Analytical technology for cleaning verification and analysis of drug purity in pharmaceutical production.

Production of pharmaceuticals is a complex process which goes beyond the synthetic reactions undertaken to produce the final drug product. In multi purpose manufacturing facilities the equipment used in the processes must be proven to be sufficiently free of residue from the previous compound so that safe manufacture of the next compound can commence. Cross contamination can pose serious health risks so cleaning verification is a process of extreme importance. Pharmaceutical products may contain impurities that originate from the synthetic stages of production, the starting materials or from in situ reactions taking place in the final drug. Some of these impurities may be genotoxic. Genotoxic impurities are a class of impurities for which awareness is currently growing in the pharmaceutical industry. Traditional analytical chemistry methods such as HPLC are currently employed for the detection and quantification of cleaning residues and genotoxic impurities. These methods can be extremely time consuming. The aims of this project are to investigate swab sampling in cleaning verification, to develop alternative analytical methods which speed up and enhance knowledge of the cleaning verification process and to begin the development of an assay system for commonly occurring genotoxic impurities

A review of microfabricated electrochemical biosensors for DNA detection

This review article presents an overview of recent work on electrochemical biosensors developed using microfabrication processes, particularly sensors used to achieve sensitive and specific detection of DNA sequences. Such devices are important as they lend themselves to miniaturisation, reproducible mass-manufacture, and integration with other previously existing technologies and production methods. The review describes the current state of these biosensors, novel methods used to produce them or enhance their sensing properties, and pathways to deployment of a complete point-of-care biosensing system in a clinical setting

Electrochemical detection of oxacillin resistance with SimpleStat : a low cost integrated potentiostat and sensor platform

Testing outside the laboratory environment, such as point of care testing, is a rapidly evolving area with advances in the integration of sample handling, measurement and sensing elements widely reported. Low cost, simple to use systems are important in this context because they provide a route to devices that can be used outside the laboratory and could be implemented in low resource settings where advanced diagnostic testing is often unavailable. Here, we present an open source highly simplified electrochemical platform, called SimpleStat, that has been programmed to perform differential pulse voltammetry and can be used to detect the presence of OXA-1 DNA sequences for oxacillin resistance. This DNA sensor can be used to specifically detect the presence of the OXA-1 gene, contrasted to the tetA gene which encodes for tetracycline resistance. These measurements were performed with both polycrystalline gold electrodes as a benchmark and electrodes integrated into the SimpleStat printed circuit

An electrochemical comparison of thiolated self‐assembled monolayer (SAM) formation and stability in solution on macro‐ and nanoelectrodes

Thiolated self-assembled monolayers (SAMs) formed on metal electrodes have been a topic of interest for many decades. One of the most common applications is in the field of biosensors, where this is a growing need for functionalising nanoelectrodes to realise more sensitive and implantable sensors. For all these applications the SAM functionalised nanoelectrodes will need to make reliable and interpretable electrochemical measurements. In this work, Electrochemical Impedance Spectroscopy (EIS) is used to monitor both the formation and subsequent stability of 6-mercaptohexan-1-ol SAMs on macro and nanoelectrodes and compares the two. To develop effective devices, it is crucial to understand both SAM formation and the resulting signal stability on nanoscale surfaces and this is done by comparing to behaviours observed at the well understood macroscale. We report an initial stochastic binding event and subsequent re-arrangement of the SAMs for both electrode types. However, this re-arrangement takes hours on the macro scale electrodes but only seconds on the nanoelectrodes. This is proposed to be due to the different structure of the SAMs on the electrodes predominantly driven by their bulk to edge ratios. After formation, the SAMs formed on both macro and nanoelectrodes exhibit significant instability over time. The reported results have practical implications for the construction of SAM based biosensors on macro and nanoscale electrodes

Development of electrochemical DNA detection methods to measure circulating tumour DNA for enhanced diagnosis and monitoring cancer

Liquid biopsies are becoming an increasingly important potential replacement for existing biopsy procedures which can be invasive, painful and compromised by tumour heterogeneity. This paper reports a simple electrochemical approach tailored towards point of care cancer detection and treatment monitoring from biofluids using a label free detection strategy. The mutations under test were the KRAS G12D and KRAS G13D mutations which are both important in the development and progression of many human cancers and whose presence correlates with poor outcomes. These common circulating tumour markers were investigated in clinical samples and amplified by standard and specialist PCR methodologies for subsequent electrochemical detection. Following pre-treatment of the sensor to give a clean surface, DNA probes developed specifically for detection of the KRAS G12D and G13D mutations were immobilized onto low cost carbon electrodes using diazonium chemistry and EDC/NHS coupling. Following functionalisation of the sensor it was possible to sensitively and specifically detect mutant KRAS G12D and G13D PCR product from cancer patients against a background of wild type KRAS DNA from the representative sample. Our findings give rise to the basis of a simple and very low cost system for measuring ctDNA biomarkers in patient samples. The current time to result of the system is 4.5 hours with considerable scope for optimisation and already compares favourably to the UK National Health Service biopsy service where patients can wait weeks for their result. The paper will report the technical developments we have made in the production of clean carbon surfaces for functionlisation, show the assay performance data for KRAS G12D and G13D with a range of PCR systems and demonstrate the potential for measuring response to treatment offered by the system

