Fluorescence lifetime imaging microscopy in the medical sciences

The steady improvement in the imaging of cellular processes in living tissue over the last 10–15 years through the use of various fluorophores including organic dyes, fluorescent proteins and quantum dots, has made observing biological events common practice. Advances in imaging and recording technology have made it possible to exploit a fluorophore's fluorescence lifetime. The fluorescence lifetime is an intrinsic parameter that is unique for each fluorophore, and that is highly sensitive to its immediate environment and/or the photophysical coupling to other fluorophores by the phenomenon Förster resonance energy transfer (FRET). The fluorescence lifetime has become an important tool in the construction of optical bioassays for various cellular activities and reactions. The measurement of the fluorescence lifetime is possible in two formats; time domain or frequency domain, each with their own advantages. Fluorescence lifetime imaging applications have now progressed to a state where, besides their utility in cell biological research, they can be employed as clinical diagnostic tools. This review highlights the multitude of fluorophores, techniques and clinical applications that make use of fluorescence lifetime imaging microscopy (FLIM).     

Materials and techniques for electrochemical biosensor design and construction

New developments in biosensor design are appearing at a high rate as these devices play increasingly important roles in daily life. This review aims to highlight recent developments in materials and techniques for electrochemical biosensor design and construction. Rapid growth in biomaterials, especially the availability and application of a vast range of polymers and copolymers associated with new sensing techniques have led to remarkable innovation in the design and construction of biosensors, significant improvements in sensor function and the emergence of new types of biosensor. Nevertheless, in vivo applications remain limited by functional deterioration due to surface fouling by biological components. However, new copolymers based upon biomembrane mimicry have been extensively investigated during the last two decades, raising hopes that the problems related to interactions between foreign surfaces and biological fluids and tissues may soon be solved.     

Recent advancement in nanosensors for neurotransmitters detection: Present and future perspective

Neurotransmitters maintain physiological condition and behaviour of the brain and body. Studies on transmitting mechanisms and change in concentration of particular neurotransmitters provide knowledge about how these substances enhance our ability to interpret complex neural pathophysiology and thus advancing us towards developing new therapeutic and diagnostic intervention. Since the role of neurotransmitters in the cognitive processes are much complex, therefore their detection-based analysis leads to expand our understanding on their functions and early disease detection. One of the most crucial aspects of neuroscience is developing profound understanding of neurotransmitter release kinetics. As compared to other techniques, employing nanobiosensors for studying neurotransmission and dynamic changes in neurons exhibit relatively fast, accurate and significant results where other techniques generally fail. Using advanced techniques, high sensitivity and specificity also furnish precise information of neurotransmitter transport at the cellular level. Over the past few years, the advanced nanomaterials in combination of various biomolecules and distinct polymers are widely used in development of biosensing platforms for in-vivo and in-vitro point of care neurotransmitters detection. The review article is focused upon summarizing present status of nanobiosensors based neurotransmitter detection for clinical samples. This firstly describes the importance of neurotransmitters, conventional methods for neurotransmitters detection; need of developing advanced sensing methods and importance of nanomaterials to develop these sensing platforms. Secondly, current status of developed electrochemical and optical nanosensors for different neurotransmitters detection is elaborated in detail. The reported work extensively explains the importance of nano based sensing platforms for understating of neurotransmitters detection. Last section of the review summarized the future perspectives of nanosensors in clinical set-up for neurotransmitter detection.     

