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.

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.

Post-Operative Monitoring of Intestinal Tissue Oxygenation Using an Implantable Microfabricated Oxygen Sensor.

Anastomotic leakage (AL) is a common and dangerous post-operative complication following intestinal resection, causing substantial morbidity and mortality. Ischaemia in the tissue surrounding the anastomosis is a major risk-factor for AL development. Continuous tissue oxygenation monitoring during the post-operative recovery period would provide early and accurate early identification of AL risk. We describe the construction and testing of a miniature implantable electrochemical oxygen sensor that addresses this need. It consisted of an array of platinum microelectrodes, microfabricated on a silicon substrate, with a poly(2-hydroxyethyl methacrylate) hydrogel membrane to protect the sensor surface. The sensor was encapsulated in a biocompatible package with a wired connection to external instrumentation. It gave a sensitive and highly linear response to variations in oxygen partial pressure in vitro, although over time its sensitivity was partially decreased by protein biofouling. Using a pre-clinical in vivo pig model, acute intestinal ischaemia was robustly and accurately detected by the sensor. Graded changes in tissue oxygenation were also measurable, with relative differences detected more accurately than absolute differences. Finally, we demonstrated its suitability for continuous monitoring of tissue oxygenation at a colorectal anastomosis over a period of at least 45 h. This study provides evidence to support the development and use of implantable electrochemical oxygen sensors for post-operative monitoring of anastomosis oxygenation.

Adhesion and Growth of Neuralized Mouse Embryonic Stem Cells on Parylene-C/SiO2 Substrates.

Neuronal patterning on microfabricated architectures has developed rapidly over the past few years, together with the emergence of soft biocompatible materials and tissue engineering scaffolds. Previously, we introduced a patterning technique based on serum and the biopolymer parylene-C, achieving highly compliant growth of primary neurons and astrocytes on different geometries. Here, we expanded this technique and illustrated that neuralized cells derived from mouse embryonic stem cells (mESCs) followed stripes of variable widths with conformity equal to or higher than that of primary neurons and astrocytes. Our results indicate the presence of undifferentiated mESCs, which also conformed to the underlying patterns to a high degree. This is an exciting and unexpected outcome, as molecular mechanisms governing cell and ECM protein interactions are different in stem cells and primary cells. Our study enables further investigations into the development and electrophysiology of differentiating patterned neural stem cells.

Predictive and Diagnostic Biomarkers of Anastomotic Leakage: A Precision Medicine Approach for Colorectal Cancer Patients.

Development of an anastomotic leak (AL) following intestinal surgery for the treatment of colorectal cancers is a life-threatening complication. Failure of the anastomosis to heal correctly can lead to contamination of the abdomen with intestinal contents and the development of peritonitis. The additional care that these patients require is associated with longer hospitalisation stays and increased economic costs. Patients also have higher morbidity and mortality rates and poorer oncological prognosis. Unfortunately, current practices for AL diagnosis are non-specific, which may delay diagnosis and have a negative impact on patient outcome. To overcome these issues, research is continuing to identify AL diagnostic or predictive biomarkers. In this review, we highlight promising candidate biomarkers including ischaemic metabolites, inflammatory markers and bacteria. Although research has focused on the use of blood or peritoneal fluid samples, we describe the use of implantable medical devices that have been designed to measure biomarkers in peri-anastomotic tissue. Biomarkers that can be used in conjunction with clinical status, routine haematological and biochemical analysis and imaging have the potential to help to deliver a precision medicine package that could significantly enhance a patient's post-operative care and improve outcomes. Although no AL biomarker has yet been validated in large-scale clinical trials, there is confidence that personalised medicine, through biomarker analysis, could be realised for colorectal cancer intestinal resection and anastomosis patients in the years to come.

The impact of tumour pH on cancer progression: strategies for clinical intervention.

Dysregulation of cellular pH is frequent in solid tumours and provides potential opportunities for therapeutic intervention. The acidic microenvironment within a tumour can promote migration, invasion and metastasis of cancer cells through a variety of mechanisms. Pathways associated with the control of intracellular pH that are under consideration for intervention include carbonic anhydrase IX, the monocarboxylate transporters (MCT, MCT1 and MCT4), the vacuolar-type H+-ATPase proton pump, and the sodium-hydrogen exchanger 1. This review will describe progress in the development of inhibitors to these targets.

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.

In vivo validation of a miniaturized electrochemical oxygen sensor for measuring intestinal oxygen tension.

