Looking towards label-free biomolecular interaction analysis in a high-throughput format: a review of new surface plasmon resonance technologies

Surface plasmon resonance (SPR) biosensors have enabled a wide range of applications in which researchers can monitor biomolecular interactions in real time. Owing to the fact that SPR can provide affinity and kinetic data, unique features in applications ranging from protein-peptide interaction analysis to cellular ligation experiments have been demonstrated. Although SPR has historically been limited by its throughput, new methods are emerging that allow for the simultaneous analysis of many thousands of interactions. When coupled with new protein array technologies, high-throughput SPR methods give users new and improved methods to analyze pathways, screen drug candidates and monitor protein-protein interactions.     

Modularized synthetic biology enabled intelligent biosensors

Biosensors that sense the concentration of a specified target and produce a specific signal output have become important technology for biological analysis. Recently, intelligent biosensors have received great interest due to their adaptability to meet sophisticated demands. Advances in developing standard modules and carriers in synthetic biology have shed light on intelligent biosensors that can implement advanced analytical processing to better accommodate practical applications. This review focuses on intelligent synthetic biology-enabled biosensors (SBBs). First, we illustrate recent progress in intelligent SBBs with the capability of computation, memory storage, and self-calibration. Then, we discuss emerging applications of SBBs in point-of-care testing (POCT) and wearable monitoring. Finally, future perspectives on intelligent SBBs are proposed.     

Microfluidic-integrated biosensors: Prospects for point-of-care diagnostics

There is a growing demand to integrate biosensors with microfluidics to provide miniaturized platforms with many favorable properties, such as reduced sample volume, decreased processing time, low cost analysis and low reagent consumption. These microfluidics-integrated biosensors would also have numerous advantages such as laminar flow, minimal handling of hazardous materials, multiple sample detection in parallel, portability and versatility in design. Microfluidics involves the science and technology of manipulation of fluids at the micro- to nano-liter level. It is predicted that combining biosensors with microfluidic chips will yield enhanced analytical capability, and widen the possibilities for applications in clinical diagnostics. The recent developments in microfluidics have helped researchers working in industries and educational institutes to adopt some of these platforms for point-of-care (POC) diagnostics. This review focuses on the latest advancements in the fields of microfluidic biosensing technologies, and on the challenges and possible solutions for translation of this technology for POC diagnostic applications. We also discuss the fabrication techniques required for developing microfluidic-integrated biosensors, recently reported biomarkers, and the prospects of POC diagnostics in the medical industry.     

Fluorescent Biosensors of Intracellular Targets from Genetically Encoded Reporters to Modular Polypeptide Probes

With the escalation of drug discovery programmes, it has become essential to visualize and monitor biological activities in healthy and pathological cells, with high spatial and temporal resolution. To this aim, the development of probes and sensors, which can report on the levels and activities of specific intracellular targets, has become essential. Together with the discovery of the Green Fluorescent Protein (GFP), and the development of GFP-based reporters, recent advances in the synthesis of small molecule fluorescent probes, and the explosion of fluorescence-based imaging technologies, the biosensor field has witnessed a dramatic expansion of fluorescence-based reporters which can be applied to complex biological samples, living cells and tissues to probe protein/protein interactions, conformational changes and posttranslational modifications. Here, we review recent developments in the field of fluorescent biosensor technology. We describe different varieties and categories of fluorescent biosensors together with an overview of the technologies commonly employed to image biosensors in cellulo and in vivo. We discuss issues and strategies related to the choice of synthetic fluorescent probes, labelling, quenching, caging and intracellular delivery of biosensors. Finally, we provide examples of some well-characterized genetically encoded FRET reporter systems, peptide and protein biosensors and describe biosensor applications in a wide variety of fields.     

Novel quartz crystal microbalance based biosensor for detection of oral epithelial cell-microparticle interaction in real-time

Recent applications of quartz crystal resonant sensor technology to monitor cell adhesion and specific ligand interaction processes has triggered the development of a new category of quartz crystal microbalance (QCM) based biosensors. In this study human oral epithelial cells (H376) were cultured on quartz sensors and their response to microspheres investigated in situ using the QCM technique. The results demonstrated that this novel biosensor was able to follow cell-microsphere interactions in real-time and under conditions of flow as would occur in the oral cavity. Unique frequency profiles generated in response to the microspheres were postulated to be due to phases of mass addition and altered cellular rigidity. Supporting microscopic evidence demonstrated that the unique frequency responses obtained to these interactions were in part due to binding between the cell surface and the microspheres. Furthermore, a cellular uptake process, in response to microsphere loading was identified and this, by influencing the rigidity of the cellular cytoskeleton, was also detectable through the frequency responses obtained.     

