Antibody fragments as probe in biosensor development

Today's proteomic analyses are generating increasing numbers of biomarkers, making it essential to possess highly specific probes able to recognize those targets. Antibodies are considered to be the first choice as molecular recognition units due to their target specificity and affinity, which make them excellent probes in biosensor development. However several problems such as difficult directional immobilization, unstable behavior, loss of specificity and steric hindrance, may arise from using these large molecules. Luckily, protein engineering techniques offer designed antibody formats suitable for biomarker analysis. Minimization strategies of antibodies into Fab fragments, scFv or even single-domain antibody fragments like VH, VL or VHHs are reviewed. Not only the size of the probe but also other issues like choice of immobilization tag, type of solid support and probe stability are of critical importance in assay development for biosensing. In this respect, multiple approaches to specifically orient and couple antibody fragments in a generic one-step procedure directly on a biosensor substrate are discussed

Covalent immobilisation of antibodies in Teflon-FEP microfluidic devices for sensitive quantification of clinically relevant protein biomarkers

This study reports for the first time sensitive colorimetric and fluorescence detection of clinically relevant protein biomarkers by sandwich immunoassays using covalent immobilisation of antibodies onto the fluoropolymer surface inside Teflon®-FEP microfluidic devices. Teflon®-FEP has outstanding optical transparency ideal for high-sensitivity colorimetric and fluorescence bioassays, however this thermoplastic is regarded as chemically inert and very hydrophobic. Covalent immobilisation can offer benefits over passive adsorption to plastic surfaces by allowing better control over antibody density, orientation and analyte binding capacity, and so we tested a range of different and novel covalent immobilisation strategies. We first functionalised the inner surface of a 10-bore, 200 µm internal diameter FEP microcapillary film with high-molecular weight polyvinyl alcohol (PVOH) without changing the outstanding optical transparency of the device delivered by the matched refractive index of FEP and water. Glutaraldehyde immobilisation was compared with use of photoactivated linkers and NHS-ester crosslinkers for covalently immobilising capture antibodies onto PVOH. Three clinically relevant sandwich ELISAs were tested, against the cytokine IL-1ß, the myocardial infarct marker cardiac troponin I (cTnI), and the chronic heart failure marker brain natriuretic peptide (BNP). Overall, glutaraldehyde immobilisation was effective for BNP assays, but yielded unacceptable background for IL-1ß and cTnI assays caused by direct binding of biotinylated detection antibody to the modified PVOH surface. We found NHS-ester groups reacted with APTES-treated PVOH coated fluoropolymer. This facilitated a novel method for capture antibody immobilisation onto fluoropolymer devices using a bifunctional NHS-maleimide crosslinker. The density of covalently immobilised capture antibodies achieved using PVOH/APTES/NHS/Maleimide approached levels seen with passive adsorption, and sensitive and quantitative assay performance was achieved using this method. Overall, PVOH coating provided an excellent surface for controlled covalent antibody immobilisation onto Teflon®-FEP for performing high-sensitivity immunoassays

Bio-functionalised nanoparticles for enzyme sensing

Inorganic nanoparticles and their accompanying diverse physical properties are now virtually in routine use as imaging tools in cell-biology. In addition to serving as excellent contrast agents, their size- and environment-dependent optical and magnetic properties can be harnessed to create enzyme biosensor devices of extremely high sensitivity, whilst circumventing the numerous technical limitations associated with traditional enzyme assays. This thesis explores new applications of noble metal and semiconductor nanoparticles (quantum dots) for the detection of a range of medically-relevant enzymes. Here, water soluble colloidal gold nanoparticles and quantum dots are modified with peptides and antibodies to afford new reagents for enzyme sensing. These bio-functionalised nanoparticle probes exhibit numerous advantages over traditional enzyme sensing systems owing to their robust and size-tuneable optical properties. These enzyme-responsive nanoparticle systems are tailored to detect the activity of protein kinase and histone acetyltransferase activity in a simple homogeneous assay format based on Förster resonance energy transfer (FRET). Furthermore, these systems can be applied to screen for putative small-molecule modulators of enzyme function. These new assays should provide the basis for the development of a variety of new enzyme detection strategies based on nanoparticlespecific optical phenomena

Bioaffinity detection of pathogens on surfaces

The demand for improved technologies capable of rapidly detecting pathogens with high sensitivity and selectivity in complex environments continues to be a significant challenge that helps drive the development of new analytical techniques. Surface-based detection platforms are particularly attractive as multiple bioaffinity interactions between different targets and corresponding probe molecules can be monitored simultaneously in a single measurement. Furthermore, the possibilities for developing new signal transduction mechanisms alongside novel signal amplification strategies aremuchmore varied. In this article, we describe some of the latest advances in the use of surface bioaffinity detection of pathogens. Three major sections will be discussed: (i) a brief overview on the choice of probe molecules such as antibodies, proteins and aptamers specific to pathogens and surface attachment chemistries to immobilize those probes onto various substrates, (ii) highlighting examples among the current generation of surface biosensors, and (iii) exploring emerging technologies that are highly promising and likely to form the basis of the next generation of pathogenic sensors

