pH-Activated Dissolvable Polymeric Coatings to Reduce Biofouling on Electrochemical Sensors.

Implantable electrochemical sensors that enable the real-time detection of significant biomarkers offer huge potential for the enhancement and personalisation of therapies; however, biofouling is a key challenge encountered by any implantable system. This is particularly an issue immediately after implantation, when the foreign body response and associated biofouling processes are at their most active in passivating a foreign object. Here, we present the development of a sensor protection and activation strategy against biofouling, based on coatings consisting of a pH-triggered, dissolvable polymer, that covered a functionalised electrode surface. We demonstrate that reproducible delayed sensor activation can be achieved, and that the length of this delay can be controlled by the optimisation of coating thickness, homogeneity and density through tuning of the coating method and temperature. Comparative evaluation of the polymer-coated and uncoated probe-modified electrodes in biological media revealed significant improvements in their anti-biofouling characteristics, demonstrating that this offers a promising approach to the design of enhanced sensing devices.

Electrofabrication of large volume di- and tripeptide hydrogels via hydroquinone oxidation.

The fabrication of protected peptide-based hydrogels on electrode surfaces can be achieved by employing the electrochemical oxidation of hydroquinone to benzoquinone, liberating protons at the electrode-solution interface. The localised reduction in pH below the dipeptide gelator molecules pKa initiates the neutralisation, self-assembly and formation of self-supporting hydrogels exclusively at the electrode surface. Previous examples have been on a nanometre to millimetre scale, using deposition times ranging from seconds to minutes. However, the maximum size to which these materials can grow and their subsequent mechanical properties have not yet been investigated. Here, we report the fabrication of the largest reported di- and tri-peptide based hydrogels using this electrochemical method, employing deposition times of two to five hours. To overcome the oxidation of hydroquinone in air, the fabrication process was performed under an inert nitrogen atmosphere. We show that this approach can be used to form multilayer gels, with the mechanical properties of each layer determined by gelator composition. We also describe examples where gel-to-crystal transitions and syneresis occur within the material.

In vivo application of an implantable tri-anchored methylene blue-based electrochemical pH sensor.

The development of robust implantable sensors is important in the successful advancement of personalised medicine as they have the potential to provide in situ real-time data regarding the status of health and disease and the effectiveness of treatment. Tissue pH is a key physiological parameter and herein, we report the design, fabrication, functionalisation, encapsulation and protection of a miniaturised, self-contained, electrochemical pH sensor system and characterisation of sensor performance. Notably for the first time in this environment the pH sensor was based on a methylene blue redox reporter which showed remarkable robustness, accuracy and sensitivity. This was achieved by encapsulation of a self-assembled monolayer containing methylene blue entrapped within a Nafion layer. Another powerful feature was the incorporation, within the same implanted device, of a fabricated on-chip Ag/AgCl reference electrode - vital in any electrochemical sensor, but often ignored. When utilised in vivo, the sensor allowed accurate tracking of externally induced pH changes within a naturally occurring ovine lung cancer model, and correlated well with single point laboratory measurements made on extracted arterial blood, whilst enabling in vivo time-dependent measurements. The sensors functioned robustly whilst implanted, and maintained in vitro function once extracted and together, these results demonstrate proof-of-concept of the ability to sense real-time intratumoral tissue pH changes in vivo.

Miniaturisation of a peptide-based electrochemical protease activity sensor using platinum microelectrodes.

Proteases are ideal target biomarkers as they have been implicated in many disease states, including steps associated with cancer progression. Electrochemical peptide-based biosensors have attracted much interest in recent years. However, the significantly large size of the electrodes typically used in most of these platforms has led to performance limitations. These could be addressed by the enhancements offered by microelectrodes, such as rapid response times, improved mass transport, higher signal-to-noise and sensitivity, as well as more localised and less invasive measurements. We present the production and characterisation of a miniaturised electrochemical biosensor for the detection of trypsin, based on 25 µm diameter Pt microelectrodes (rather than the ubiquitous Au electrodes), benchmarked by establishing the equivalent Pt macroelectrode response in terms of quantitative response to the protease, the kinetics of cleavage and the effects of non-specific protein binding and temperature. Interestingly, although there was little difference between Au and Pt macroelectrode response, significant differences were observed between the responses of the Pt macroelectrode and microelectrode systems indicative of increased reproducibility in the microelectrode SAM structure and sensor performance between the electrodes, increased storage stability and a decrease in the cleavage rate at functionalised microelectrodes, which is mitigated by measurement at normal body temperature. Together, these results demonstrate the robustness and sensitivity of the miniaturised sensing platform and its ability to operate within the clinically-relevant concentration ranges of proteases in normal and disease states. These are critical features for its translation into implantable devices.