Developments in microscale and nanoscale sensors for biomedical sensing

The widespread use of point of care testing in biomedical and clinical applications is a major aim of the electrochemical field. A large number of groups are working on lab-on-a-chip systems or sensor arrays which are underpinned by electrochemical detection methodologies. Miniaturized transducers have the potential to be adopted in such systems for diagnosis of a range of diseases in both clinical and nonclinical settings. In this review, we will present the current trends and state of the art for a selection of miniaturized sensing elements (microelectrodes, nanoelectrodes, and field-effect transistors) and provide an impression of current technologies, their associated performance characteristics, and also considering the major barriers to adoption and how they might be surmounted in future so these technologies can fulfil their early promise

Effect of microchannel dimensions in electrochemical impedance spectroscopy using gold microelectrode

Microfluidic chip systems have been an area of interest for lab-on-a-chip and organ-on-a-chip studies in recent years. These chips have many advantages such as high efficiency, low sample consumption, fast analysis, durability and low cost. Today, electrochemical sensors are frequently applied in microfluidic chips because of their potential for label-free detection and low-cost production. A commonly employed electrochemical technique is electrochemical impedance spectroscopy (EIS), which captures changes in phase and amplitude as signal passes through the system under test. In the utilization of microelectrodes within microfluidic channels, noise becomes a problem in EIS measurements. In this study, EIS measurements were performed using microfluidic chips with various dimensions of width while the properties and dimensions of the microelectrodes were kept constant. It was found that the results of cyclic voltammetry (CV) cleaning and EIS experiments deteriorated when smaller than 1 mm wide-microchannels were integrated onto 100 µm wide microelectrodes. These finding sets the basics for on-chip electrochemistry experiments using microfluidic integrated microelectrodes and therefore is fundamentally important in future on-chip EIS measurements

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 ssDNA 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

Fabrication of a graphite-paraffin carbon paste electrode and demonstration of its use in electrochemical detection strategies

Electrochemical detection methods hold many advantages over their optical counterparts, such as operation in complex sample matrices, low-cost and high volume manufacture and possible equipment miniaturisation. Despite these advantages, the use of electrochemical detection is currently limited in the clinical setting. There is a wide range of potential electrode materials, selected for optimal signal-to-noise ratios and reproducibility when detecting target analytes. The use of carbon paste electrodes (CPEs) for electrochemical detection can be limited by their analytical performance, however they remain very attractive due to their low cost and biocompatibility. This paper presents the fabrication of an easy-to-make and use graphite powder/paraffin wax paste combined with a substrate produced via additive manufacturing and confirms its functionality for both direct and indirect electrochemical measurements. The produced CPEs enable the direct voltammetric detection of hexaammineruthenium(III) chloride and dopamine at an experimental limit of detection (ELoD) of 62.5 µM. The key inflammatory biomarker Interleukin-6 through an enzyme-linked immunosorbant assay (ELISA) was also quantified, yielding a clinically-relevant ELoD of 150 pg/ml in 10% human serum. The performance of low-cost and easy-to-use CPEs obtained in 0.5 hours is showcased in this study, demonstrating the platform’s potential uses for point-of-need electroanalytical applications

Accelerating the development of implantable neurochemical biosensors by using existing clinically applied depth electrodes

In this study, an implantable stereo-electroencephalography (sEEG) depth electrode was functionalised with an enzyme coating for enzyme-based biosensing of glucose and L-glutamate. This was done because personalised medicine could benefit from active real-time neurochemical monitoring on small spatial and temporal scales to further understand and treat neurological disorders. To achieve this, the sEEG depth electrode was characterised using cyclic voltammetry (CV), differential pulse voltammetry (DPV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS) using several electrochemical redox mediators (potassium ferri/ferrocyanide, ruthenium hexamine chloride, and dopamine). To improve performance, the Pt sensors on the sEEG depth electrode were coated with platinum black and a crosslinked gelatin-enzyme film to enable enzymatic biosensing. This characterisation work showed that producing a useable electrode with a good electrochemical response showing the expected behaviour for a platinum electrode was possible. Coating with Pt black improved the sensitivity to H2O2 over unmodified electrodes and approached that of well-defined Pt macro disc electrodes. Measured current showed good dependence on concentration, and the calibration curves report good sensitivity of 29.65 nA/cm2/μM for glucose and 8.05 nA/cm2/μM for L-glutamate with a stable, repeatable, and linear response. These findings demonstrate that existing clinical electrode devices can be adapted for combined electrochemical and electrophysiological measurement in patients and obviate the need to develop new electrodes when existing clinically approved devices and the associated knowledge can be reused. This accelerates the time to use and application of in vivo and wearable biosensing for diagnosis, treatment, and personalised medicine