Recent advances in the fields of electronics and microfabrication techniques have led to the development of implantable medical devices for use within the field of precision medicine. Monitoring visceral surface tissue O2 tension (PTo2) by means of an implantable sensor is potentially useful in many clinical situations, including the perioperative management of patients undergoing intestinal resection and anastomosis. This concept could provide a means by which treatment could be tailored to individual patients. This study describes the in vivo validation of a novel, miniaturized electrochemical O2 sensor to provide real-time data on intestinal PTo2. A single O2 sensor was placed onto the serosal surface of the small intestine of anesthetized rats that were exposed to ischemic (superior mesenteric artery occlusion) and hypoxemic (alterations in inspired fractional O2 concentrations) insults. Control experiments demonstrated that the sensors can function and remain stable in an in vivo environment. Intestinal PTo2 decreased following superior mesenteric artery occlusion and with reductions in inspired O2 concentrations. These results were reversible after reinstating blood flow or by increasing inspired O2 concentrations. We have successfully developed an anesthetized rat intestinal ischemic and hypoxic model for validation of a miniaturized O2 sensor to provide real-time measurement of intestinal PTo2. Our results support further validation of the sensors in physiological conditions using a large animal model to provide evidence of their use in clinical applications where monitoring visceral surface tissue O2 tension is important.NEW & NOTEWORTHY This is the first report of real-time continuous measurements of intestinal oxygen tension made using a microfabricated O2 sensor. Using a developed rodent model, we have validated this sensor's ability to accurately measure dynamic and reversible changes in intestinal oxygenation that occur through ischemic and hypoxemic insults. Continuous monitoring of local intestinal oxygenation could have value in the postoperative monitoring of patients having undergone intestinal surgery.

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.

Horizon scanning implanted biosensors in personalising breast cancer management: First pilot study of breast cancer patients views.

AIMS: This study aimed to explore breast cancer patients' understanding and acceptability of implanted biosensors (BS) within the primary tumour to personalise adjuvant radiotherapy, and to determine optimal design and number of BS, and evaluate potential clinical benefits as well as concerns about tolerance, toxicity, dwell time, and confidentiality of data. PATIENTS AND METHODS: A total of 32 patients treated by surgery (29 breast conserving, 3 mastectomy), postoperative radiotherapy and systemic therapy for early breast cancer, were recruited from a posttreatment radiotherapy clinic at a cancer centre. Patients participated in semistructured interviews. Interview transcripts were analysed using qualitative methods. RESULTS: Participants were aged 39 to 87 years, with a median age of 62 years. Most (N = 23[72%]) were unfamiliar with biosensors. The majority (N = 29[90.6%]) were supportive of the technology's potential use in future breast cancer treatment and were willing to accept biosensors (N = 28[88%]) if they were endorsed by their breast cancer consultant. Only 3 patients expressed concerns, predominantly about uncertainties on their role in the diagnostic and treatment pathway. Patients were flexible about the size and shape of BS, but had a preference for small size (N = 28 [87.5%]). Most (N = 22[69%]) would accept implantation of more than 5 BS and were flexible (N = 22[69%]) about indefinite dwell time. Patients had a strong preference for wireless powering of the BS (N = 28[87.5%]). Few had concerns about loss of confidentiality of data collected. All patients considered biosensors to be potentially of important clinical benefit. CONCLUSIONS: While knowledge of biosensors was limited, patients were generally supportive of biosensors implanted within the primary tumour to collect data that might personalise and improve breast cancer radiotherapy in future.

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.

Investigating parylene-HT as a substrate for human cell patterning.

We demonstrate, for the first time, how parylene-HT on SiO2 substrates can be used as a human cell patterning platform. We demonstrate this platform with hNT astrocytes, derived from the human NTera2.D1 cell line. We show how hNT astrocytes are attracted to Parylene-HT and repelled by the SiO2 and are shown to adopt a similar morphology as that attained on standard tissue culture polystyrene. Furthermore, parylene-HT was capable of patterning the astrocytes achieving a ratio of 8:1 for cells on parylene compared to SiO2. Thus, as parylene-HT has similar physical properties to parylene-C with the addition of UV and thermal resistance, parylene-HT represents a desirable alternative substrate for human cell patterning.

Towards a 'siliconeural computer': technological successes and challenges.

Electronic signals govern the function of both nervous systems and computers, albeit in different ways. As such, hybridizing both systems to create an iono-electric brain-computer interface is a realistic goal; and one that promises exciting advances in both heterotic computing and neuroprosthetics capable of circumventing devastating neuropathology. 'Neural networks' were, in the 1980s, viewed naively as a potential panacea for all computational problems that did not fit well with conventional computing. The field bifurcated during the 1990s into a highly successful and much more realistic machine learning community and an equally pragmatic, biologically oriented 'neuromorphic computing' community. Algorithms found in nature that use the non-synchronous, spiking nature of neuronal signals have been found to be (i) implementable efficiently in silicon and (ii) computationally useful. As a result, interest has grown in techniques that could create mixed 'siliconeural' computers. Here, we discuss potential approaches and focus on one particular platform using parylene-patterned silicon dioxide.

Cell patterning on photolithographically defined parylene-C: SiO2 substrates.

