Development of a Minimally Invasive Microneedle-Based Sensor for Continuous Monitoring of β-Lactam Antibiotic Concentrations in Vivo.

Antimicrobial resistance poses a global threat to patient health. Improving the use and effectiveness of antimicrobials is critical in addressing this issue. This includes optimizing the dose of antibiotic delivered to each individual.

Rapid, low cost prototyping of transdermal devices for personal healthcare monitoring.

The next generation of devices for personal healthcare monitoring will comprise molecular sensors to monitor analytes of interest in the skin compartment. Transdermal devices based on microneedles offer an excellent opportunity to explore the dynamics of molecular markers in the interstitial fluid, however good acceptability of these next generation devices will require several technical problems associated with current commercially available wearable sensors to be overcome. These particularly include reliability, comfort and cost.

Microneedle Enzyme Sensor Arrays for Continuous In Vivo Monitoring.

Microneedle enzyme sensors by virtue of their minimally invasive and hence pain-free penetration of skin allow for the measurement of metabolites, biomarkers, and drugs in the interstitial fluid that bathes the dermal tissue. Such devices if they are to be adopted widely into clinical practice need to be capable of delivering reliable measurements over extended periods of time (days) and to be fabricated by low-cost, scalable methods. Using injection molding of the base structures in polycarbonate, metal film deposition by sputtering and enzyme immobilization by electrodeposition can meet these requirements.

Evaluation of a minimally invasive glucose biosensor for continuous tissue monitoring.

We describe here a minimally invasive glucose biosensor based on a microneedle array electrode fabricated from an epoxy-based negative photoresist (SU8 50) and designed for continuous measurement in the dermal compartment with minimal pain. These minimally invasive, continuous monitoring sensor devices (MICoMS) were produced by casting the structures in SU8 50, crosslinking and then metallising them with platinum or silver to obtain the working and reference electrodes, respectively. The metallised microneedle array electrodes were subsequently functionalised by entrapping glucose oxidase in electropolymerised polyphenol (PP) film.

Application of Gold Nanorods for Photothermal Therapy in Ex Vivo Human Oesophagogastric Adenocarcinoma.

Gold nanoparticles are chemically fabricated and tuned to strongly absorb near infrared (NIR) light, enabling deep optical penetration and therapy within human tissues, where sufficient heating induces tumour necrosis. In our studies we aim to establish the optimal gold nanorod (GNR) concentration and laser power for inducing hyperthermic effects in tissues and test this photothermal effect on ex vivo human oesophagogastric adenocarcinoma. The ideal GNR concentration and NIR laser power that would elicit sufficient hyperthermia for tumour necrosis was pre-determined on porcine oesophageal tissues.

Enzymatic cyanide degradation by cell-free extract of Rhodococcus UKMP-5M.

The cell-free extract of locally isolated Rhodococcus UKMP-5M strain was used as an alternative to develop greener and cost effective cyanide removal technology. The present study aims to assess the viability of the cell-free extract to detoxify high concentrations of cyanide which is measured through the monitoring of protein concentration and specific cyanide-degrading activity. When cyanide-grown cells were subjected to grinding in liquid nitrogen which is relatively an inexpressive and fast cell disruption method, highest cyanide-degrading activity of 0.

Dip-pen patterning of poly(9,9-dioctylfluorene) chain-conformation-based nano-photonic elements.

Metamaterials are a promising new class of materials, in which sub-wavelength physical structures, rather than variations in chemical composition, can be used to modify the nature of their interaction with electromagnetic radiation. Here we show that a metamaterials approach, using a discrete physical geometry (conformation) of the segments of a polymer chain as the vector for a substantial refractive index change, can be used to enable visible wavelength, conjugated polymer photonic elements. In particular, we demonstrate that a novel form of dip-pen nanolithography provides an effective means to pattern the so-called β-phase conformation in poly(9,9-dioctylfluorene) thin films.

High efficiency acetylcholinesterase immobilization on DNA aptamer modified surfaces.

We report here the in vitro selection of DNA aptamers for electric eel acetylcholinesterase (AChE). One selected aptamer sequence (R15/19) has a high affinity towards the enzyme (Kd=157±42 pM). Characterization of the aptamer showed its binding is not affected by low ionic strength (~20 mM), however significant reduction in affinity occurred at high ionic strength (~1.

Biodegradation of cyanide by acetonitrile-induced cells of Rhodococcus sp. UKMP-5M.

A Rhodococcus sp. UKMP-5M isolate was shown to detoxify cyanide successfully, suggesting the presence of an intrinsic property in the bacterium which required no prior cyanide exposure for induction of this property. However, in order to promote growth, Rhodococcus sp.

Smart plastic antibody material (SPAM) tailored on disposable screen printed electrodes for protein recognition: application to myoglobin detection.

This work introduces two major changes to the conventional protocol for designing plastic antibodies: (i) the imprinted sites were created with charged monomers while the surrounding environment was tailored using neutral material; and (ii) the protein was removed from its imprinted site by means of a protease, aiming at preserving the polymeric network of the plastic antibody. To our knowledge, these approaches were never presented before and the resulting material was named here as smart plastic antibody material (SPAM). As proof of concept, SPAM was tailored on top of disposable gold-screen printed electrodes (Au-SPE), following a bottom-up approach, for targeting myoglobin (Myo) in a point-of-care context.

Streptavidin binding bifunctional aptamers and their interaction with low molecular weight ligands.

This paper describes the measurement of the binding affinities of two bifunctional RNA aptamers to their respective ligands. The aptamers comprise either a theophylline or malachite green binding sequence fused to a streptavidin binding sequence. These bifunctional aptamers are shown to bind simultaneously to both the small ligand and to streptavidin whether in free solution or on gold surfaces.