Advances in cell-based biosensors using three-dimensional cell-encapsulating hydrogels

Cell-based biosensors (CBBs) have emerged as promising biotechnical tools whereby various cell types can be used as basic sensing units to detect external stimuli. Specifically, CBBs have been applied in environmental monitoring, drug screening, clinical diagnosis and biosecurity. For these applications, CBBs offer several advantages over conventional molecular-based biosensors or living animal-based approaches, such as the capability to better mimic physiological situations, to enhance detection specificity and sensitivity, and to detect unknown compounds and toxins. On the other hand, existing CBBs suffer from several limitations, such as weak cell-substrate attachment, two-dimensional (2D) cell microenvironment, and limited shelf life. An emerging method for scaffold-free three-dimensional (3D) cell culture uses hydrogels to encapsulate cells. Advances in novel biomaterials and nano/microscale technologies have enabled encapsulation of cells in hydrogels to fabricate 3D CBBs, which hold great potential for addressing the limitation in existing 2D CBBs. Here, we present an overview of the emerging hydrogel-based CBBs, their applications in pathogen/toxin detection, drug screening and screening of cell-biomaterials interaction, and the associated challenges and potential solutions.     

Live-cell imaging of cAMP dynamics

Spatial and temporal compartmentalization of cAMP (and its target proteins) is central to the ability of this second messenger to govern cellular activity over timescales ranging from milliseconds to several hours. Recent years have witnessed a burgeoning of methodologies that enable researchers to directly monitor rapid subcellular cAMP dynamics, which are unobtainable by traditional cAMP assays. In this review, we examine cAMP biosensors that are currently available for measuring cAMP at the single-cell level, compare their various operating principles and discuss their applications.     

Translocation biosensors to study signal-specific nucleo-cytoplasmic transport, protease activity and protein-protein interactions

Regulated nucleo-cytoplasmic transport is crucial for cellular homeostasis and relies on protein interaction networks. In addition, the spatial division into the nucleus and the cytoplasm marks two intracellular compartments that can easily be distinguished by microscopy. Consequently, combining the rules for regulated nucleo-cytoplasmic transport with autofluorescent proteins, we developed novel cellular biosensors composed of glutathione S-transferase, mutants of green fluorescent protein and rational combinations of nuclear import and export signals. Addition of regulatory sequences resulted in three classes of biosensors applicable for the identification of signal-specific nuclear export and import inhibitors, small molecules that interfere with protease activity and compounds that prevent specific protein-protein interactions in living cells. As a unique feature, our system exploits nuclear accumulation of the cytoplasmic biosensors as the reliable readout for all assays. Efficacy of the biosensors was systematically investigated and also demonstrated by using a fully automated platform for high throughput screening (HTS) microscopy and assay analysis. The introduced modular biosensors not only have the potential to further dissect nucleo-cytoplasmic transport pathways but also to be employed in numerous screening applications for the early stage evaluation of potential drug candidates.     

Microbial biosensors: a review

A microbial biosensor is an analytical device which integrates microorganism(s) with a physical transducer to generate a measurable signal proportional to the concentration of analytes. In recent years, a large number of microbial biosensors have been developed for environmental, food, and biomedical applications. Starting with the discussion of various sensing techniques commonly used in microbial biosensing, this review article concentrates on the summarization of the recent progress in the fabrication and application of microbial biosensors based on amperometry, potentiometry, conductometry, voltammetry, microbial fuel cell, fluorescence, bioluminescence, and colorimetry, respectively. Prospective strategies for the design of future microbial biosensors will also be discussed.     

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

Förster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo^1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest^3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells⁵, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium⁶, cAMP^7-8, inositol phosphates⁹ and cGMP^10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a β-adrenergic receptor agonist and blocker.     

Development of biosensors for cancer clinical testing

Biosensors are devices that combine a biochemical recognition/binding element (ligand) with a signal conversion unit (transducer). Biosensors are already used for several clinical applications, for example for electrochemical measurement of blood glucose concentrations. Application of biosensors in cancer clinical testing has several potential advantages over other clinical analysis methods including increased assay speed and flexibility, capability for multi-target analyses, automation, reduced costs of diagnostic testing and a potential to bring molecular diagnostic assays to community health care systems and to underserved populations. They have the potential for facilitating Point of Care Testing (POCT), where state-of-the-art molecular analysis is carried out without requiring a state-of-the-art laboratory. However, not many biosensors have been developed for cancer-related testing. One major challenge in harnessing the potential of biosensors is that cancer is a very complex set of diseases. Tumors vary widely in etiology and pathogenesis. Oncologists rely heavily on histological characterization of tumors and a few biomarkers that have demonstrated clinical utility to aid in patient management decisions. New genomic and proteomic molecular tools are being used to profile tumors and produce "molecular signatures." These signatures include genetic and epigenetic signatures, changes in gene expression, protein profiles and post-translational modifications of proteins. These molecular signatures provide new opportunities for utilizing biosensors. Biosensors have enormous potential to deliver the promise of new molecular diagnostic strategies to patients. This article describes some of the basic elements of cancer biology and cancer biomarkers relevant for the development of biosensors for cancer clinical testing, along with the challenges in using this approach.     