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based 'lab on a chip' technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2 wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Patterning human neuronal networks on photolithographically engineered silicon dioxide substrates functionalized with glial analogues.

Interfacing neurons with silicon semiconductors is a challenge being tackled through various bioengineering approaches. Such constructs inform our understanding of neuronal coding and learning and ultimately guide us toward creating intelligent neuroprostheses. A fundamental prerequisite is to dictate the spatial organization of neuronal cells. We sought to pattern neurons using photolithographically defined arrays of polymer parylene-C, activated with fetal calf serum. We used a purified human neuronal cell line [Lund human mesencephalic (LUHMES)] to establish whether neurons remain viable when isolated on-chip or whether they require a supporting cell substrate. When cultured in isolation, LUHMES neurons failed to pattern and did not show any morphological signs of differentiation. We therefore sought a cell type with which to prepattern parylene regions, hypothesizing that this cellular template would enable secondary neuronal adhesion and network formation. From a range of cell lines tested, human embryonal kidney (HEK) 293 cells patterned with highest accuracy. LUHMES neurons adhered to pre-established HEK 293 cell clusters and this coculture environment promoted morphological differentiation of neurons. Neurites extended between islands of adherent cell somata, creating an orthogonally arranged neuronal network. HEK 293 cells appear to fulfill a role analogous to glia, dictating cell adhesion, and generating an environment conducive to neuronal survival. We next replaced HEK 293 cells with slower growing glioma-derived precursors. These primary human cells patterned accurately on parylene and provided a similarly effective scaffold for neuronal adhesion. These findings advance the use of this microfabrication-compatible platform for neuronal patterning.

Parapapillary atrophy and optic disc region assessment (PANDORA): retinal imaging tool for assessment of the optic disc and parapapillary atrophy.

We describe a computer-aided measuring tool, named parapapillary atrophy and optic disc region assessment (PANDORA), for automated detection and quantification of both the parapapillary atrophy (PPA) and the optic disc (OD) regions in two-dimensional color retinal fundus images. The OD region is segmented using a combination of edge detection and ellipse fitting methods. The PPA region is identified by the presence of bright pixels in the temporal zone of the OD, and it is segmented using a sequence of techniques, including a modified Chan-Vese approach, thresholding, scanning filter, and multiseed region growing. PANDORA has been tested with 133 color retinal images (82 with PPA; 51 without PPA) drawn randomly from the Lothian Birth Cohort (LBC) database, together with a "ground truth" estimate from an ophthalmologist. The PPA detection rate is 89.47% with a sensitivity of 0.83 and a specificity of 1. The mean accuracy in defining the OD region is 81.31% (SD=10.45) when PPA is present and 95.32% (SD=4.36) when PPA is absent. The mean accuracy in defining the PPA region is 73.57% (SD=11.62). PANDORA demonstrates for the first time how to quantify the OD and PPA regions using two-dimensional fundus images, enabling ophthalmologists to study ocular diseases related to PPA using a standard fundus camera.

Spike-timing-dependent plasticity with weight dependence evoked from physical constraints.

Analogue and mixed-signal VLSI implementations of Spike-Timing-Dependent Plasticity (STDP) are reviewed. A circuit is presented with a compact implementation of STDP suitable for parallel integration in large synaptic arrays. In contrast to previously published circuits, it uses the limitations of the silicon substrate to achieve various forms and degrees of weight dependence of STDP. It also uses reverse-biased transistors to reduce leakage from a capacitance representing weight. Chip results are presented showing: various ways in which the learning rule may be shaped; how synaptic weights may retain some indication of their learned values over periods of minutes; and how distributions of weights for synapses convergent on single neurons may shift between more or less extreme bimodality according to the strength of correlational cues in their inputs.

Controlled adhesion and growth of long term glial and neuronal cultures on Parylene-C.

This paper explores the long term development of networks of glia and neurons on patterns of Parylene-C on a SiO(2) substrate. We harvested glia and neurons from the Sprague-Dawley (P1-P7) rat hippocampus and utilized an established cell patterning technique in order to investigate cellular migration, over the course of 3 weeks. This work demonstrates that uncontrolled glial mitosis gradually disrupts cellular patterns that are established early during culture. This effect is not attributed to a loss of protein from the Parylene-C surface, as nitrogen levels on the substrate remain stable over 3 weeks. The inclusion of the anti-mitotic cytarabine (Ara-C) in the culture medium moderates glial division and thus, adequately preserves initial glial and neuronal conformity to underlying patterns. Neuronal apoptosis, often associated with the use of Ara-C, is mitigated by the addition of brain derived neurotrophic factor (BDNF). We believe that with the right combination of glial inhibitors and neuronal promoters, the Parylene-C based cell patterning method can generate structured, active neural networks that can be sustained and investigated over extended periods of time. To our knowledge this is the first report on the concurrent application of Ara-C and BDNF on patterned cell cultures.