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

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

Morpholino oligonucleotides in responsive hydrogels for microRNA sensing

In recent years, microRNA (miRNA) has garnered a high level of interest in the field of biosensor development. MiRNA are a class of small, circulating RNA sequences that are essential for healthy control of protein expression. The variation of levels of specific miRNAs has been linked with over 150 diseases since the turn of the millennium, including cancers, cardiovascular diseases, parasitic infections and neurological disorders. The improved prognosis from early detection is stark, but established methods of miRNA detection suffer from poor sensitivity, low throughput, and require specialised laboratory equipment and trained staff to perform the time-consuming techniques. A simple, cheap and sensitive miRNA point of care sensor would be an invaluable tool in healthcare. This thesis presents the continued optimisation of a miRNA sensing hydrogel with oligonucleotide crosslinks that are selectively cleaved in the presence of the target miRNA sequence. This selective reduction in crosslink density was transduced by a change in the swelling profile of the hydrogel and intelligent crosslink design used to control the swelling response for detecting a miRNA sequence, a short RNA (sRNA) sequence, or a small molecule using an aptamer. Morpholino oligonucleotides (MOs), an uncharged DNA analogue, were functionalised with an acrylamide moiety and used as responsive crosslinks for miRNA sequence detection in a world’s first MO crosslinked hydrogel. The MO crosslinks offered significant improvements over DNA crosslinked hydrogels through improved thermal stability, no salt requirement and 1000-fold improved sensitivity, facilitating a wider range of sensing conditions. Analysis was also achieved using a mobile phone camera and laptop, demonstrating portability. Carbon nanoparticles (CNP) were suspended in the hydrogels to act as a conductive component. As the hydrogel swells the distance between the particles is increased until there is no conductive pathway, resulting in an increase in the hydrogel’s resistance. Numerous reproducibility challenges were identified with regards to gel delamination and CNP leaching partly due to inefficient UV photoinitiation of the pigment composite pre-gel solution. SEM imaging identified inconsistent composite homogeneity with areas of higher CNP and gel density in DNA crosslinked composites, while MO crosslinked composites were homogenous and less conductive. Inkjet printing of the composite material using an electrostatic dispersion as the conductive component was made possible using MO crosslinks with no salt and ammonium persulfate with TEMED in place of UV initiation. Optimised synthesis resulted in homogenous conductive composites far more robust and reproducible than the UV initiated CNP composite. However, MO solution viscosity resulted in improper aspiration and inaccurate deposition. Potential solutions and improvements are suggested, facilitated by the improvements offered by MO crosslinks

Cellulose-Based Biosensing Platforms

Cellulose empowers measurement science and technology with a simple, low-cost, and highly transformative analytical platform. This book helps the reader to understand and build an overview of the state of the art in cellulose-based (bio)sensing, particularly in terms of the design, fabrication, and advantageous analytical performance. In addition, wearable, clinical, and environmental applications of cellulose-based (bio)sensors are reported, where novel (nano)materials, architectures, signal enhancement strategies, as well as real-time connectivity and portability play a critical role

Novel Electrochemical Biosensors for Clinical Assays

Biosensors, i.e., devices where biological molecules or bio(mimetic)structures are intimately coupled to a chemo/physical transducer for converting a biorecognition event into a measurable signal, have recently gained a wide (if not huge) academic and practical interest for the multitude of their applications in analysis, especially in the field of bioanalysis, medical diagnostics, and clinical assays. Indeed, thanks to their very simple use (permitting sometimes their application at home), the minimal sample pretreatment requirement, the higher selectivity, and sensitivity, biosensors are an essential tool in the detection and monitoring of a wide range of medical conditions from glycemia to Alzheimer’s disease as well as in the monitoring of drug responses. Soon, we expect that their importance and use in clinical diagnostics will expand rapidly so as to be of critical importance to public health in the coming years. This Special Issue would like to focus on recent research and development in the field of biosensors as analytical tools for clinical assays and medical diagnostics