A Dual Killing Strategy: Photocatalytic Generation of Singlet Oxygen with Concomitant PtIV Prodrug Activation.

A ruthenium-based mitochondrial-targeting photosensitiser that undergoes efficient cell uptake, enables the rapid catalytic conversion of PtIV prodrugs into their active PtII counterparts, and drives the generation of singlet oxygen was designed. This dual mode of action drives two orthogonal cancer-cell killing mechanisms with temporal and spatial control. The designed photosensitiser was shown to elicit cell death of a panel of cancer cell lines including those showing oxaliplatin-resistance.

Electrodrugs: an electrochemical prodrug activation strategy.

The term electroceutical has been used to describe implanted devices that deliver electrical stimuli to modify biological function. Herein, we describe a new concept in electroceuticals, demonstrating for the first time the electrochemical activation of metal-based prodrugs. This is illustrated by the controlled activation of Pt(iv) prodrugs into their active Pt(ii) forms within a cellular context allowing selectivity and control of where, when and how much active drug is generated.

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.

Functionalised microscale nanoband edge electrode (MNEE) arrays: the systematic quantitative study of hydrogels grown on nanoelectrode biosensor arrays for enhanced sensing in biological media.

Nanoelectrodes and nanoelectrode arrays show enhanced diffusion and greater faradaic current densities and signal-to-noise ratios compared to macro and microelectrodes, which can lead to enhanced sensing and detection. One example is the microsquare nanoband edge electrode (MNEE) array system, readily formed through microfabrication and whose quantitative response has been established electroanalytically. Hydrogels have been shown to have applications in drug delivery, tissue engineering, and anti-biofouling; some also have the ability to be grown electrochemically. Here, we combine these two emerging technologies to demonstrate the principles of a hydrogel-coated nanoelectrode array biosensor that is resistant to biofouling. We first electrochemically grow and analyze hydrogels on MNEE arrays. The structure of these gels is shown by imaging to be electrochemically controllable, reproducible and structurally hierarchical. This structure is determined by the MNEE array diffusion fields, consistent with the established hydrogel formation reaction, and varies in structural scale from nano (early time, near electrode growth) to micro (for isolated elements in the array) to macro (when there is array overlap) with distance from the electrode, forming a hydrogel mesh of increasing density on progression from solution to electrode. There is also increased hydrogel structural density observed at electrode corners, attributable to enhanced diffusion. The resulting hydrogel structure can be formed on (and is firmly anchored to/through) an established clinically relevant biosensing layer without compromising detection. It is also shown to be capable, through proof-of-principle model protein studies using bovine serum albumin (BSA), of preventing protein biofouling whilst enabling smaller molecules such as DNA to pass through the hydrogel matrix and be sensed. Together, this demonstrates a method for developing reproducible, quantitative electrochemical nanoelectrode biosensors able to sense selectively in real-world sample matrices through the tuning of their interfacial properties.

A Microelectrode Array with Reproducible Performance Shows Loss of Consistency Following Functionalization with a Self-Assembled 6-Mercapto-1-hexanol Layer.

