Piezoelectric microsensors for semiochemical communication

Chemical communication plays vital role in the mediating the behaviour of an organism living in the “odour space”. The mechanisms by which odours are generated and detected by the organism has evolved over thousands of years and thus the potential advantages of translating this system into a fully functional communication system has opened new avenues in the area of multi-disciplinary research. This formed the basis of the Biosynthetic Infochemical Communications project – iCHEM whose central aim was to develop a new class of communication technology based on the biosynthesis pathways of the moth, S. littoralis. This novel infochemical communication system would consist of a “chemoemitter” unit which would generate a precise mix of infochemicals which after travelling through the odour space would be detected by a complementary tuned detector – the “chemoreceiver” unit comprising of a ligand specific detection element and an associated biophysical model functioning similar to the antennal lobe neuron of the moth. This combined novel system will have the capability of communicating by the help of chemicals only, in the vapour or liquid phase. For the work presented in this thesis, the novel concept of infochemical communication has been examined in the vapour and liquid phase by employing piezoelectric microsensors. This has been achieved and demonstrated throughout the thesis by employing chemo-specific acoustic wave microsensors. For vapour phase assessment, quartz crystal microbalance, were coated with different organic polymer coatings and incorporated in a prototype infochemical communication system detecting encoded volatiles. For liquid phase assessment, shear horizontal surface acoustic wave (SH-SAW) microsensors were specifically designed and immobilised within Sf9 insect cells. This GPCR based whole cell biosensing system was then employed to detect ligand specific activations thus acting as a precursor to the development of a fully functionalised OR based signalling system, thus contributing to the growing field of communication and labelling technology

Towards a cell-based chemo receiver for artificial insect olfaction

Infochemical communication is ubiquitous amongst all living organisms, and particularly important in insects. Because smell being the most common basic means of chemical communication, infochemical blends must be constantly decoded in order to proclaim their readiness to mate, to mark out territorial boundaries, to warn off intruders and predators or, in some cases, to locate food or predators with millisecond precision. The central challenge of the thesis was to mimic nature in both cellular and molecular levels on to a technological platform that aids in the development of a new class of technology employing chemicals alone to communicate over space and time. This thesis describes a body of work conducted in the development of a miniaturised, smart and label-free cell-based chemoreceiver for artificial insect olfaction, as part of the development of a novel biomimetic infochemical communication system. A surface acoustic wave based microsensor has been utilized to engineer and develop a chemoreceiver system that mimics the cellular and molecular mechanisms occurring during infochemical detection and decoding in insects. Successful recovery of ratiometric information with the aid of polymer-based gas-phase measurements, established the concept of chemical communication. Thus, small scale, high-throughput infochemical communication has been realized by a combination of precise spatiotemporal signal generation using fruit volatiles and insect sex pheromones with highly sensitive detection and signal processing. This was followed by the investigation of the feasibility of using the prototype cell-based biosensor system in a static mode for artificial insect olfaction applications, mimicking the cellular detection in the receptor/antenna apparatus of insects. Finally, as part of the development of a compact and low-power portable chemoreceiver system, the discrete sensor drive and interface circuitry was deployed in an analogue VLSI chip, thereby overcoming the associated measurement complexity and equipment cost, in addition to extending the reach and functionality of point of use technologie