Evanescent wave biosensors

Optical biosensors, based on evanescent wave technology, are analytical devices that measure the interactions between biomolecules in real time, without the need for any labels. Specific ligands are immobilized to a sensor surface, and a solution of receptor or antibody is injected over the top. Binding is measured by recording changes in the refractive index, caused by the molecules interacting near the sensor surface within the evanescent field. Evanescent wave-based biosensors are being used to study an increasing number of applications in the life sciences, including the binding and dissociation kinetics of antibodies and receptor-ligand pairs, protein-DNA and DNA-DNA interactions, epitope mapping, phage display libraries, and whole cell- and virus-protein interactions. There are currently four commercially available avanescent wave biosensors on the market. This article describes the technology behind their sensing techniques, as well as the range of applications in which they are employed.     

Applications of laccases and tyrosinases (phenoloxidases) immobilized on different supports: a review

This review summarizes all the research efforts that have been spent to immobilize laccase and tyrosinase for various applications, including synthetic and analytical purposes, bioremediation, wastewater treatment, and must and wine stabilization. All immobilization procedures used in these areas are discussed. Considerations on the efficacy of immobilized copper oxidases and products, in addition to their kinetic parameters are also discussed. The available data indicate that the immobilization of laccase into cationic polymer cross-linked with epichlorohydrin appears to be a promising procedure for industrial applications. The development of laccase and tyrosinase-based biosensors to monitor a wide range of compounds appears to be at a mature stage of technology.     

Principles of affinity-based biosensors

Despite the amount of resources that have been invested by national and international academic, government, and commercial sectors to develop affinity-based biosensor products, little obvious success has been realized through commercialization of these devices for specific applications (such as the enzyme biosensors for blood glucose analysis). Nevertheless, the fastest growing area in the biosensors research literature continues to involve advances in affinity-based biosensors and biosensor-related methods. Numerous biosensor techniques have been reported that allow researchers to better study the kinetics, structure, and (solid/liquid) interface phenomena associated with protein-ligand binding interactions. In addition, potential application areas for which affinity-based biosensor techniques show promise include clinical/diagnostics, food processing, military/antiterrorism, and environmental monitoring. The design and structural features of these devices—composed of a biological affinity element interfaced to a signal transducer—primarily determine their operational characteristics. This paper although not intended as a comprehensive review, will outline the principles of affinity biosensors with respect to potential application areas.     

New insights into the molecular mechanisms of biomembrane structural changes and interactions by optical biosensor technology

Biomolecular-membrane interactions play a critical role in the regulation of many important biological processes such as protein trafficking, cellular signalling and ion channel formation. Peptide/protein–membrane interactions can also destabilise and damage the membrane which can lead to cell death. Characterisation of the molecular details of these binding-mediated membrane destabilisation processes is therefore central to understanding cellular events such as antimicrobial action, membrane-mediated amyloid aggregation, and apoptotic protein induced mitochondrial membrane permeabilisation. Optical biosensors have provided a unique approach to characterising membrane interactions allowing quantitation of binding events and new insight into the kinetic mechanism of these interactions. One of the most commonly used optical biosensor technologies is surface plasmon resonance (SPR) and there have been an increasing number of studies reporting the use of this technique for investigating biophysical analysis of membrane-mediated events. More recently, a number of new optical biosensors based on waveguide techniques have been developed, allowing membrane structure changes to be measured simultaneously with mass binding measurements. These techniques include dual polarisation interferometry (DPI), plasmon waveguide resonance spectroscopy (PWR) and optical waveguide light mode spectroscopy (OWLS). These techniques have expanded the application of optical biosensors to allow the analysis of membrane structure changes during peptide and protein binding. This review provides a theoretical and practical overview of the application of biosensor technology with a specific focus on DPI, PWR and OWLS to study biomembrane-mediated events and the mechanism of biomembrane disruption. This article is part of a Special Issue entitled: Lipid–protein interactions.     

