Portable oxidative stress sensor: dynamic and non-invasive measurements of extracellular H₂O₂ released by algae

Reactive oxygen species (ROS) generated by aerobic organisms are essential for physiological processes such as cell signaling, apoptosis, immune defense and oxidative stress mechanisms. Unbalanced oxidant/antioxidant budgets are involved in many diseases and, therefore, the sensitive measurement of ROS is of great interest. Here, we present a new device for the real-time monitoring of oxidative stress by measuring one of the most stable ROS, namely hydrogen peroxide (H2O2). This portable oxidative stress sensor contains the heme protein cytochrome c (cyt c) as sensing element whose spectral response enables the detection of H2O2 down to a detection limit of 40 nM. This low detection limit is achieved by introducing cyt c in a random medium, enabling multiscattering that enhances the optical trajectory through the cyt c spot. A contact microspotting technique is used to produce reproducible and reusable cyt c spots which are stable for several days. Experiments in static and microfluidic regimes, as well as numerical simulations demonstrate the suitability of the cyt c/H2O2 reaction system for the real-time sensing of the kinetics of biological processes without H2O2 depletion in the measurement chamber. As an example, we detect the release of H2O2 from the green alga Chlamydomonas reinhardtii exposed to either 180 nM functionalized CdSe/ZnS core shell quantum dots, or to 10 mg/l TiO2 nanoparticles. The continuous measurement of extracellular H2O2 by this optical sensor with high sensitivity is a promising new means for real-time cytotoxicity tests, the investigation of oxidative stress and other physiological cell processes

New insights into ROS dynamics: a multi-layered microfluidic chip for ecotoxicological studies on aquatic microorganisms

Reactive oxygen species (ROS) play an important role in the life of every cell, including cellular defense and signaling mechanisms. Continuous and quantitative ROS sensing can provide valuable information about the cell state, but it remains a challenge to measure. Here, we introduce a multi-layered microfluidic chip with an integrated optical sensor for the continuous sensitive detection of extracellular hydrogen peroxide (H2O2), one of the most stable ROS. This platform includes hydraulically controlled microvalves and microsieves, which enable the precise control of toxicants and complex exposure sequences. In particular, we use this platform to study the dynamics of toxicity-induced ROS generation in the green microalga Chlamydomonas reinhardtii during short-term exposures, recovery periods, and subsequent re-exposures. Two cadmium-based toxicants with distinct internalization mechanisms are used as stress inducers: CdSe/ZnS quantum dots (Qdots) and ionic cadmium (Cd2+). Our results show the quantitative dynamics of ROS generation by the model microalga, the recovery of cell homeostasis after stress events and the cumulative nature of two consecutive exposures. The dissolution of quantum dots and its possible influence on toxicity and H2O2 depletion is discussed. The obtained insights are relevant from ecotoxicological and physiological perspectives

Non-invasive continuous monitoring of pro-oxidant effects of engineered nanoparticles on aquatic microorganisms

Engineered nanomaterials (ENMs) are key drivers for the development of highly sophisticated new technologies. As all new attainments, the rapidly increasing used of ENMs raise concerns about their safety for the environment and humans. There is growing evidence showing that if engineered nanomaterials are released into the environment, there is a possibility that they could cause harm to aquatic microorganisms. Among the divers effects triggering their toxicity the ability of ENMs to generate reactive oxygen species (ROS) capable of oxidizing biomolecules is currently considered a central mechanism of toxicity. Therefore, development of sensitive tools for quantification of the ROS generation and oxidative stress are highly sought. After briefly introducing ENMs-induced ROS generation and oxidative stress in the aquatic microorganisms (AMOs), this overview paper focuses on a new optical biosensor allowing sensitive and dynamic measurements of H2O2 in real-time using multiscattering enhanced absorption spectroscopy. Its principle is based on sensitive absorption measurements of the heme protein cytochrome c whose absorption spectrum alters with the oxidation state of constituent ferrous FeII and ferric FeIII. For biological applications cytochrome c was embedded in porous random media resulting in an extended optical path length through multiple scattering of light, which lowers the limit of detection to a few nM of H2O2. The sensor was also integrated in a microfluidic system containing micro-valves and sieves enabling more complex experimental conditions. To demonstrate its performance, abiotic absorption measurements of low concentrations of dye molecules and 10 nm gold particles were carried out achieving limits of detection in the low nM range. Other biologically relevant reactive oxygen species can be measured at sub-μM concentrations, which was shown for glucose and lactate through enzymatic reactions producing H2O2. In ecotoxicological investigations H2O2 excreted by aquatic microorganisms exposed to various stressors were measured. Pro-oxidant effects of nano-TiO2 and nano-CuO towards green alga Chlamydomonas reinhardtii were explored in various exposure media and under different light illuminations. Dynamics of Cd2+ induced effects on photosynthetic activity, sensitisation and recovery of cells of C. reinhardtii was also studied