Novel Biosensing Approaches for Detection of Exosomal Proteins

Exosomes are endocytic lipid membrane-bound bodies that have been shown to carry proteins associated with cancer and neurodegenerative disease. This has led to an exponential rise in research that looks to incorporate exosomal proteins as disease biomarkers within diagnostic assays. Furthermore, ubiquitous presence of exosomes in nearly all biological fluids creates the possibly of minimally invasive liquid biopsies for the patient. However, the heterogeneity of exosomes and complexity of biological source materials requires a consideration of optimal isolation protocols. More importantly, the development of effective exosome based assays is limited by the scarcity of translational characterization approaches that are capable of determining their molecular composition and physical properties in physiological fluids. The key objectives of this doctoral research was to establish a robust exosome isolation protocol from complex media, prior to sensing the exosomes on an immunosensor transduced by acoustic wave and electrochemical measurements. This work also looked to enhance these platforms from their current baseline performance, through the implementation of various surface structure modifications. A size exclusion chromatography approach was developed for the isolation of exosomes expressing CD63, Alix, CD81 and CD9 proteins, and allowed them to be extracted effectively from cell culture media, human serum and urine. Isolated exosomes were subsequently detected on a quartz crystal microbalance with dissipation (QCM-D) monitoring, after the optimisation of an affinity-based immunofunctionalisation approach. This technique displayed high sensitivity and specificity towards exosomal CD63 at clinically relevant concentrations in complex media. The QCM-D sensor was also used as a working electrode, as part of an electrochemical cell, to enable additional impedance spectroscopy analysis of exosome binding in tandem with the QCM-D response, collectively termed EQCM-D. The combination of these approaches offers a label-free, sensitive and real-time approach to exosome detection. The sensitivity of the EQCM-D platform was improved through surface formation of tuneable gold nanoparticle arrays from selective impregnation of block-copolymer templates, taking advantage of their segregation behaviour. This presented a versatile approach to tune sensor surfaces in order to improve ligand orientation and subsequent analyte binding. Similar advancements were made on silica detection surfaces through the formation of silica inverse opal crystals, with differing thicknesses, using a single-step co-assembly approach that combines a sol-gel matrix with poly(methyl methacrylate) (PMMA) spheres. Porous networks atop the sensors increased the internal surface area significantly, translating to a higher binding capacity of exosomes notwithstanding a higher degree of artefact entrapment. The results achieved through this work offer a potential for multi-modal analysis of exosomal proteins in diagnostics, underpinned by acoustic wave methodologies and nanostructured materials

The evaluation of an optical biosensor for at-line monitoring and control of a bioprocess

The evaluation of an optical biosensor to produce real-time data regarding the specific product of interest, which may ultimately permit feed-back control, is discussed. Improvement in the understanding of many bioprocess operations may benefit greatly from the availability of such data. The ability to make control decisions based upon specific product data could lead to increased levels of productivity and sample integrity. The optical biosensor, which utilises a resonant mirror to monitor biomolecular interactions, was configured to permit the monitoring of the recombinant antibody fragment, D1.3 Fv. The Fv fragment is derived from the monoclonal D1.3, which is specific for hen egg lysozyme (HEL) and is able to discriminate turkey egg lysozyme (TEL). During operation both HEL and TEL were immobilised to the surfaces of two biosensor devices, permitting their use to monitor D1.3 Fv during its expression by Escherichia coli. It was demonstrated that HEL provided specific data, whilst TEL provided an appropriate control response. The combined response towards each surface permitted quantification of D1.3 Fv within fermenter broths. Analysis of biosensor data, which had traditionally been performed as an off-line process using either exponential curve fitting or linearisation routines (derivative analysis) to determine the kinetic constants describing the biomolecular interaction under investigation, was shown to be too cumbersome where data was required in real-time. An alternative analytical method based on linear regression of the initial binding curve data was developed, which has been demonstrated off-line, to produce analytical results from biosensor data typically less than 5 seconds after sample addition. Using the routine based upon linear regression the dynamic response of the sensor for purified D1.3 Fv was shown to cover the range 0.2 to 150 μg mL -1, which encompasses the total bioprocess for the production of D 1.3 Fv. The sensor has been demonstrated to provide specific product data during the course of fermentation, indicating the presence of product and permitting the production of a profile depicting the onset of product formation as well the maximum titre. Based upon such data decisions regarding the fate of the fermentation may be taken, for example indicating the optimal point to harvest in order to achieve maximum yields. Preliminary data also indicate the potential of the sensor to monitor the purification of D1.3 Fv by immuno-affinity chromatography. Highlighting such events as column saturation and indicating product location within the eluted fractions

Development and characterisation of affinity devices for cell detection and separation

