Sub-second striatal dopamine dynamics assessed by simultaneous fast-scan cyclic voltammetry and fluorescence biosensor

Fast-scan cyclic voltammetry (FSCV) is an electrochemical method used to detect dopamine on a subsecond time scale. Recordings using FSCV in freely behaving animals revolutionized the study of behaviors associated with motivation and learning. Despite this advance, FSCV cannot distinguish between catecholamines, which limits its use to brain regions where dopamine is the predominant neurotransmitter. It has also been difficult to detect dopamine in vivo in some striatal subregions with FSCV. Recently, fluorescent biosensors for dopamine were developed, allowing for discrimination between catecholamines. However, the performance of these biosensors relative to FSCV has not been determined. Thus, we compared fluorescent photometry responses of the dopamine biosensor, dLight, with FSCV. We also used dLight photometry to assess changes in tonic and phasic dopamine, which has not been possible with FSCV. Finally, we examined dopamine dynamics during Pavlovian conditioning in striatal subregions, including the dorsolateral striatum where dopamine measurements are challenging with FSCV.

Fluorescent Biosensors Based on Silicon Nanowires

Nanostructures are arising as novel biosensing platforms promising to surpass current performance in terms of sensitivity, selectivity, and affordability of standard approaches. However, for several nanosensors, the material and synthesis used make the industrial transfer of such technologies complex. Silicon nanowires (NWs) are compatible with Si-based flat architecture fabrication and arise as a hopeful solution to couple their interesting physical properties and surface-to-volume ratio to an easy commercial transfer. Among all the transduction methods, fluorescent probes and sensors emerge as some of the most used approaches thanks to their easy data interpretation, measure affordability, and real-time in situ analysis. In fluorescent sensors, Si NWs are employed as substrate and coupled with several fluorophores, NWs can be used as quenchers in stem-loop configuration, and have recently been used for direct fluorescent sensing. In this review, an overview on fluorescent sensors based on Si NWs is presented, analyzing the literature of the field and highlighting the advantages and drawbacks for each strategy.

Genetically Encoded Fluorescent Biosensors for Biomedical Applications

One of the challenges of modern biology and medicine is to visualize biomolecules in their natural environment, in real-time and in a non-invasive fashion, so as to gain insight into their physiological behavior and highlight alterations in pathological settings, which will enable to devise appropriate therapeutic strategies. Genetically encoded fluorescent biosensors constitute a class of imaging agents that enable visualization of biological processes and events directly in situ, preserving the native biological context and providing detailed insight into their localization and dynamics in cells. Real-time monitoring of drug action in a specific cellular compartment, organ, or tissue type; the ability to screen at the single-cell resolution; and the elimination of false-positive results caused by low drug bioavailability that is not detected by in vitro testing methods are a few of the obvious benefits of using genetically encoded fluorescent biosensors in drug screening. This review summarizes results of the studies that have been conducted in the last years toward the fabrication of genetically encoded fluorescent biosensors for biomedical applications with a comprehensive discussion on the challenges, future trends, and potential inputs needed for improving them.

A Co-Culture-Based Multiparametric Imaging Technique to Dissect Local H2O2 Signals with Targeted HyPer7

Multispectral live-cell imaging is an informative approach that permits detecting biological processes simultaneously in the spatial and temporal domain by exploiting spectrally distinct biosensors. However, the combination of fluorescent biosensors with distinct spectral properties such as different sensitivities, and dynamic ranges can undermine accurate co-imaging of the same analyte in different subcellular locales. We advanced a single-color multiparametric imaging method, which allows simultaneous detection of hydrogen peroxide (H2O2) in multiple cell locales (nucleus, cytosol, mitochondria) using the H2O2 biosensor HyPer7. Co-culturing of endothelial cells stably expressing differentially targeted HyPer7 biosensors paved the way for co-imaging compartmentalized H2O2 signals simultaneously in neighboring cells in a single experimental setup. We termed this approach COMPARE IT, which is an acronym for co-culture-based multiparametric imaging technique. Employing this approach, we detected lower H2O2 levels in mitochondria of endothelial cells compared to the cell nucleus and cytosol under basal conditions. Upon administering exogenous H2O2, the cytosolic and nuclear-targeted probes displayed similarly slow and moderate HyPer7 responses, whereas the mitochondria-targeted HyPer7 signal plateaued faster and reached higher amplitudes. Our results indicate striking differences in mitochondrial H2O2 accumulation of endothelial cells. Here, we present the method’s potential as a practicable and informative multiparametric live-cell imaging technique.