Biofunctionalised electrospun scaffolds for cartilage tissue engineering

The field of tissue engineering has advanced and evolved to focus on biomimetic strategies to meet the rise in demands of tissue replacements for surgical reconstruction. One of the key strategies focuses on developing growth factor delivery systems, by incorporating growth factors into tissue scaffolds. While growth factors are crucial cell-inducing components, their limitations such as short half-lives and dose related adverse effects remain a challenge. To overcome these challenges, this thesis is focused on the development of a novel biomimetic tissue scaffold concept incorporating cell-mediated activation of growth factors for cartilage regeneration. The latent transforming growth factor-β1 (TGF-β1) was selected as a model latent protein due to its well established effects on cartilage as well as its ubiquity in many other tissue types. The thesis first focused on the development and characterisation of the tissue scaffold. A non-woven fibrous scaffold was fabricated by electrospinning, surface modified using ammonia plasma, followed by scaffold surface biofunctionalisation with the latent TGF-β1. Physiochemical characterisation revealed that: (1) the scaffold architecture closely resembled that of the native cartilage extracellular matrix; (2) the scaffold surface was chemically modified for subsequent biofunctionalisation reaction; and (3) the latent TGF-β1 was incorporated onto the scaffold and the active TGF-β1 was detected upon acid and enzymatic activation. Biological effects of the biofunctionalised scaffold were assessed using human nasal chondrocytes in a serum free environment and compared with conventional TGF-β1 supplementation on non-biofunctionalised scaffolds (as control). The biofunctionalised scaffold group induced a lower cell metabolic activity and significantly higher gene expression of cartilage specific transcription factor Sox9 after 14 days. The second part of the thesis evaluated the chondrogenic efficacy of the biofunctionalised scaffolds, using the aforementioned chondrocytes and human mesenchymal stem cells (MSC), in an in-vivo rat model. Cell-scaffold constructs were implanted into subcutaneous pockets of athymic rats for six weeks. Gene expression and immunohistochemistry showed that the biofunctionalised group induced significant chondrogenic differentiation in chondrocytes and type II collagen production when compared to controls. Interestingly, a converse response was observed in MSC where the control group induced relatively higher chondrogenic potentials that the biofunctionalised group. This thesis demonstrates that the latent TGF-β1 biofunctionalised scaffolds induced chondrocytic differentiation in chondrocytes and more importantly the proof of concept of cell-mediated activation of growth factors as a novel approach for functional tissue regeneration

ELISA in the multiplex era: potentials and pitfalls

Multiplex immunoassays confer several advantages over widely adopted singleplex immunoassays including increased efficiency at a reduced expense, greater output per sample volume ratios and higher throughput predicating more resolute, detailed diagnostics and facilitating personalised medicine. Nonetheless, to date, relatively few protein multiplex immunoassays have been validated for in vitro diagnostics in clinical/point-of-care settings. This review article will outline the challenges, which must be ameliorated prior to the widespread integration of multiplex immunoassays in clinical settings: (i) biomarker validation; (ii) standardisation of immunoassay design and quality control (calibration and quantification); (iii) availability, stability, specificity and cross-reactivity of reagents; (iv) assay automation and the use of validated algorithms for transformation of raw data into diagnostic results. A compendium of multiplex immunoassays applicable to in vitro diagnostics and a summary of the diagnostic products currently available commercially are included, along with an analysis of the relative states of development for each format (namely planar slide based, suspension and planar/microtitre plate based) with respect to the aforementioned issues

Recent Progress in Optical Sensors for Biomedical Diagnostics

In recent years, several types of optical sensors have been probed for their aptitude in healthcare biosensing, making their applications in biomedical diagnostics a rapidly evolving subject. Optical sensors show versatility amongst different receptor types and even permit the integration of different detection mechanisms. Such conjugated sensing platforms facilitate the exploitation of their neoteric synergistic characteristics for sensor fabrication. This paper covers nearly 250 research articles since 2016 representing the emerging interest in rapid, reproducible and ultrasensitive assays in clinical analysis. Therefore, we present an elaborate review of biomedical diagnostics with the help of optical sensors working on varied principles such as surface plasmon resonance, localised surface plasmon resonance, evanescent wave fluorescence, bioluminescence and several others. These sensors are capable of investigating toxins, proteins, pathogens, disease biomarkers and whole cells in varied sensing media ranging from water to buffer to more complex environments such as serum, blood or urine. Hence, the recent trends discussed in this review hold enormous potential for the widespread use of optical sensors in early-stage disease prediction and point-of-care testing devices.DFG, 428780268, Biomimetische Rezeptoren auf NanoMIP-Basis zur Virenerkennung und -entfernung mittels integrierter Ansätz

Electrochemical immunosensor based on polythionine/gold nanoparticles for the determination of Aflatoxin B1

An aflatoxin B1 (AFB1) electrochemical immunosensor was developed by the immobilisation of aflatoxin B1-bovine serum albumin (AFB1-BSA) conjugate on a polythionine (PTH)/gold nanoparticles (AuNP)-modified glassy carbon electrode (GCE). The surface of the AFB1-BSA conjugate was covered with horseradish peroxidase (HRP), in order to prevent non-specific binding of the immunosensors with ions in the test solution. The AFB1 immunosensor exhibited a quasi-reversible electrochemistry as indicated by a cyclic voltammetric (CV) peak separation (ΔEp) value of 62 mV. The experimental procedure for the detection of AFB1 involved the setting up of a competition between free AFB1 and the immobilised AFB1-BSA conjugate for the binding sites of free anti-aflatoxin B1 (anti-AFB1) antibody. The immunosensor’s differential pulse voltammetry (DPV) responses (peak currents) decreased as the concentration of free AFB1 increased within a dynamic linear range (DLR) of 0.6 - 2.4 ng/mL AFB1 and a limit of detection (LOD) of 0.07 ng/mL AFB1. This immunosensing procedure eliminates the need for enzyme-labeled secondary antibodies normally used in conventional ELISA–based immunosensors