This thesis seeks to demonstrate technologies capable of improving the purity of pluripotent-derived cells. Such cells offer an enormous opportunity for medical science. However, it is anticipated that a higher proportion of undifferentiated pluripotent cells will remain when manufacturing at scale. As indicated in the literature, the in vivo transplantation of undifferentiated cells is a threat to patient safety and is considered a limiting factor for large-scale manufacture. Pluripotent undifferentiated cells can be identified and removed based on an affinity interaction with the SSEA-4 antigen, which is down-regulated on differentiated cells. Using CD20+ and CD20- (HLA-A2+) lymphocyte cells as a cost-effective alternative, where the CD20+ cells are target impurities and the HLA-A2+ cells are product-designated cells, this thesis presents two technologies to minimise the proportion of undifferentiated cells during manufacture. These technologies rely upon shear stress-induced affinity separation to differentiate between cells with and without target antigens. A small-scale model is used to identify a range of shear stresses (0 25 dynes/cm2) with which these differences can be elucidated. These technologies are: (1) a quartz crystal microbalance (QCM) biosensor to detect pluripotent cell differentiation over a multi-day period, particularly during process development. The limit of quantitation (LoQ) was estimated to be 5,000 cells, which would enable the measurement of target cell purities in excess of 4 %. Findings provide the basis for such a system, but also highlight the technical challenges of development, in particular variability. (2) two affinity membranes (hollow fibre and flat sheet) were used to deplete cells positive for the target antigen, demonstrating a possible downstream-purification tool in instances where clinical purity does not suffice. The shear stress-induced detachment of adsorbed cells incubated for 30 minutes was investigated over 1 25 dynes/cm2 and 1 10 dynes/cm2 for the hollow fibre and flat sheet membranes respectively. Measured output included cell purity, which showed an increase in the relative change in purity (RCP) of 0.2 0.5 for the hollow fibre modules at 5 dynes/cm2 (n = 5) and was as high as 11.8 at 10 dynes/cm2 for the flat sheet membrane cassette (n = 1). A loss of cell membrane integrity, where up to 5 % in hollow fibres and up to 55 % in the flat sheet membrane were observed

Fundamentals of SARS-CoV-2 Biosensors

COVID-19 diagnostic strategies based on advanced techniques are currently essential topics of interest, with crucial roles in scientific research. This book integrates fundamental concepts and critical analyses that explore the progress of modern methods for the detection of SARS-CoV-2

The development of a novel native prothrombin assay for the more efficient management of oral anticoagulation therapy

Disturbances of the natural balance between pro-coagulant and anticoagulant systems, due to hereditary or acquired factors may result in haemorrhagic or thrombotic diseases. Currently the INR/ISI coagulation monitoring system, introduced in 1983 by the World Health Organisation (WHO), is that of choice for most anticoagulation management clinics. Patients undergoing anticoagulation therapy must regularly attend specialised outpatient clinics for the close monitoring and maintenance of their INR. The automated laboratory caters for rapid online simultaneous analysis of multiple blood samples, resulting in the calculation of a patient’s INR from the recorded prothrombin time. The insensitivity of the prothrombin time test has been well documented, requiring a reduction in the prothrombin concentration of 45 % prior to the materialisation of clinically significant prothrombin times. The project aims to employ three avenues of biotechnology to aid in the development of an immuno or molecular imprinted polymer (MIP) based anticoagulation assay. The project will utilise computational molecular modelling in an attempt to visualise the tertiary structure of human prothrombin, which will allow the rational selection of antigenic sites for molecular imprinting or antibody production. An aqueous, prothrombin-imprinted homo-polymer was grafted to the gold surface of a surface plasmon resonance biosensor (Biacore). The Biacore allowed the real time monitoring of imprinted polymer binding characteristics to i homogeneous protein solutions. As a direct comparison of two technologies, molecular imprinting and immuno technology, polyclonal antibodies showing specificity towards the same prothrombin antigen were immobilised onto Biacore chips. The imprinted polymer graft and polyclonal antibody based assays recognised homogeneous solutions of prothrombin at a concentration range of 0.01 nM to 14.2 nM and 0.01 nM to 0.5 nM respectively. A randomised preliminary clinical trial was initiated to compare the two assays’ ability to differentiate plasma samples with a variety of INR values. The results thus far show promise for the development of a new anticoagulation assay using molecularly imprinted polymer technology. The ultimate aim for this project is to develop a consistently more accurate point-of-care anticoagulation therapy monitoring kit, incorporating this new technology, which can replace or be used concomitantly with the INR/ISI system currently in use. This thesis raises more questions regarding the efficacy of oral anticoagulation therapy (OACT) and argues for and against the necessity of a novel OACT management assay.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Development of electrochemical assays and biosensors for detection of Zika virus

