quantum-efficiency, form of BRET chemistry.

Functional coupling of a nematode chemoreceptor to the yeast pheromone response pathway.

Sequencing of the Caenorhabditis elegans genome revealed sequences encoding more than 1,000 G-protein coupled receptors, hundreds of which may respond to volatile organic ligands. To understand how the worm's simple olfactory system can sense its chemical environment there is a need to characterise a representative selection of these receptors but only very few receptors have been linked to a specific volatile ligand. We therefore set out to design a yeast expression system for assigning ligands to nematode chemoreceptors.

Drosophila olfactory receptors as classifiers for volatiles from disparate real world applications.

Olfactory receptors evolved to provide animals with ecologically and behaviourally relevant information. The resulting extreme sensitivity and discrimination has proven useful to humans, who have therefore co-opted some animals' sense of smell. One aim of machine olfaction research is to replace the use of animal noses and one avenue of such research aims to incorporate olfactory receptors into artificial noses.

Oligomerisation of C. elegans olfactory receptors, ODR-10 and STR-112, in yeast.

It is widely accepted that vertebrate G-Protein Coupled Receptors (GPCRs) associate with each other as homo- or hetero-dimers or higher-order oligomers. The C. elegans genome encodes hundreds of olfactory GPCRs, which may be expressed in fewer than a dozen chemosensory neurons, suggesting an opportunity for oligomerisation.

Micromolar biosensing of nitric oxide using myoglobin immobilized in a synthetic silk film.

In this work we investigate the use of coiled-coil silk proteins, produced in recombinant Escherichia coli, as a new material for immobilizing biosensors. Myoglobin was embedded in transparent honeybee silk protein films. Immobilized myoglobin maintained a high affinity for nitric oxide (KD NO=52 µM) and good sensitivity with a limit of detection of 5 µM.

Real-time, continuous detection of maltose using bioluminescence resonance energy transfer (BRET) on a microfluidic system.

We have previously shown that a genetically encoded bioluminescent resonance energy transfer (BRET) biosensor, comprising maltose binding protein (MBP) flanked by a green fluorescent protein (GFP(2)) at the N-terminus and a variant of Renilla luciferase (RLuc2) at the C-terminus, has superior sensitivity and limits of detection for maltose, compared with an equivalent fluorescent resonance energy transfer (FRET) biosensor. Here, we demonstrate that the same MBP biosensor can be combined with a microfluidic system for detection of maltose in water or beer. Using the BRET-based biosensor, maltose in water was detected on a microfluidic chip, either following a pre-incubation step or in real-time with similar sensitivity and dynamic range to those obtained using a commercial 96-well plate luminometer.

Feature selection for chemical sensor arrays using mutual information.

We address the problem of feature selection for classifying a diverse set of chemicals using an array of metal oxide sensors. Our aim is to evaluate a filter approach to feature selection with reference to previous work, which used a wrapper approach on the same data set, and established best features and upper bounds on classification performance. We selected feature sets that exhibit the maximal mutual information with the identity of the chemicals.

Comparison of static and microfluidic protease assays using modified bioluminescence resonance energy transfer chemistry.

Background: Fluorescence and bioluminescence resonance energy transfer (F/BRET) are two forms of Förster resonance energy transfer, which can be used for optical transduction of biosensors. BRET has several advantages over fluorescence-based technologies because it does not require an external light source. There would be benefits in combining BRET transduction with microfluidics but the low luminance of BRET has made this challenging until now.

Comparisons of contact chemoreception and food acceptance by larvae of polyphagous Helicoverpa armigera and oligophagous Bombyx mori.

We compared food choice and the initial response to deterrent treated diet between fifth instars of Helicoverpa armigera, a polyphagous generalist pest, and Bombyx mori, an oligophagous specialist beneficial. Bombyx mori was more behaviorally sensitive to salicin than to caffeine. The relative sensitivities were reversed for H.

Advantages of substituting bioluminescence for fluorescence in a resonance energy transfer-based periplasmic binding protein biosensor.

A genetically encoded maltose biosensor was constructed, comprising maltose binding protein (MBP) flanked by a green fluorescent protein (GFP(2)) at the N-terminus and a Renilla luciferase variant (RLuc2) at the C-terminus. This Bioluminescence resonance energy transfer(2) (BRET(2)) system showed a 30% increase in the BRET ratio upon maltose binding, compared with a 10% increase with an equivalent fluorescence resonance energy transfer (FRET) biosensor. BRET(2) provides a better matched Förster distance to the known separation of the N and C termini of MBP than FRET.

Effect of enhanced Renilla luciferase and fluorescent protein variants on the Förster distance of Bioluminescence resonance energy transfer (BRET).

Bioluminescence resonance energy transfer (BRET) is an important tool for monitoring macromolecular interactions and is useful as a transduction technique for biosensor development. Förster distance (R(0)), the intermolecular separation characterized by 50% of the maximum possible energy transfer, is a critical BRET parameter. R(0) provides a means of linking measured changes in BRET ratio to a physical dimension scale and allows estimation of the range of distances that can be measured by any donor-acceptor pair.

Comparison of enhanced bioluminescence energy transfer donors for protease biosensors.

Bioluminescence energy transfer (BRET) is a powerful tool for the study of protein-protein interactions and conformational changes within proteins. We directly compared two recently developed variants of Renilla luciferase (RLuc), RLuc2 and RLuc8, as BRET donors using an in vitro thrombin assay. The comparison was carried out by placing a thrombin-specific cleavage sequence between the donor luciferase and a green fluorescent protein (GFP(2)) acceptor.

Topological and functional characterization of an insect gustatory receptor.

Insect gustatory receptors are predicted to have a seven-transmembrane structure and are distantly related to insect olfactory receptors, which have an inverted topology compared with G-protein coupled receptors, including mammalian olfactory receptors. In contrast, the topology of insect gustatory receptors remains unknown. Except for a few examples from Drosophila, the specificity of individual insect gustatory receptors is also unknown.

Greatly enhanced detection of a volatile ligand at femtomolar levels using bioluminescence resonance energy transfer (BRET).

Our goal is to develop a general transduction system for G-protein coupled receptors (GPCRs). GPCRs are present in most eukaryote cells and transduce diverse extracellular signals. GPCRs comprise not only the largest class of integral membrane receptors but also the largest class of targets for therapeutic drugs.