Emerging Technologies for the Electrochemical Detection of Bacteria

Infections are a huge economic liability to the health care system, although real‐time detection can allow early treatment protocols to avoid some of this cost and patient morbidity and mortality. Pseudomonas aeruginosa (PA) is a drug‐resistant gram‐negative bacterium found ubiquitously in clinical settings, accounting for up to 27% of hospital acquired infections. PA secretes a vast array of molecules, ranging from secondary metabolites to quorum sensing molecules, of which many can be exploited to monitor bacterial presence. In addition to electrochemical immunoassays to sense bacteria via antigen–antibody interactions, PA pertains a distinct redox‐active virulence factor called pyocyanin (PYO), allowing a direct electrochemical detection of the bacteria. There has been a surge of publications relating to the electrochemical tracing of PA via a myriad of novel biosensing techniques, materials, and methodologies. In addition to indirect methods, research approaches where PYO has been sensitively detected using surface modified electrodes are reviewed and compared with conventional PA‐sensing methodologies. This review aims at presenting indirect and direct electrochemical methods currently developed using various surface modified electrodes, materials, and electrochemical configurations on their electrocatalytic effects on sensing of PA and in particular PYO.     

In situ sensor techniques in modern bioprocess monitoring

New reactor concepts as multi-parallel screening systems or disposable bioreactor systems for decentralized and reproducible production increase the need for new and easy applicable sensor technologies to access data for process control. These sophisticated reactor systems require sensors to work with the lowest sampling volumes or, even better, to measure directly in situ, but in situ sensors are directly incorporated into a reactor or fermenter within the sterility barrier and have therefore to stand the sterilization procedures. Consequently, these in situ sensor technologies should enable the measurement of multi-analytes simultaneously online and in real-time at a low price for the robust sensing element. Current research therefore focuses on the implementation of noninvasive spectroscopic and optical technologies, and tries to employ them through fiber optics attached to disposable sensing connectors. Spectroscopic methods reach from ultraviolet to infrared and further comprising fluorescence and Raman spectroscopy. Also, optic techniques like microscopy are adapted for the direct use in bioreactor systems (Ulber et al. in Anal Bioanal Chem 376:342–348, 2003) as well as various electrochemical methods (Joo and Brown in Chem Rev 108:638–651, 2008). This review shows the variety of modern in situ sensing principles in bioprocess monitoring with emphasis on spectroscopic and optical techniques and the progress in the adaption to latest reactor concepts.     

Techniques for detecting protein-protein interactions in living cells: principles,limitations, and recent progress

Detecting protein-protein interactions(PPIs) provides fundamental information for understanding biochemical processes such as the transduction of signals from one cellular location to another; however, traditional biochemical techniques cannot provide sufficient spatio-temporal information to elucidate these molecular interactions in living cells. Over the past decade, several new techniques have enabled the identification and characterization of PPIs. In this review, we summarize three main techniques for detecting PPIs in vivo, focusing on their basic principles and applications in biological studies. We place a special emphasis on their advantages and limitations, and, in particular, we introduced some uncommon new techniques, such as single-molecule FRET(smFRET), FRET-fluorescence lifetime imaging microscopy(FRET-FLIM), cytoskeleton-based assay for protein-protein interaction(CAPPI) and single-molecule protein proximity index(smPPI), highlighting recent improvements to the established techniques. We hope that this review will provide a valuable reference to enable researchers to select the most appropriate technique for detecting PPIs.     

Nanostructured material-based optical and electrochemical detection of amoxicillin antibiotic

The penicillin derivative amoxicillin (AMX) plays an important role in treating various types of infections caused by bacteria. However, excessive use of AMX may have negative health effects. Therefore, it is of utmost importance to detect and quantify the AMX in pharmaceutical drugs, biological fluids, and environmental samples with high sensitivity. Therefore, this review article provides valuable and up-to-date information on nanostructured material-based optical and electrochemical sensors to detect AMX in various biological and chemical samples. The role of using different nanostructured materials on the performance of important optical sensors such as colorimetric sensors, fluorescence sensors, surface-enhanced Raman scattering sensors, chemiluminescence/electroluminescence sensors, optical immunosensors, optical fibre-based sensors, and several important electrochemical sensors based on different electrode types have been discussed. Moreover, nanocomposites, polymer, and MXenes-based electrochemical sensors have also been discussed, in which such materials are being used to further enhance the sensitivity of these sensors. Furthermore, nanocomposite-based photo-electrochemical sensors and the market availability of biosensors including AMX have also been discussed briefly. Finally, the conclusion, challenges, and future perspectives of the above-mentioned sensing techniques for AMX detection are presented.     

