Eva González-Fernández

Impedimetric aptasensor for tobramycin detection in human serum.

An RNA aptamer is proposed as a recognition element for the detection of tobramycin in human serum. A displacement assay was developed using faradaic-electrochemical impedance spectroscopy (F-EIS) as a detection technique. Two modified aptamers, a partially (ATA) and a fully O-methylated aptamer (FATA) were evaluated and compared. The affinity constant, K(D), for both aptamers was estimated by F-EIS resulting virtually identical within the experimental error. The selectivity towards other aminoglycosides was also studied. The analytical characteristics were evaluated in aqueous solution using both aptamers and FATA was selected for human serum experiments. Using a 1:0.5 dilution of the serum, a linear range between 3 μM and 72.1 μM was obtained, which included the therapeutic range of the antibiotic.

Methylene blue not ferrocene: Optimal reporters for electrochemical detection of protease activity

Electrochemical peptide-based biosensors are attracting significant attention for the detection and analysis of proteins. Here we report the optimisation and evaluation of an electrochemical biosensor for the detection of protease activity using self-assembled monolayers (SAMs) on gold surfaces, using trypsin as a model protease. The principle of detection was the specific proteolytic cleavage of redox-tagged peptides by trypsin, which causes the release of the redox reporter, resulting in a decrease of the peak current as measured by square wave voltammetry. A systematic enhancement of detection was achieved through optimisation of the properties of the redox-tagged peptide; this included for the first time a side-by-side study of the applicability of two of the most commonly applied redox reporters used for developing electrochemical biosensors, ferrocene and methylene blue, along with the effect of changing both the nature of the spacer and the composition of the SAM. Methylene blue-tagged peptides combined with a polyethylene-glycol (PEG) based spacer were shown to be the best platform for trypsin detection, leading to the highest fidelity signals (characterised by the highest sensitivity (signal gain) and a much more stable background than that registered when using ferrocene as a reporter). A ternary SAM (T-SAM) configuration, which included a PEG-based dithiol, minimised the non-specific adsorption of other proteins and was sensitive towards trypsin in the clinically relevant range, with a Limit of Detection (LoD) of 250pM. Kinetic analysis of the electrochemical response with time showed a good fit to a Michaelis-Menten surface cleavage model, enabling the extraction of values for kcat and KM. Fitting to this model enabled quantitative determination of the solution concentration of trypsin across the entire measurement range. Studies using an enzyme inhibitor and a range of real world possible interferents demonstrated a selective response to trypsin cleavage. This indicates that a PEG-based peptide, employing methylene blue as redox reporter, and deposited on an electrode as a ternary SAM configuration, is a suitable platform to develop clinically-relevant and quantitative electrochemical peptide-based protease biosensing.

Implantable Microsystems for Personalised Anticancer Therapy

The Implantable Microsystems for Personalised Anti-Cancer Therapy (IMPACT) project aims to produce an implantable wireless sensor device for monitoring tumour physiology. Real-time measurements will be used to improve radiotherapy by allowing treatment to be responsively delivered at the most effective time and location. We are developing miniaturised microfabricated sensors for measuring local oxygen concentration and pH within the tumour, using technologies that are amenable to integration on CMOS. In addition, we have established proof of concept for a range of electrochemical biosensors that can respond to enzyme biomarkers. Together these sensors will allow comprehensive monitoring of tissue physiology before and after radiotherapy treatment. For clinical use, the complete system will be equipped with circuits for wireless power and communications and packaged in biocompatible materials. This is a very challenging application for sensors integrated on CMOS. Here we provide a brief background to medical aspects of the work and describe our progress towards solving the engineering challenges it has presented.

Test structures for optimizing polymer electrolyte performance in a microfabricated electrochemical oxygen sensor

Test structures were produced for optimizing the design and fabrication of a patterned solid polymer electrolyte in an electrochemical oxygen sensor. Measurements showed that choice of photoresist developer and the underlying insulator material affected durability of the polymer structures. Test electrodes covered by the polymer were effective at supporting electrochemical oxygen detection.

Design and fabrication of microelectrodes for electrical impedance tomography of cell spheroids

Three-dimensional (3D) tumour spheroids exhibit great potential as an in vitro for pharmaceutical drug testing. Spheroids represent more physiologically relevant systems compared to 2D cell culture models. In this work, MCF7 spheroids are cultured in Corning® spheroid microplates to investigate cell morphology and proliferation. Platinum microelectrodes designed to enable electrical impedance tomography (EIT) imaging were fabricated on glass substrates. EIT measurements were performed on 250 μm radius circular electrode tips for 0.71 mm MCF7 spheroid at 20 kHz using time difference imaging method. Initial results indicate the capability of EIT to image cell spheroids.

Electrochemical sensing of human neutrophil elastase and polymorphonuclear neutrophil activity

Human neutrophil elastase (HNE) is a serine protease, produced by polymorphonuclear neutrophils (PMNs), whose uncontrolled production has been associated with various inflammatory disease states as well as tumour proliferation and metastasis. Here we report the development and characterisation of an electrochemical peptide-based biosensor, which enables the detection of clinically relevant levels of HNE. The sensing platform was characterised in terms of its analytical performance, enzymatic cleavage kinetics and cross-reactivity and applied to the quantitative detection of protease activity from PMNs from human blood.

Data associated with manuscript: Effect of spacer length on the performance of peptide-based electrochemical biosensors for protease detection

Peptide-based electrochemical biosensors typically consist of a short peptide sequence, labelled with a redox reporter and modified with a thiol-containing moiety to allow immobilisation onto a gold electrode surface. A spacer is often introduced between the thiol group and the peptide with the aim of promoting enzyme accessibility as well as conferring flexibility onto the probe. Herein we report a systematic study of the effect of polyethylene glycol (PEG)-based spacer length on the performance of such biosensors in order to gain a deeper understanding of their role and optimise a peptide-based electrochemical sensor. Thus a specific peptide endowed with varying PEG spacers (PEG-4, PEG-6, PEG-8 and PEG-12) were synthesised and interrogated by the addition of both a target enzyme (trypsin) and BSA in order to evaluate their analytical performance. An alkyl-based spacer was also assessed in order to compare the effect of the nature of the spacer. All of the proposed probes supported efficient protease detection; however, PEG-6 provided enhanced antifouling properties, which highlights the vital role of the spacer in the design of peptide-based probes.