Visualizing endogenous Rho activity with an improved localization-based, genetically encoded biosensor

Rho GTPases are regulatory proteins, which orchestrate cell features such as morphology, polarity and movement. Therefore, probing Rho GTPase activity is key to understanding processes such as development and cell migration. Localization-based reporters for active Rho GTPases are attractive probes to study Rho GTPase-mediated processes, in real time with subcellular resolution in living cells and tissue. Until now, relocation Rho biosensors seem to only be useful in certain organisms and have not been characterized well. In this paper, we systematically examined the contribution of the fluorescent protein and Rho binding peptides, on the performance of localization-based sensors. To test the performance, we compared relocation efficiency and specificity in cell-based assays. We identified several improved localization-based, genetically encoded, fluorescent biosensors for detecting endogenous Rho activity. This enables a broader application of Rho relocation biosensors, which was demonstrated by using the improved biosensor to visualize Rho activity during several cellular processes, such as cell division, migration and G protein-coupled receptor signaling. Due to the improved avidity of the new biosensors for Rho activity, cellular processes regulated by Rho can be better understood.

Shedding light on developmental ERK signaling with genetically encoded biosensors

ABSTRACT The extracellular signal-regulated kinase (ERK) pathway governs cell proliferation, differentiation and migration, and therefore plays key roles in various developmental and regenerative processes. Recent advances in genetically encoded fluorescent biosensors have unveiled hitherto unrecognized ERK activation dynamics in space and time and their functional importance mainly in cultured cells. However, ERK dynamics during embryonic development have still only been visualized in limited numbers of model organisms, and we are far from a sufficient understanding of the roles played by developmental ERK dynamics. In this Review, we first provide an overview of the biosensors used for visualization of ERK activity in live cells. Second, we highlight the applications of the biosensors to developmental studies of model organisms and discuss the current understanding of how ERK dynamics are encoded and decoded for cell fate decision-making.

Nanomaterial-Based Dual-Emission Ratiometric Fluorescent Sensors for Biosensing and Cell Imaging

Owing to the unique optophysical properties of nanomaterials and their self-calibration characteristics, nanomaterial-based (e.g., polymer dots (Pdots) quantum dots (QDs), silicon nanorods (SiNRs), and gold nanoparticle (AuNPs), etc.) ratiometric fluorescent sensors play an essential role in numerous biosensing and cell imaging applications. The dual-emission ratiometric fluorescence technique has the function of effective internal referencing, thereby avoiding the influence of various analyte-independent confounding factors. The sensitivity and precision of the detection can therefore be greatly improved. In this review, the recent progress in nanomaterial-based dual-emission ratiometric fluorescent biosensors is systematically summarized. First, we introduce two general design approaches for dual-emission ratiometric fluorescent sensors, involving ratiometric fluorescence with changes of one response signal and two reversible signals. Then, some recent typical examples of nanomaterial-based dual-emission ratiometric fluorescent biosensors are illustrated in detail. Finally, probable challenges and future outlooks for dual-emission ratiometric fluorescent nanosensors for biosensing and cell imaging are rationally discussed.

Biocontrol of Biofilm Formation: Jamming of Sessile-Associated Rhizobial Communication by Rhodococcal Quorum-Quenching

Biofilms are complex structures formed by a community of microbes adhering to a surface and/or to each other through the secretion of an adhesive and protective matrix. The establishment of these structures requires a coordination of action between microorganisms through powerful communication systems such as quorum-sensing. Therefore, auxiliary bacteria capable of interfering with these means of communication could be used to prevent biofilm formation and development. The phytopathogen Rhizobium rhizogenes, which causes hairy root disease and forms large biofilms in hydroponic crops, and the biocontrol agent Rhodococcus erythropolis R138 were used for this study. Changes in biofilm biovolume and structure, as well as interactions between rhizobia and rhodococci, were monitored by confocal laser scanning microscopy with appropriate fluorescent biosensors. We obtained direct visual evidence of an exchange of signals between rhizobia and the jamming of this communication by Rhodococcus within the biofilm. Signaling molecules were characterized as long chain (C14) N-acyl-homoserine lactones. The role of the Qsd quorum-quenching pathway in biofilm alteration was confirmed with an R. erythropolis mutant unable to produce the QsdA lactonase, and by expression of the qsdA gene in a heterologous host, Escherichia coli. Finally, Rhizobium biofilm formation was similarly inhibited by a purified extract of QsdA enzyme.