Application of ionic liquids in electrochemical sensing systems

Since 1992, when the room temperature ionic liquids (ILs) based on the 1-alkyl-3-methylimidazolium cation were reported to provide an attractive combination of an electrochemical solvent and electrolyte, ILs have been widely used in electrodeposition, electrosynthesis, electrocatalysis, electrochemical capacitor, and lithium batteries. However, it has only been in the last few years that electrochemical biosensors based on carbon ionic liquid electrodes (CILEs) and IL-modified macrodisk electrodes have been reported. However, there are still a lot of challenges in achieving IL-based sensitive, selective, and reproducible biosensors for high speed analysis of biological and environmental compounds of interest. This review discusses the principles of operation of electrochemical biosensors based on CILEs and IL/composite-modified macrodisk electrodes. Subsequently, recent developments and major strategies for enhancing sensing performance are discussed. Key challenges and opportunities of IL-based biosensors to further development and use are considered. Emphasis is given to direct electron-transfer reaction and electrocatalysis of hemeproteins and enzyme-modified composite electrodes.     

Recent advances in intravital microscopy for preclinical research

Intravital microscopy (IVM) has revolutionized our understanding of single-cell behavior in complex tissues by enabling real-time observation of molecular and cellular processes in their natural environment. In preclinical research, IVM has emerged as a standard tool for mechanistic studies of therapy response and the rational design of new treatment strategies. Technological developments keep expanding the imaging depth and quality that can be achieved in living tissue, and the maturation of imaging modalities such as fluorescence and phosphorescence lifetime imaging facilitates co-registration of individual cell dynamics with metabolic tissue states. Correlation of IVM with mesoscopic and macroscopic imaging modalities further promotes the translation of mechanistic insights gained by IVM into clinically relevant information. This review highlights some of the recent advances in IVM that have made the transition from experimental optical techniques to practical applications in basic and preclinical research.     

Fabrication and characterization of a radiation sensor based on bacteriorhodopsin

Available techniques of X-ray detection have been under development due to specific shortcomings such as finite lifetime, low sensitivity, and post-processing requirements. Here we report on the fabrication of an X-ray sensor based on bacteriorhodopsin (BR) with a radius of r=3mm as the sensing area on a flexible substrate. The flexible X-ray detector can be placed on the targeted area for real-time monitoring of radiation dosage. We show that BR sensor is a potential candidate for such a powerful sensing device. For this purpose, we measure the electrical current generated by the BR sensor under different radiation dosages, energies and dose rates. This averaged current is in the range of nanoampere and is proportional to the dose rate of the received X-ray. The current also increases with the increase of radiation energy. BR radiation sensor can be readily miniaturized and is relatively easy to fabricate. The capability for real-time data collection and reusability are other advantages of this radiation sensor.     

Electrochemical arrays coupled with magnetic separators for immunochemistry

Electrochemical methods are increasingly applied to immunoassays, because they overcome problems associated with other modes of detection. In particular, with respect to conventional immunoassays, electrochemical immunosensors show versatility, reliability, and fast analysis time. In immunosensor strategy, the antigen or antibody can be immobilized directly onto the surface of the electrochemical transducer that will finally be used to reveal the amount of the affinity reaction. However, the use of the electrode surface as a solid phase as well as an electrochemical transducer presents some problems: a shielding of the surface by biospecifically bound antibody molecules can cause hindrance in the electron transfer, resulting in a reduced voltammetric signal. Thus, as an alternative solid phase, magnetic beads because of their low toxicity and high biocompatibility have gained much attention in chemistry, associated with various analytical techniques, due to their suitability for immobilization of biomolecules. Magnetic micro- or nanobeads can be separated easily and quickly by magnetic forces and will be used together with bioaffine ligands, e.g., antibodies or proteins with a high affinity to the target. The special advantages of magnetic separation techniques are the fast and simple handling of a sample vial and the opportunity to deal with large sample volumes without the need for time-consuming centrifugation steps. This also makes biomagnetic separation ideal for automated assay/analysis systems which will play a very important role in the near future. This review presents some examples of immunochemical assay developed using magnetic beads as a solid phase coupled with electrochemical detection techniques, in particular, using electrochemical arrays as transducers. Applications related to static measurements, together with in-flow detection systems are presented.     