2D and 3D optical imaging of SERS nanotags intracellularly

Adoption of a multi-marker nanotag approach will led to better disease characterisation whilst simultaneously enabling targeting of multiple disease markers or organelles. The employed nanotag method controllably aggregated nanoparticles with 1,6-hexamethylene diamine (1,6-HMD), before polymer coating with polyvinylpyrrolidone (PVP) and labelling with small molecule reporters; 4-mercaptopyridine (MPY), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 4-nitrobenzenethiol (NBT) and 2-naphthalenethiol (2-NPT). Within a multiple component suspension reporters were identified by their unique peak and when present within single cells or populations they were additionally identified using component direct classical least squares (DCLS). Within a single cell three of the four components (MPY, DTNB and NBT) were positively identified. 2D SERS imaging can monitor nanotag uptake but it provides no conclusive evidence of cellular inclusion. The simultaneous determination of cellular uptake and nanotag identification was however achieved using combined 3D Raman and SERS imaging. Three of the four components were detected within a single cell and by combining 2D sections from the 3D images it was possible to determine their intracellular location. Determination of intracellular localisation was achieved using principal component analysis (PCA) since it resulted in the resolution of a subcellular compartment. However, the ultimate success of the system will only be realised when active targeting is demonstrated. Nanotags were functionalised with peptide sequences specific for the endoplasmic reticulum (ER) and trans-Golgi network (TGN). Both nanotag systems were found to locate within lipid rich regions of the cell but they could not be positively confirmed as the ER or TGN. To identify these structures and confirm localisation, further chemometric methods must be investigated including hierarchical cluster analysis (HCA). In conclusion, the SERS nanotags were suitable imaging agents for 2 and 3D cell interrogation. 3D imaging simultaneously permitted organelle resolution and the intracellular localisation of the SERS nanotags. Targeting systems were developed and in future work their localisation within organelles will be confirmed by the application of advanced chemometric methods.Adoption of a multi-marker nanotag approach will led to better disease characterisation whilst simultaneously enabling targeting of multiple disease markers or organelles. The employed nanotag method controllably aggregated nanoparticles with 1,6-hexamethylene diamine (1,6-HMD), before polymer coating with polyvinylpyrrolidone (PVP) and labelling with small molecule reporters; 4-mercaptopyridine (MPY), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 4-nitrobenzenethiol (NBT) and 2-naphthalenethiol (2-NPT). Within a multiple component suspension reporters were identified by their unique peak and when present within single cells or populations they were additionally identified using component direct classical least squares (DCLS). Within a single cell three of the four components (MPY, DTNB and NBT) were positively identified. 2D SERS imaging can monitor nanotag uptake but it provides no conclusive evidence of cellular inclusion. The simultaneous determination of cellular uptake and nanotag identification was however achieved using combined 3D Raman and SERS imaging. Three of the four components were detected within a single cell and by combining 2D sections from the 3D images it was possible to determine their intracellular location. Determination of intracellular localisation was achieved using principal component analysis (PCA) since it resulted in the resolution of a subcellular compartment. However, the ultimate success of the system will only be realised when active targeting is demonstrated. Nanotags were functionalised with peptide sequences specific for the endoplasmic reticulum (ER) and trans-Golgi network (TGN). Both nanotag systems were found to locate within lipid rich regions of the cell but they could not be positively confirmed as the ER or TGN. To identify these structures and confirm localisation, further chemometric methods must be investigated including hierarchical cluster analysis (HCA). In conclusion, the SERS nanotags were suitable imaging agents for 2 and 3D cell interrogation. 3D imaging simultaneously permitted organelle resolution and the intracellular localisation of the SERS nanotags. Targeting systems were developed and in future work their localisation within organelles will be confirmed by the application of advanced chemometric methods

Utilization of yeast pheromones and hydrophobin-based surface engineering for novel whole-cell sensor applications

Whole-cell sensors represent an emerging branch in biosensor development since they obviate the need for enzyme/antibody purification and provide the unique opportunity to assess global parameters such as genotoxicity and bioavailability. Yeast species such as Saccharomyces cerevisiae are ideal hosts for whole-cell sensor applications. However, current approaches almost exclusively rely on analyte-induced expression of fluorescent proteins or luciferases that imply issues with light scattering and/or require the supply of additional substrates. In this study, the yeast α-factor mating pheromone, a peptide pheromone involved in cell-cell communication in Saccharomyces cerevisiae, was utilized to create the whole-cell sensor read-out signal, in particular by employing engineered sensor cells that couple the response to a user-defined environmental signal to α-factor secretion. Two novel immunoassays - relying on hydrophobin-based surface engineering - were developed to quantify the α-factor. Hydrophobins are amphiphilic fungal proteins that self-assemble into robust monolayers at hydrophobic surfaces. Two recombinant hydrophobins, either lacking (EAS) or exposing the α-factor pheromone (EAS-α) upon self-assembly, were used to functionalize polystyrene supports. In a first approach (competitive immunoassay), pheromone-specific antibodies initially bound to the functionalized surface (due to the α-factor exposed by the hydrophobin layer) were competitively detached by soluble α-factor. In a second approach, the antibodies were first premixed with pheromone-containing samples and subsequently applied to functionalized surfaces, allowing for the attachment of antibodies that still carried available binding sites (inverse immunoassay). Both immunoassays enabled quantitative assessment of the yeast pheromone in a unique but partially overlapping dynamic range and allowed for facile tuning of the assay sensitivity by adjustment of the EAS-α content of the hydrophobin layer. With a limit of detection of 0.1 nM α-factor, the inverse immunoassay proved to be the most sensitive pheromone quantification assay currently available. Due to the high stability of hydrophobin monolayers, functionalized surfaces could be reused for multiple consecutive measurements. Favorably, both immunoassays proved to be largely robust against the changes in the sample matrix composition, allowing for pheromone quantification in complex sample matrices such as yeast culture supernatants. Hence, these immunoassays could also be applied to study the pheromone secretion of wild-type and engineered Saccharomyces cerevisiae strains. Additionally, a proof-of-concept whole-cell sensor for thiamine was developed by combining the hydrophobin-based immunoassays with engineered sensor cells of Schizosaccharomyces pombe modulating the secretion of the α-factor pheromone in response to thiamine. Since this read-out strategy encompasses intrinsic signal amplification and enables flexible choice of the transducer element, it could contribute to the development of miniaturized, portable whole-cell sensors for on-site application