For analytical applications involving label-free biosensors and multiple measurements, i.e., across an electrode array, it is essential to develop complete sensor systems capable of functionalization and of producing highly consistent responses. To achieve this, a multi-microelectrode device bearing twenty-four equivalent 50 µm diameter Pt disc microelectrodes was designed in an integrated 3-electrode system configuration and then fabricated. Cyclic voltammetry and electrochemical impedance spectroscopy were used for initial electrochemical characterization of the individual working electrodes. These confirmed the expected consistency of performance with a high degree of measurement reproducibility for each microelectrode across the array. With the aim of assessing the potential for production of an enhanced multi-electrode sensor for biomedical use, the working electrodes were then functionalized with 6-mercapto-1-hexanol (MCH). This is a well-known and commonly employed surface modification process, which involves the same principles of thiol attachment chemistry and self-assembled monolayer (SAM) formation commonly employed in the functionalization of electrodes and the formation of biosensors. Following this SAM formation, the reproducibility of the observed electrochemical signal between electrodes was seen to decrease markedly, compromising the ability to achieve consistent analytical measurements from the sensor array following this relatively simple and well-established surface modification. To successfully and consistently functionalize the sensors, it was necessary to dilute the constituent molecules by a factor of ten thousand to support adequate SAM formation on microelectrodes. The use of this multi-electrode device therefore demonstrates in a high throughput manner irreproducibility in the SAM formation process at the higher concentration, even though these electrodes are apparently functionalized simultaneously in the same film formation environment, confirming that the often seen significant electrode-to-electrode variation in label-free SAM biosensing films formed under such conditions is not likely to be due to variation in film deposition conditions, but rather kinetically controlled variation in the SAM layer formation process at these microelectrodes.

Integration of Electrodeposited Ni-Fe in MEMS with Low-Temperature Deposition and Etch Processes.

This article presents a set of low-temperature deposition and etching processes for the integration of electrochemically deposited Ni-Fe alloys in complex magnetic microelectromechanical systems, as Ni-Fe is known to suffer from detrimental stress development when subjected to excessive thermal loads. A selective etch process is reported which enables the copper seed layer used for electrodeposition to be removed while preserving the integrity of Ni-Fe. In addition, a low temperature deposition and surface micromachining process is presented in which silicon dioxide and silicon nitride are used, respectively, as sacrificial material and structural dielectric. The sacrificial layer can be patterned and removed by wet buffered oxide etch or vapour HF etching. The reported methods limit the thermal budget and minimise the stress development in Ni-Fe. This combination of techniques represents an advance towards the reliable integration of Ni-Fe components in complex surface micromachined magnetic MEMS.

Correction: Impedimetric measurement of DNA-DNA hybridisation using microelectrodes with different radii for detection of methicillin resistant Staphylococcus aureus (MRSA).

Correction for 'Impedimetric measurement of DNA-DNA hybridisation using microelectrodes with different radii for detection of methicillin resistant Staphylococcus aureus (MRSA)' by Poh Quan Li et al., Analyst, 2017, 142, 1946-1952.

Impedimetric measurement of DNA-DNA hybridisation using microelectrodes with different radii for detection of methicillin resistant Staphylococcus aureus (MRSA).

Due to their electroanalytical advantages, microelectrodes are a very attractive technology for sensing and monitoring applications. One highly important application is measurement of DNA hybridisation to detect a wide range of clinically important phenomena, including single nucleotide polymorphisms (SNPs), mutations and drug resistance genes. The use of electrochemical impedance spectroscopy (EIS) for measurement of DNA hybridisation is well established for large electrodes but as yet remains relatively unexplored for microelectrodes due to difficulties associated with electrode functionalisation and impedimetric response interpretation. To shed light on this, microelectrodes were initially fabricated using photolithography and characterised electrochemically to ensure their responses matched established theory. Electrodes with different radii (50, 25, 15 and 5 µm) were then functionalised with a mixed film of 6-mercapto-1-hexanol and a thiolated single stranded DNA capture probe for a specific gene from the antibiotic resistant bacterium MRSA. The complementary oligonucleotide target from the mecA MRSA gene was hybridised with the surface tethered ssDNA probe. The EIS response was evaluated as a function of electrode radius and it was found that charge-transfer (RCT) was more significantly affected by hybridisation of the mecA gene than the non-linear resistance (RNL) which is associated with the steady state current. The discrimination of mecA hybridisation improved as electrode radius reduced with the RCT component of the response becoming increasingly dominant for smaller radii. It was possible to utilise these findings to produce a real time measurement of oligonucleotide binding where changes in RCT were evident one minute after nanomolar target addition. These data provide a systematic account of the effect of microelectrode radius on the measurement of hybridisation, providing insight into critical aspects of sensor design and implementation for the measurement of clinically important DNA sequences. The findings open up the possibility of developing rapid, sensitive DNA based measurements using microelectrodes.