Unsupervised fluorescence lifetime imaging microscopy for high content and high throughput screening

Proteomics and cellomics clearly benefit from the molecular insights in cellular biochemical events that can be obtained by advanced quantitative microscopy techniques like fluorescence lifetime imaging microscopy and F?rster resonance energy transfer imaging. The spectroscopic information detected at the molecular level can be combined with cellular morphological estimators, the analysis of cellular localization, and the identification of molecular or cellular subpopulations. This allows the creation of powerful assays to gain a detailed understanding of the molecular mechanisms underlying spatiotemporal cellular responses to chemical and physical stimuli. This work demonstrates that the high content offered by these techniques can be combined with the high throughput levels offered by automation of a fluorescence lifetime imaging microscope setup capable of unsupervised operation and image analysis. Systems and software dedicated to image cytometry for analysis and sorting represent important emerging tools for the field of proteomics, interactomics, and cellomics. These techniques could soon become readily available both to academia and the drug screening community by the application of new all-solid-state technologies that may results in cost-effective turnkey systems. Here the application of this screening technique to the investigation of intracellular ubiquitination levels of alpha-synuclein and its familial mutations that are causative for Parkinson disease is shown. The finding of statistically lower ubiquitination of the mutant alpha-synuclein forms supports a role for this modification in the mechanism of pathological protein aggregation.     

Nanostructured biomolecular detectors: pushing performance at the nanoscale

Nanomaterial-based biosensing strategies offer a number of advantages over traditional molecular diagnostic and cellular analysis techniques, including signal amplification, improved sensitivity and speed, and versatile sensing schemes that can be tailored to a desired target. In this article, we highlight a variety of nanomaterial-based sensors, and discuss the advantages of different nanomaterials compositions and probes of different biomolecular classes. Recent advances in the development of optical, electrical, or electrochemical transduction mechanisms are covered, with special regard to breakthroughs in sensitivity. The works reviewed herein emphasize the improvements that nanomaterials offer in the realm of diagnostic assays and make a solid case for further advancement with automation and multiplexing.     

Strategies to extend the lifetime of bioelectrochemical enzyme electrodes for biosensing and biofuel cell applications

Enzymes are powerful catalysts for biosensor and biofuel cell electrodes due to their unique substrate specificity. This specificity is defined by the amino acid chain's complex three-dimensional structure based on non-covalent forces, being also responsible for the very limited enzyme lifetime of days to weeks. Many electrochemical applications, however, would benefit from lifetimes over months to years. This mini-review provides a critical overview of strategies and ideas dealing with the problem of short enzyme lifetime, which limits the overall lifetime of bioelectrochemical electrodes. The most common approaches aim to stabilize the enzyme itself. Various immobilization techniques have been used to reduce flexibility of the amino acid chain by introducing covalent or non-covalent binding forces to external molecules. The enzyme can also be stabilized using genetic engineering methods to increase the binding forces within the protein or by optimizing the environment in order to reduce destabilizing interactions. In contrast, renewing the inactivated catalyst decouples overall system lifetime from the limited enzyme lifetime and thereby promises theoretically unlimited electrode lifetimes. Active catalyst can be supplied by exchanging the electrolyte repeatedly. Alternatively, integrated microorganisms can display the enzymes on their surface or secrete them to the electrolyte, allowing unattended power supply for long-term applications.     