Applications of the green fluorescent protein as a molecular marker in environmental microorganisms.

In this review, we examine numerous applications of the green fluorescent protein (GFP) marker gene in environmental microbiology research. The GFP and its variants are reviewed and applications in plant-microbe interactions, biofilms, biodegradation, bacterial-protozoan interactions, gene transfer, and biosensors are discussed. Methods for detecting GFP-marked cells are also examined. The GFP is a useful marker in environmental microorganisms, allowing new research that will increase our understanding of microorganisms in the environment.     

Use of a novel micro-fluidic device to create arrays for multiplex analysis of large and small molecular weight compounds by surface plasmon resonance

There is an increasing demand to develop biosensor monitoring devices capable of biomarker profiling for predicting animal adulteration and detecting multiple chemical contaminants or toxins in food produce. Surface plasmon resonance (SPR) biosensors are label free detection systems that monitor the binding of specific biomolecular recognition elements with binding partners. Essential to this technology are the production of biochips where a selected binding partner, antibody, biomarker protein or low molecular weight contaminant, is immobilised. A micro-fluidic immobilisation device allowing the covalent attachment of up to 16 binding partners in a linear array on a single surface has been developed for compatibility with a prototype multiplex SPR analyser. The immobilisation unit and multiplex SPR analyser were respectively evaluated in their ability to be fit-for-purpose for binding partner attachment and detection of high and low molecular weight molecules. The multiplexing capability of the dual technology was assessed using phycotoxin concentration analysis as a model system. The parent compounds of four toxin groups were immobilised within a single chip format and calibration curves were achieved. The chip design and SPR technology allowed the compartmentalisation of the binding interactions for each toxin group offering the added benefit of being able to distinguish between toxin families and perform concentration analysis. This model is particularly contemporary with the current drive to replace biological methods for phycotoxin screening.     

Applications of genetically-encoded biosensors for the construction and control of biosynthetic pathways

Cells are filled with biosensors, molecular systems that measure the state of the cell and respond by regulating host processes. In much the same way that an engineer would monitor a chemical reactor, the cell uses these sensors to monitor changing intracellular environments and produce consistent behavior despite the variable environment. While natural systems derive a clear benefit from pathway regulation, past research efforts in engineering cellular metabolism have focused on introducing new pathways and removing existing pathway regulation. Synthetic biology is a rapidly growing field that focuses on the development of new tools that support the design, construction, and optimization of biological systems. Recent advances have been made in the design of genetically-encoded biosensors and the application of this class of molecular tools for optimizing and regulating heterologous pathways. Biosensors to cellular metabolites can be taken directly from natural systems, engineered from natural sensors, or constructed entirely in vitro. When linked to reporters, such as antibiotic resistance markers, these metabolite sensors can be used to report on pathway productivity, allowing high-throughput screening for pathway optimization. Future directions will focus on the application of biosensors to introduce feedback control into metabolic pathways, providing dynamic control strategies to increase the efficient use of cellular resources and pathway reliability.     

Magnetic relaxation switches capable of sensing molecular interactions

Highly sensitive, efficient, and high-throughput biosensors are required for genomic and proteomic data acquisition in complex biological samples and potentially for in vivo applications. To facilitate these studies, we have developed biocompatible magnetic nanosensors that act as magnetic relaxation switches (MRS) to detect molecular interactions in the reversible self-assembly of disperse magnetic particles into stable nanoassemblies. Using four different types of molecular interactions (DNA-DNA, protein-protein, protein-small molecule, and enzyme reactions) as model systems, we show that the MRS technology can be used to detect these interactions with high efficiency and sensitivity using magnetic relaxation measurements including magnetic resonance imaging (MRI). Furthermore, the magnetic changes are detectable in turbid media and in whole-cell lysates without protein purification. The developed magnetic nanosensors can be used in a variety of biological applications such as in homogeneous assays, as reagents in miniaturized microfluidic systems, as affinity ligands for rapid and high-throughput magnetic readouts of arrays, as probes for magnetic force microscopy, and potentially for in vivo imaging.     

Fluorescent labels in biosensors for pathogen detection

Infectious diseases caused by pathogens have become a life-threatening problem for millions of people around the world in recent years. Therefore, the need of efficient, fast, low-cost and user-friendly biosensing systems to monitor pathogen has increased enormously in the last few years. This paper presents an overview of different fluorescent labels and the utilization of fluorescence-based biosensor techniques for rapid, direct, sensitive and real-time identification of bacteria. In these biosensors, organic dyes, nanomaterials and rare-earth elements are playing an increasing role in the design of biosensing systems with an interest for applications in bacterial analysis.