Advances in electroanalysis, sensing and monitoring in molten salts.

Microelectrodes have a number of advantages over macroelectrodes for quantitative electroanalysis and monitoring, including reduced iR drop, a high signal-to-noise ratio and reduced sensitivity to convection. Their use in molten salts has been generally precluded by the combined materials challenges of stresses associated with thermal cycling and physical and corrosive chemical degradation at the relatively high temperatures involved. We have shown that microfabrication, employing high precision photolithographic patterning in combination with the controlled deposition of materials, can be used to successfully address these challenges. The resulting molten salt compatible microelectrodes (MSMs) enable prolonged quantitative microelectrode measurements in molten salts (MSs). This paper reports the fabrication of novel MSM disc electrodes, chosen because they have an established ambient analytical response. It includes a detailed set of electrochemical characterisation studies which demonstrate both their enhanced capability over macroelectrodes and over commercial glass pulled microelectrodes, and their ability to extract quantitative electroanalytical information from MS systems. MSM measurements are then used to demonstrate their potential for shedding new light on the fundamental properties of, and processes in, MSs, such as mass transport, charge transfer reaction rates and the selective plating/stripping and alloying reactions of liquid Bi and other metals; this will underpin the development of enhanced MS industrial processes, including pyrochemical spent nuclear fuel reprocessing.

Nanoband array electrode as a platform for high sensitivity enzyme-based glucose biosensing.

We describe a novel glucose biosensor based on a nanoband array electrode design, manufactured using standard semiconductor processing techniques, and bio-modified with glucose oxidase immobilized at the nanoband electrode surface. The nanoband array architecture allows for efficient diffusion of glucose and oxygen to the electrode, resulting in a thousand-fold improvement in sensitivity and wide linear range compared to a conventional electrode. The electrode constitutes a robust and manufacturable sensing platform.

Label- and amplification-free electrochemical detection of bacterial ribosomal RNA.

Current approaches to molecular diagnostics rely heavily on PCR amplification and optical detection methods which have restrictions when applied to point of care (POC) applications. Herein we describe the development of a label-free and amplification-free method of pathogen detection applied to Escherichia coli which overcomes the bottleneck of complex sample preparation and has the potential to be implemented as a rapid, cost effective test suitable for point of care use. Ribosomal RNA is naturally amplified in bacterial cells, which makes it a promising target for sensitive detection without the necessity for prior in vitro amplification. Using fluorescent microarray methods with rRNA targets from a range of pathogens, an optimal probe was selected from a pool of probe candidates identified in silico. The specificity of probes was investigated on DNA microarray using fluorescently labeled 16S rRNA target. The probe yielding highest specificity performance was evaluated in terms of sensitivity and a LOD of 20 pM was achieved on fluorescent glass microarray. This probe was transferred to an EIS end point format and specificity which correlated to microarray data was demonstrated. Excellent sensitivity was facilitated by the use of uncharged PNA probes and large 16S rRNA target and investigations resulted in an LOD of 50 pM. An alternative kinetic EIS assay format was demonstrated with which rRNA could be detected in a species specific manner within 10-40min at room temperature without wash steps.

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.

Enhanced electroanalysis in lithium potassium eutectic (LKE) using microfabricated square microelectrodes.

Molten salts (MSs) are an attractive medium for chemical and electrochemical processing and as a result there is demand for MS-compatible analysis technologies. However, MSs containing redox species present a challenging environment in which to perform analytical measurements because of their corrosive nature, significant thermal convection and the high temperatures involved. This paper outlines the fabrication and characterization of microfabricated square microelectrodes (MSMs) designed for electrochemical analysis in MS systems. Their design enables precise control over electrode dimension, the minimization of stress because of differential thermal expansion through design for high temperature operation, and the minimization of corrosive attack through effective insulation. The exemplar MS system used for characterization was lithium chloride/potassium chloride eutectic (LKE), which has potential applications in pyrochemical nuclear fuel reprocessing, metal refining, molten salt batteries and electric power cells. The observed responses for a range of redox ions between 400 and 500 °C (673 and 773 K) were quantitative and typical of microelectrodes. MSMs also showed the reduced iR drop, steady-state diffusion-limited response, and reduced sensitivity to convection seen for microelectrodes under ambient conditions and expected for these electrodes in comparison to macroelectrodes. Diffusion coefficients were obtained in close agreement with literature values, more readily and at greater precision and accuracy than both macroelectrode and previous microelectrode measurements. The feasibility of extracting individual physical parameters from mixtures of redox species (as required in reprocessing) and of the prolonged measurement required for online monitoring was also demonstrated. Together, this demonstrates that MSMs provide enhanced electrode devices widely applicable to the characterization of redox species in a range of MS systems.