Plant cell organelle proteomics in response to abiotic stress

Proteomics is one of the finest molecular techniques extensively being used for the study of protein profiling of a given plant species experiencing stressed conditions. Plants respond to a stress by alteration in the pattern of protein expression, either by up-regulating of the existing protein pool or by the synthesizing novel proteins primarily associated with plants antioxidative defense mechanism. Improved protein extraction protocols and advance techniques for identification of novel proteins have been standardized in different plant species at both cellular and whole plant level for better understanding of abiotic stress sensing and intracellular stress signal transduction mechanisms. In contrast, an in-depth proteome study of subcellular organelles could generate much detail information about the intrinsic mechanism of stress response as it correlates the possible relationship between the protein abundance and plant stress tolerance. Although a wealth of reviews devoted to plant proteomics are available, review articles dedicated to plant cell organelle proteins response under abiotic stress are very scanty. In the present review, an attempt has been made to summarize all significant contributions related to abiotic stresses and their impacts on organelle proteomes for better understanding of plants abiotic stress tolerance mechanism at protein level. This review will not only provide new insights into the plants stress response mechanisms, which are necessary for future development of genetically engineered stress tolerant crop plants for the benefit of humankind, but will also highlight the importance of studying changes in protein abundance within the cell organelles in response to abiotic stress.     

Characterisation of an antibody coated microcantilever as a potential immuno-based biosensor

In this study, we investigated the activity, stability, lifetime and re-usability of monoclonal antibodies to myoglobin covalently immobilised onto microfabricated cantilever surfaces. These sensing surfaces are of interest to us in the development of novel cantilever-based immunosensors. For such sensors the antibody layer represents the sensing element while the microcantilever acts as a mechanical transducer. A procedure for producing re-usable biological coatings has been tested with different independent techniques. An Enzyme Linked Immunosorbent Assay (ELISA) was used to determine the presence of an active antibody coating, and to monitor the lifetime and stability of the immobilised antibody. Through this analysis, the activity of the immobilised antibody layer was found to be more stable with the introduction of sucrose, as a stabilising agent. Sucrose was applied to the immobilised antibody layer after each regeneration step. The immobilised antibody was found to have a stable active lifetime for up to 7 weeks. Fluorescence microscopy was used to give information on the distribution of the coating on the gold and silicon nitride sides of the cantilever. Atomic Force Microscopy was used to determine the presence of the biological coating on the cantilever and to obtain information on the surface morphology of the biological element of the sensor. The combined results provide valuable information on the development of an optimised sensing element and demonstrate a set of methods to use for future sensor-to-sensor characterisation. Preliminary experimental results showing the antibody activity against myoglobin, detected with a microcantilever based sensor prototype confirmed the motivations and potentialities of the proposed immunosensing technique.     

Viral manipulation of DNA repair and cell cycle checkpoints

Recognition and repair of DNA damage is critical for maintaining genomic integrity and suppressing tumorigenesis. In eukaryotic cells, the sensing and repair of DNA damage are coordinated with cell cycle progression and checkpoints, in order to prevent the propagation of damaged DNA. The carefully maintained cellular response to DNA damage is challenged by viruses, which produce a large amount of exogenous DNA during infection. Viruses also express proteins that perturb cellular DNA repair and cell cycle pathways, promoting tumorigenesis in their quest for cellular domination. This review presents an overview of strategies employed by viruses to manipulate DNA damage responses and cell cycle checkpoints as they commandeer the cell to maximize their own viral replication. Studies of viruses have identified key cellular regulators and revealed insights into molecular mechanisms governing DNA repair, cell cycle checkpoints, and transformation.     

Big roles for small GTPases in the control of directed cell movement

Small GTPases are involved in the control of diverse cellular behaviours, including cellular growth, differentiation and motility. In addition, recent studies have revealed new roles for small GTPases in the regulation of eukaryotic chemotaxis. Efficient chemotaxis results from co-ordinated chemoattractant gradient sensing, cell polarization and cellular motility, and accumulating data suggest that small GTPase signalling plays a central role in each of these processes as well as in signal relay. The present review summarizes these recent findings, which shed light on the molecular mechanisms by which small GTPases control directed cell migration.