Pro-oxidant effects of nano-TiO₂ on Chlamydomonas reinhardtii during short-term exposure

This study sheds light on the short-term dynamics of pro-oxidant processes related to the exposure of C. reinhardtii microalgae to nano-TiO2 using a) conventional fluorescent probes for cellular pro-oxidant process and b) a recently developed cytochrome c biosensor for the continuous quantification of H2O2. The main aims are to investigate nano-TiO2 toxicity and the modifying factors thereof based on the paradigm of oxidative stress and to explore the utility of extracellular H2O2 as a potential biomarker of the observed cellular responses. This is the first study to provide continuous quantitative data on abiotic and biotic nano-TiO2-driven H2O2 generation to systematically investigate the link between extracellular and cellular pro-oxidant responses. Acute exposures of 1 h were performed in two different exposure media (MOPS and lake water), with particle concentrations from 10 mg L-1 to 200 mg L-1, with and without UV pre-illumination. Abiotic and biotic extracellular H2O2 were continuously measured with the biosensor and complemented with endpoints for abiotic ROS (H2DCF-DA), oxidative stress (CellROX® Green) and damage (propidium iodide) measured by flow cytometry at the beginning and end of exposure. Results showed that extracellular and intracellular pro-oxidant processes differed and that extracellular H2O2 cannot per se serve as a predictor of cellular oxidative stress or damage. The main predictors best describing the cellular responses included “exposure medium, “exposure time”, “UV treatment” as well as “exposure concentration”

Ionic Strength-Mediated Phase Transitions of Surface-Adsorbed DNA on Single-Walled Carbon Nanotubes

Single-stranded DNA oligonucleotides have unique, and in some cases sequence-specific molecular interactions with the surface of carbon nanotubes that remain the subject of fundamental study. In this work, we observe and analyze a generic, ionic strength-mediated phase transition exhibited by over 25 distinct oligonucleotides adsorbed to single-walled carbon nanotubes (SWCNTs) in colloidal suspension. The phase transition occurs as monovalent salts are used to modify the ionic strength from 500 mM to 1 mM, causing a reversible reduction in the fluorescence quantum yield by as much as 90%. The phase transition is only observable by fluorescence quenching within a window of pH and in the presence of dissolved O₂, but occurs independently of this optical quenching. The negatively charged phosphate backbone increases (decreases) the DNA surface coverage on an areal basis at high (low) ionic strength, and is well described by a two-state equilibrium model. The resulting quantitative model is able to describe and link, for the first time, the observed changes in optical properties of DNA-wrapped SWCNTs with ionic strength, pH, adsorbed O₂, and ascorbic acid. Cytosine nucleobases are shown to alter the adhesion of the DNA to SWCNTs through direct protonation from solution, decreasing the driving force for this phase transition. We show that the phase transition also changes the observed SWCNT corona phase, modulating the recognition of riboflavin. These results provide insight into the unique molecular interactions between DNA and the SWCNT surface, and have implications for molecular sensing, assembly, and nanoparticle separations

Memristor Circuits for Colloidal Robotics: Temporal Access to Memory, Sensing, and Actuation

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/172294/1/aisy202100205.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/172294/2/aisy202100205-sup-0001-SuppData-S1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/172294/3/aisy202100205_am.pd