Abstract:The continuous measurement of uptake or release of biomarkers provides invaluable information for understanding and monitoring the metabolism of cells. In this work, a multiscattering-enhanced optical biosensor for the multiplexed, non-invasive, and continuous detection of hydrogen peroxide (H2O2), lactate and glucose is presented. The sensing scheme is based on optical monitoring of the oxidation state of the metalloprotein cytochrome c (cyt c). The analyte of interest is enzymatically converted into H2O2 lea… Show more
“…For the sake of completeness, it should be mentioned that, due to diffusion issues, interferences were observed when placing the sensing elements for the substrate and H 2 O 2 in the same chamber. This problem was solved by adapting the microfluidic configuration to separate the sensing elements [40]. Finally, LODs as low as 240 and 110 nM for lactate and glucose, respectively, were achieved for the configuration at hand.Fig.…”
Section: Portable Setup and Microfluidic Chipmentioning
confidence: 99%
“… OECD Generation of intracellular ROS by HA [ 33 ] CdTe/CdS C. reinhardtii MES, MOPS, HEPES Oxdative stress [ 34 ] Al 2 O 3 , SiO 2 , ZnO and TiO 2 Chlorella sp. SE ROS may not be the dominant mechanism for algal growth inhibition [ 35 ] Ag C. vulgaris, Dunaliella tertiolecta Growth medium BG-11 ROS induced lipid peroxidation and a decrease of cell viability [ 36 ] Pt C. reinhardtii P. subcapitata ISO 8692 medium and 4-fold diluted tris-acetate-phosphate medium Substantial oxidative stress and negligible membrane damage; significant growth inhibition [ 30 ] Coated and uncoated CuO C. reinhardtii High salt medium ROS formation may be the primary toxicity mechanism [ 37 ] CeO 2 P. subcapitata Standard US EPA The oxidative activity is mediated by OH and initiation of lipid peroxidation [ 38 ] Core–shell CuO C. reinhardtii High salt growth medium ROS are responsible for chlorophyll deterioration, significant decrease of PSII primary photochemistry [ 39 ] CuO C. reinhardtii Various media, lake water Oxidative stress and damage of membrane integrity [ …”
Section: Enms and Oxidative Stress In Aquatic Microorganismsmentioning
confidence: 99%
“…Koman et al presented a detection scheme for sensitive and real-time detection of those metabolites [ 40 ]. Taking advantage of the above presented multiscattering approach they were detected with sub-micromolar LODs.…”
Section: Sensitive Real-time Detection Of H
2
O
mentioning
confidence: 99%
“…Furthermore, glucose and H 2 O 2 and lactate and H 2 O 2 were simultaneously measured adding glucose (GOx) and lactate oxidase (LOx), respectively, for the enzymatic conversion into H 2 O 2 [Eq. ( 4 )] [ 40 ]. Thus, to avoid that the fast conversion already takes place in the solution the oxidase was incorporated inside the sensing element.…”
Section: Portable Setup and Microfluidic Chipmentioning
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.
“…For the sake of completeness, it should be mentioned that, due to diffusion issues, interferences were observed when placing the sensing elements for the substrate and H 2 O 2 in the same chamber. This problem was solved by adapting the microfluidic configuration to separate the sensing elements [40]. Finally, LODs as low as 240 and 110 nM for lactate and glucose, respectively, were achieved for the configuration at hand.Fig.…”
Section: Portable Setup and Microfluidic Chipmentioning
confidence: 99%
“… OECD Generation of intracellular ROS by HA [ 33 ] CdTe/CdS C. reinhardtii MES, MOPS, HEPES Oxdative stress [ 34 ] Al 2 O 3 , SiO 2 , ZnO and TiO 2 Chlorella sp. SE ROS may not be the dominant mechanism for algal growth inhibition [ 35 ] Ag C. vulgaris, Dunaliella tertiolecta Growth medium BG-11 ROS induced lipid peroxidation and a decrease of cell viability [ 36 ] Pt C. reinhardtii P. subcapitata ISO 8692 medium and 4-fold diluted tris-acetate-phosphate medium Substantial oxidative stress and negligible membrane damage; significant growth inhibition [ 30 ] Coated and uncoated CuO C. reinhardtii High salt medium ROS formation may be the primary toxicity mechanism [ 37 ] CeO 2 P. subcapitata Standard US EPA The oxidative activity is mediated by OH and initiation of lipid peroxidation [ 38 ] Core–shell CuO C. reinhardtii High salt growth medium ROS are responsible for chlorophyll deterioration, significant decrease of PSII primary photochemistry [ 39 ] CuO C. reinhardtii Various media, lake water Oxidative stress and damage of membrane integrity [ …”
Section: Enms and Oxidative Stress In Aquatic Microorganismsmentioning
confidence: 99%
“…Koman et al presented a detection scheme for sensitive and real-time detection of those metabolites [ 40 ]. Taking advantage of the above presented multiscattering approach they were detected with sub-micromolar LODs.…”
Section: Sensitive Real-time Detection Of H
2
O
mentioning
confidence: 99%
“…Furthermore, glucose and H 2 O 2 and lactate and H 2 O 2 were simultaneously measured adding glucose (GOx) and lactate oxidase (LOx), respectively, for the enzymatic conversion into H 2 O 2 [Eq. ( 4 )] [ 40 ]. Thus, to avoid that the fast conversion already takes place in the solution the oxidase was incorporated inside the sensing element.…”
Section: Portable Setup and Microfluidic Chipmentioning
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.
“…58 Recent works also describe the application of optical biosensors for glucose and lactate for cell culture applications. 60 Compared to electrochemical biosensors, until now, they have not played an equally important role in metabolic monitoring in cell culture.…”
Microsensor systems for cell metabolism are essential tools for investigation and standardization in cell culture. Electrochemical and optical read-out schemes dominate, which enable the marker-free, continuous, online recording of transient effects and deliver information beyond microscopy and end-point tests. There has been much progress in microfluidics and microsensors, but the translation of both into standard cell culture procedures is still limited. Within this critical review, we discuss different cell culture formats ranging from standard culture vessels to dedicated microfluidic platforms. Key aspects are the appropriate supply of cells, mass transport of metabolites to the sensors and generation of stimuli. Microfluidics enable the transition from static to dynamic conditions in culture and measurement. We illustrate the parameters oxygen (respiration), pH (acidification), glucose and lactate (energy metabolism) as well as short-lived reactive species (ROS/RNS) from the perspective of microsensor integration in 2D and 3D cell culture. We discuss different sensor principles and types, along with their limitations, microfabrication technologies and materials. The state-of-the-art of microsensor platforms for cell culture is discussed with respect to sensor performance, the number of parameters and timescale of application. That includes the advances from 2D culture to the increasingly important 3D approaches, with specific requirements for organotypic microtissues, spheroids and solid matrix cultures. We conclude on the current progress, potential, benefits and limitations of cell culture monitoring systems from monolayer culture to organ-on-chip systems.
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