Selection of DNA aptamers with affinity for pro-gastrin-releasing peptide (proGRP), a tumor marker for small cell lung cancer.

Aptamers are single-strand oligonucleotides that are generated by the systemic evolution of ligands by exponential enrichment (SELEX) technique and that can bind to target molecules specifically. However, only a few aptamers have been developed to date against tumor markers. To utilize aptamers for tumor diagnosis, a variety of aptamers are required.

Microfluidic device to investigate factors affecting performance in biosensors designed for transdermal applications.

In this work we demonstrate a novel microfluidic based platform to investigate the performance of 3D out-of-plane microspike array based glucose and lactate biosensors. The microspike array was bonded with a glass slide and modified with glucose oxidase or lactate oxidase using covalent coupling chemistry. An epoxy-polyurethane based membrane was used to extend the linear working range (from 0 to 25 mM of substrate) of these biosensors.

Nucleic acid aptamers: ideal reagents for point-of-care diagnostics?

Nucleic acid aptamers have many of the properties that make for effective reagents in point-of-care diagnostic devices and whilst superficially similar to antibodies as affinity reagents the scope for engineering them to fit this role is considerable. Synthesis of aptamers allows for the incorporation of functionality for both immobilisation and electrochemical signalling in a way that is compatible with the 'strip' type sensors familiar in enzyme sensors, such as those for glucose. Control of the structure of DNA aptamers through Watson-Crick base pairing allows for different electrochemical assay formats, whilst ferrocenes provide a versatile redox label and insights into the interactions between the aptamer and its target are obtained through both cyclic and square-wave voltammetries.

A simple method for controlled immobilization of proteins on modified SAMs.

This paper describes a method for modifying self-assembled monolayers (SAMs) with the nitrilotriacetic acid (NTA) group for subsequent immobilization of hexahistidine tagged proteins. The method has two important improvements over previous ones; firstly it avoids the need to carry out a complex synthesis of the chelator alkanethiols prior to deposition because the reactions are performed in situ on a preassembled SAM. This in situ approach also avoids phase segregation of alkanethiols with different functional groups, especially bulky ones such as NTA and tri(ethylene glycol), since a simple SAM is employed as the starting material.

CE-based sample quality assessment prior to 2-D gel electrophoresis: towards the standardization of gel-based proteomics.

2-DE remains one of the most commonly used separation techniques for complex protein mixtures. This article describes a new approach to 2-DE sample assessment using SDS capillary gel electrophoresis (in Beckman Coulter sieving medium) combined with multi-pixel detection. The performance of this platform was investigated using protein samples prepared for 2-DE.

Towards microfluidic technology-based MALDI-MS platforms for drug discovery: a review.

Background: Microfluidic methods have found applications in various disciplines. It has been predicted that the microfluidic technology would be useful in performing routine steps in drug discovery ranging from target identification to lead optimisation in which the number of compounds evaluated in this regard determines the success of combinatorial screening. The sheer size of the parameter space that can be explored often poses an enormous challenge.

Biocompatible ion selective electrode for monitoring metabolic activity during the growth and cultivation of human cells.

Ammonia is the main nitrogenous waste product of cellular metabolism and if accumulated in culture media may limit cell growth and affect the quality of cultured cell lines. Therefore, it is crucial to control levels of this metabolite during the in vitro expansion of human cells. This paper describes the successful application of ion selective electrodes (ISE) to continuously monitor ammonium concentrations in a perfused cell bioreactor.

Proteomics, nanotechnology and molecular diagnostics.

Sequencing of the human genome opened the way to the exploration of the proteome and this has lead to the identification of large numbers of proteins in complex biological samples. The identification of diagnostic patterns in samples taken from patients to aid diagnosis is in the early stages of development. The solution to many of the technical challenges in proteomics and protein based molecular diagnostics will be found in new applications of nanomaterials.

Individually addressable microelectrode array for monitoring oxygen and nitric oxide release.

We have fabricated a six individual addressable gold working electrode microarray. The device is wirebonded to an eight-pin DIL package that can be easily interconnected to an external multi-channel potentiostat. A polyion complex film coating on the electrode surface provides a suitable coating for the growth of cells.

Intelligent bioprocessing for haemotopoietic cell cultures using monitoring and design of experiments.

The need for successful ex-vivo expansion and directed differentiation of haematopoietic stem cells (HSCs) for therapeutic applications has increased over the past decade. Haematopoietic cell cultures are complex and full characterisation of the process environment has yet to be achieved. The complexity and transient nature of HSC cultures make the identification, maintenance and control of optimal operating conditions challenging.

Discrimination between single Escherichia coli cells using time-resolved confocal spectroscopy.

We describe a technique for rapidly discriminating between single-cell populations within a flowing microfluidic stream. Single-cell time-correlated single-photon counting (scTCSPC) as well as photon burst spectroscopy are used to characterize individual Escherichia coli cells expressed with either green, cyano, or yellow fluorescent protein. The approach utilizes standard confocal fluorescence microscopy incorporating femtoliter detection volumes.

Construction of a high sensitive Escherichia coli alkaline phosphatase reporter system for screening affinity peptides.

An enzyme-linker-peptide fusion protein reporter system was constructed for sensitive analysis of affinity of peptide ligands to their receptor. An E. coli alkaline phosphatase (EAP) mutant enzyme with high catalytic activity was selected as the reporter protein.

Directed evolution of the 5'-untranslated region of the phoA gene in Escherichia coli simultaneously yields a stronger promoter and a stronger Shine-Dalgarno sequence.

Directed evolution has been widely applied for gene improvement through random mutagenesis of coding sequences. Through error-prone PCR both in the coding sequence and the regulatory sequence of E. coli alkaline phosphatase, the cellular enzyme activity has been efficiently enhanced.