2019 Spring.Includes bibliographical references.Zika virus (ZIKV) emerged as a significant public health concern after the 2015-2016 outbreak in South and Central America. Severe neurological complications and birth defects in adults and children respectively underscore the need for quick and accurate diagnosis so that proper medical observation and intervention can be done. Electrochemical assays and biosensors are attractive as alternative diagnostic tools due to their sensitivity and ease of miniaturization. This dissertation describes three novel electrochemical assays and biosensors to detect ZIKV specific nucleic acid, antibodies, and virus particles. A nuclease protection ELISA (NP-ELISA) was developed for nucleic acid detection by enzymatic readout. The assay was validated using synthetic complementary oligos for absorbance, chemiluminescence, and electrochemical enzymatic readout. Two horseradish peroxidase substrates, 3,3',5,5'-Tetramethylbenzidine (TMB) and hydroquinone, were characterized electrochemically and compared for electrochemical assay use. Electrochemical TMB readout demonstrated better sensitivity compared to all tested detection modalities with a limit of detection of 3.72×103 molecules mL-1, which compares well to the amount of ZIKV RNA in clinical samples and to other approved assays like the CDC's Trioplex assay. For serological analysis, a capacitive microwire biosensor was developed and validated using immunized mouse sera to detect a ZIKV antibody response. Measurements were taken through a wide serial dilution range of 1:1018 to 1:103 and two dilutions (1:1012 and 1:106) were used for analysis for optimal sensitivity. A statistically significant immune response was detected four days after immunization at a 1:1012 dilution and was specific for ZIKV when compared with Chikungunya virus (CHIKV). These results indicate that serological analysis can be performed four days earlier with the wire sensor compared to ELISAs using ultra-dilute samples. The sensor also was used to differentiate between IgG and IgM antibodies and compared well with ELISA results. Lastly, an impedance array sensor was designed and validated for detection of ZIKV particles. The array allows for simultaneous handling of many electrodes, which increases throughput compared to other biosensor designs. The sensor demonstrated good sensitivity with an LOD of 22.4 focus forming units (FFU) which compares well to other reported sensors. In addition, it was optimized for specificity and tested using Sindbis virus (SINV) as a negative control. These novel platforms comprise new advancements in biosensor technology by simplifying existing assays, increasing sensitivity, and providing a new platform for handheld measurements

Development and application of affinity-mass spectrometry to identify protein-carbohydrate interactions

Affinity mass spectrometry using selective and pressure-enhanced proteolytic excision and extraction combined with MALDI and ESI-MS has been applied to the identification of epitope binding sites of GalNac and blood group oligosaccharides in lectins. The epitope peptides identified comprise all essential amino acids involved in carbohydrate recognition, in complete agreement with available X-ray structures. Coupling of a specific peptide glycoprobe to the gold surface of a biosensor chip was successfully employed for identification of carbohydrate epitopes and affinity measurements.Die druckunterstützte proteolytische Exzision, kombiniert mit MALDI und ESI-MS, wurde zur Identifizierung der Epitop-Bindungsstellen von GalNac und Blutgruppen-Oligosacchariden in Lektinen eingesetzt. Die identifizierten Epitoppeptide umfassen alle wesentlichen Aminosäuren, die an der Kohlenhydraterkennung beteiligt sind, und stimmen vollständig mit den verfügbaren Röntgenstrukturen überein. Die Kopplung einer spezifischen Peptid-Glykoprobe an die Goldoberfläche eines Biosensorchips wurde erfolgreich für die Identifizierung von Kohlenhydrat-Epitopen und Affinitätsmessungen eingesetzt

Selected Papers from the 1st International Electronic Conference on Biosensors (IECB 2020)

The scope of this Special Issue is to collect some of the contributions to the First International Electronic Conference on Biosensors, which was held to bring together well-known experts currently working in biosensor technologies from around the globe, and to provide an online forum for presenting and discussing new results. The world of biosensors is definitively a versatile and universally applicable one, as demonstrated by the wide range of topics which were addressed at the Conference, such as: bioengineered and biomimetic receptors; microfluidics for biosensing; biosensors for emergency situations; nanotechnologies and nanomaterials for biosensors; intra- and extracellular biosensing; and advanced applications in clinical, environmental, food safety, and cultural heritage fields