Nanotechnology for single-cell bacterial activity monitoring

PIEZO1 is critical to numerous physiological processes, transducing diverse mechanical stimuli into electrical and chemical signals. Recent studies underscore the importance of visualizing endogenous PIEZO1 activity and localization to understand its functional roles. To enable physiologically and clinically relevant studies on human PIEZO1, we genetically engineered human induced pluripotent stem cells (hiPSCs) to express a HaloTag fused to endogenous PIEZO1. Combined with advanced imaging, our chemogenetic platform allows precise visualization of PIEZO1 localization dynamics in various cell types. Furthermore, the PIEZO1-HaloTag hiPSC technology facilitates the non-invasive monitoring of channel activity across diverse cell types using Ca2+-sensitive HaloTag ligands, achieving temporal resolution approaching that of patch clamp electrophysiology. Finally, we use lightsheet microscopy on hiPSC-derived neural organoids to achieve molecular scale imaging of PIEZO1 in three-dimensional tissue. Our advances establish a platform for studying PIEZO1 mechanotransduction in human systems, with potential for elucidating disease mechanisms and targeted drug screening.

We studied the effects of natural sunlight on heterotrophic marine bacterioplankton in short-term experiments. We used a single-cell level approach involving flow cytometry combined with physiological probes and microautoradiography to determine sunlight effects on the activity and integrity of the cells. After 4 h of sunlight exposure, most bacterial cells maintained membrane integrity and viability as assessed by the simultaneous staining with propidium iodide and SYBR green I. In contrast, a significant inhibition of heterotrophic bacterial activity was detected, measured by 5-cyano-2,3 ditolyl tetrazolium chloride reduction and leucine incorporation. We applied microautoradiography combined with catalyzed reporter deposition-fluorescence in situ hybridization to test the sensitivity of the different bacterial groups naturally occurring in the Northwestern Mediterranean to sunlight. Members of the Gammaproteobacteria and Bacteroidetes groups appeared to be highly resistant to solar radiation, with small changes in activity after exposure. On the contrary, Alphaproteobacteria bacteria were more sensitive to radiation as measured by the cell-specific incorporation of labeled amino acids, leucine, and ATP. Within Alphaproteobacteria, bacteria belonging to the Roseobacter group showed higher resistance than members of the SAR11 cluster. The activity of Roseobacter was stimulated by exposure to photosynthetic available radiation compared to the dark treatment. Our results suggest that UV radiation can significantly affect the in situ single-cell activity of bacterioplankton and that naturally dominating phylogenetic bacterial groups have different sensitivity to natural levels of incident solar radiation.

BackgroundBacterial biofilms are communities of surface-associated microorganisms living in cellular clusters or micro-colonies, encapsulated in a complex matrix composed of an extracellular polymeric substance, separated by open water channels that act as a circulatory system that enable better diffusion of nutrients and easier removal of metabolic waste products. The monitoring of biofilms can provide important information on fundamental biofilm-related processes. That information can shed light on the bacterial processes and enable scientists to find ways of preventing future bacterial infections. Various approaches in use for biofilm analysis are based on microscopic, spectrochemical, electrochemical, and piezoelectrical methods. All these methods provide significant progress in understanding the bio-process related to biofilm formation and eradication, nevertheless, the development of novel approaches for the real-time monitoring of biochemical, in particular metabolic activity, of bacterial species during the formation, life and eradication of biofilms is of great potential importance.ResultsHere, detection and monitoring of the metabolic activity of bacterial biofilms in high-ionic-strength solutions were enabled as a result of novel surface modification by an active redox system, composed of 9,10-dihydroxyanthracene/9,10-anthraquinone, on the oxide layer of the SiNW, yielding a chemically-gated FET array. With the use of enzymatic reactions of oxidases, metabolites can be converted to H2O2 and monitored by the nanosensors. Here, the successful detection of glucose metabolites in high-ionic-strength solutions, such as bacterial media, without pre-processing of small volume samples under different conditions and treatments, has been demonstrated. The biofilms were treated with antibiotics differing in their mechanisms of action and were compared to untreated biofilms. Further examination of biofilms under antibiotic treatment with SiNW-FET devices could shed light on the bioprocess that occurs within the biofilm. Moreover, finding proper treatment that eliminates the biofilm could be examined by the novel nanosensor as a monitoring tool.ConclusionsTo summarize, the combination of redox-reactive SiNW-FET devices with micro-fluidic techniques enables the performance of rapid, automated, and real-time metabolite detection with the use of minimal sample size, noninvasively and label-free. This novel platform can be used as an extremely sensitive tool for detection and establishing medical solutions for bacterial-biofilm eradication and for finding a proper treatment to eliminate biofilm contaminations. Moreover, the sensing system can be used as a research tool for further understanding of the metabolic processes that occur within the bacterial biofilm population.

Automatic monitoring of protease activity during fermentation

Electrophysical sensor systems for in vitro monitoring of bacterial metabolic activity

In this publication an alternative approach to investigations of bacterial growth is proposed. Contrary to the conventional physical methods it is based on enzyme activity detection. The procedure for real-time and on-line monitoring of microbial ureolytic activity (applied as a model experimental biosystem) in the flow analysis format is presented. The developed fully-mechanized bioanalytical flow system is composed of solenoid micropumps and microvalves actuated by Arduino microcontroller. The photometric detection based on Nessler reaction is performed using dedicated flow-through optoelectronic detector made of paired light emitting diodes. The developed bioanalytical system allows discrete assaying of microbial urease in the wide range of activity up to 5.4 U mL−1 with detection limit below 0.44 U mL−1, a high sensitivity in the linear range of response (up to 200 mV U−1 mL and relatively high throughput (9 detection per hour). The proposed differential procedure of measurements (i.e. a difference between peaks register for sample with and without external addition of urea is treated as an analytical signal) allows elimination of interfering effects from substrate and products of biocatalysed reaction as well as other components of medium used for microbial growth. The developed bioanalytical system was successfully applied for the control of growth of urease-positive bacteria strains (Proteus vulgaris, Klebsiella pneumoniae and Paracoccus yeei) including examination of effects from various microbial cultivation conditions like temperature, composition of culture medium and amount of substrate required for induction of bacterial enzymatic activity. The developed bioanalytical flow system can be applied for metabolic activity-based estimation of parameters of lag and log phases of microbial growth as well as for detection of decline phase.

Monitoring the characteristics of wastewater can enable a better choice of the options available for its treatment and also provide a rational basis for the surveillance of the spread of waterborne diseases. Our work focused on bringing down the costs associated with the estimation of the biological and physico-chemical parameters of wastewater at a common wavelength of 600 nm. We demonstrated the advantages of using the Dye Reduction-based Electron-transfer Activity Monitoring (DREAM) assay to estimate bacterial activity as an alternative to the bacterial colony count method. This assay relies on the colorimetric measurement of the extent of reduction of methylene blue in response to bacterial electron transfer. We also present the results of estimating COD and turbidity, using the standard methods of wastewater analysis, at the same wavelength of 600 nm using samples having known values of chemical oxygen demand (COD) and turbidity. Our results open up the possibility of replacing expensive spectrophotometers with LED-based, microcontroller enabled colorimeters that can be easily automated. The results of our study set the stage for the establishment of a greater number of wastewater analysis laboratories in economically backward regions which can in turn support measures to improve sanitation facilities.