Nanoscale electrode arrays produced with microscale lithographic techniques for use in biomedical sensing applications.

A novel technique for the production of nanoscale electrode arrays that uses standard microfabrication processes and micron-scale photolithography is reported here in detail. These microsquare nanoband edge electrode (MNEE) arrays have been fabricated with highly reproducible control of the key array dimensions, including the size and pitch of the individual elements and, most importantly, the width of the nanoband electrodes. The definition of lateral features to nanoscale dimensions typically requires expensive patterning techniques that are complex and low-throughput. However, the fabrication methodology used here relies on the fact that vertical dimensions (i.e. layer thicknesses) have long been manufacturable at the nanoscale using thin film deposition techniques that are well established in mainstream microelectronics. The authors report for the first time two aspects that highlight the particular suitability of these MNEE array systems for probe monolayer biosensing. The first is simulation, which shows the enhanced sensitivity to the redox reaction of the solution redox couple. The second is the enhancement of probe film functionalisation observed for the probe film model molecule, 6-mercapto-1-hexanol compared with microsquare electrodes. Such surface modification for specific probe layer biosensing and detection is of significance for a wide range of biomedical and other sensing and analytical applications.

Comparison of the performance of an array of nanoband electrodes with a macro electrode with similar overall area.

The performance of two electrode architectures with broadly similar overall active electrode areas are examined. The first is an electrode comprising a single contiguous area (a disc) and the second is an electrode in which the cumulative electrode area is dispersed over a wide area as a 50 nm thickness platinum nanoband. A direct comparison of the electrochemical performance of these two electrodes has been made. The relatively simple nanoband electrode architecture is shown to have benefits, including two orders of magnitude greater mass transport limited currents, the ability to measure faster electrode kinetics (by a similar factor), a three orders of magnitude lowering of the Limit of Detection and a significantly reduced susceptibility to hydrodynamic perturbations. The consequences and implications of these performance characteristics on the uses of such a nanoband electrode have been considered.

Cyclic denaturation and renaturation of double-stranded DNA by redox-state switching of DNA intercalators.

Hybridization of complementary nucleic acid strands is fundamental to nearly all molecular bioanalytical methods ranging from polymerase chain reaction and DNA biosensors to next generation sequencing. For nucleic acid amplification methods, controlled DNA denaturation and renaturation is particularly essential and achieved by cycling elevated temperatures. Although this is by far the most used technique, the management of rapid temperature changes requires bulky instrumentation and intense power supply. These factors so far precluded the development of true point-of-care tests for molecular diagnostics. To overcome this limitation we explored the possibility of using electrochemical means to control reversible DNA hybridization by using the electroactive intercalator daunomycin (DM). We show that redox-state switching of DM altered its properties from DNA binding to nonbinding, under otherwise constant conditions, and thus altered the thermodynamic stability of duplex DNA. The operational principle was demonstrated using complementary synthetic 20mer and 40mer DNA oligonucleotides. Absorbance-based melting curve analysis revealed significantly higher melting temperatures for DNA in the presence of oxidized compared to chemically reduced DM. This difference was exploited to drive cyclic electrochemically controlled denaturation and renaturation. Analysis with in situ UV-vis and circular dichroism spectroelectrochemistry, as two independent techniques, indicated that up to 80% of the DNA was reversibly hybridized. This remarkable demonstration of electrochemical control of five cycles of DNA denaturation and renaturation, under otherwise constant conditions, could have wide-ranging implications for the future development of miniaturized analytical systems for molecular diagnostics and beyond.