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We postulate that significant environmental effects such as altered motor functions, physiological alterations reflected in heart rate, effects on development and reproduction can occur at sub-lethal concentrations well below than LC10. Behavioral studies can, therefore, provide a valuable integrative link between physiological and ecological effects. Despite the advantages of behavioral analysis development of behavioral toxicity, biotests is greatly hampered by the lack of dedicated laboratory automation, in particular, user-friendly and automated video microscopy systems.
In this work we present a proof-of-concept development of an optical system capable of tracking embryonic vertebrates behavioral responses using automated and vastly miniaturized time-resolved video-microscopy. We have employed miniaturized CMOS cameras to perform high definition video recording and analysis of earliest vertebrate behavioral responses. The main objective was to develop a biocompatible embryo positioning structures that were suitable for high-throughput imaging as well as video capture and video analysis algorithms. This system should support the development of sub-lethal and behavioral markers for accelerated environmental monitoring.
The main limitation, however, of virtually all polymers that are used to both manufacture LOC devices as well as to provide fluidic interconnects is their significant hydrophobicity. Conventionally the hydrophobic properties were postulated to impede wetting and priming of the polymeric chip-based devices and tubing interconnects. Such issues were often solved with plasma treatment or ethanol priming to help wet the polymeric substrata and also reduce the nucleation and persistence of air bubbles.
In this work, we present evidence that use of certain hydrophobic polymers is a significant impediment in performing ecotoxicity tests of organic chemicals on biomicrofluidic devices. We report on electrostatic interaction between polymers and toxicants that lead to non-covalent adsorption and rapid depletion of chemicals from the tested media. This introduces a significant bioanalytical bias irrespectively of the fact that microfluidic tests are preformed under continuous perfusion.
Compared with other rapid prototyping technologies such as soft lithography and infrared laser micromachining in PMMA, SLA and MJM systems can enable user-friendly production of prototypes, superior feature reproduction quality and comparable levels of optical transparency. Prospectively they can revolutionize fabrication of microfluidic devices with complex geometric features and eliminate the need to use clean room environment and conventional microfabrication techniques.
In this work we demonstrate preliminary data on toxicity profiling of a panel of common polymers used in 3D printing applications. The main motivation of our work was to evaluate toxicity profiles of most commonly used polymers using standardized biotests according to OECD guidelines for testing of chemic risk assessment. Our work for the first time provides a multispecies view of potential dangers and limitation for building biocompatible devices using FDM, SLA and MJM additive manufacturing systems. Our work shows that additive manufacturing holds significant promise for fabricating LOC and MEMS but requires caution when selecting systems and polymers due to toxicity exhibited by some 3D printing polymers.
We developed an agarose based mounting system that holds the embryos in defined positions along removable strips. Homeostasis and temperature control is ensured by channels circulating buffer and heated water. This allows to image up to 120 larvae simultaneously for more than two days. Its flexibility and the low-volume, high larvae ratio will allow screening of small compound libraries. Taken together, we offer a low cost, highly adaptable solution for long term in-vivo imaging.
In this work, we present a pilot development of miniaturized Lab-on-a-Chip (LOC) devices for studying toxin avoidance behaviors of small aquatic crustaceans. As an investigative tool, LOCs represent a new direction that may miniaturize and revolutionize behavioral ecotoxicology. Specifically our innovative microfluidic prototype: (i) enables convening “caging” of specimens for real-time videomicroscopy; (ii) eliminates the evaporative water loss thus providing an opportunity for long-term behavioral studies; (iii) exploits laminar fluid flow under low Reynolds numbers to generate discrete domains and gradients enabling for the first time toxin avoidance studies on small aquatic crustaceans; (iv) integrates off-the-chip mechatronic interfaces and video analysis algorithms for single animal movement analysis. We provide evidence that by merging innovative bioelectronic and biomicrofluidic technologies we can deploy inexpensive and reliable systems for culture, electronic tracking and complex computational analysis of behavior of bioindicator organisms.
In this work, we present development of a miniaturized Lab-on-a-Chip (LOC) platform for automation and enhancement of acute ecotoxicity test based on immobilization of a freshwater crustacean Daphnia magna (Daphtoxkit-FTM). Daphnids’ immobilization in response to sudden changes in environment parameters is fast, unambiguous, and easy to record optically. We also for the first time demonstrate that LOC system enables studies of sub-lethal ecotoxic effects using behavioral responses of Daphnia magna as sentinels of water pollution.
The system working principle incorporated a high definition (HD) time-resolved video data analysis to dynamically assess impact of the reference toxicant on swimming behavior of D. magna. Our system design combined: (i) microfluidic device for caging of Daphnia sp.; (ii) mechatronic interface for fluidic actuation; (iii) video data acquisition; and (iv) algorithms for animal movement tracking and analysis.
Non-invasive and real-time visualisation of metabolic activities in living small organisms such as zebrafish embryo and larvae has not yet been attempted due to profound analytical limitations of existing technologies. Significant progress in the development of physico-optical oxygen sensors using luminescence quenching by molecular oxygen has recently been made. Sensing using such microsensors is, however, still performed in small glass chambers that hold single specimens and thus not amenable for high-throughput data acquisition.
In this work, we present a proof-of-concept approach by using microfluidic Lab-on-a-Chip (LOC) technologies combined with sophisticated optoelectronic sensors. The LOC device is capable of immobilising live zebrafish embryos with continuous flow perfusion, while the sensor uses innovative Fluorescence Ratiometric Imaging (FRIM) technology that can kinetically quantify the temporal patterns of aqueous oxygen gradients at a very fine scale based on signals coming from an optical sensor referred to as a sensor foil. By embedding the sensor foil onto the microfluidic living embryo array system, we demonstrated in situ FRIM on developing zebrafish embryos. Future integration of microfluidic chip-based technologies with FRIM technology represents a noteworthy direction to miniaturise and revolutionise research on metabolism and physiology in vivo.
Traditional marine ecotoxicity testing is inherently labor intensive, requiring extensive manual procedures both to set up the tests and more importantly to collect experimental readouts. Moreover, static test procedures offer poor control of water parameters such as toxicant concentration and dissolved oxygen, which are important considerations in evaluating environmental impacts of aquatic pollution. So far only minimal levels of automation have been adopted in ecotoxicology. Our current work attempts to address the current limitations by capitalizing on latest advances in microfluidics, 3D printing and laser micromachining technologies to develop highly customized, low cost and high-throughput devices.
Here we for the first time introduce a proof-of-concept laboratory automation system to perform ecotoxicity tests on the marine amphipod Allorchestes compressa in a microfluidic environment. Our innovative system incorporated a microperfusion Lab-on-a-Chip device that enabled the biotests to be run in both closed- or open-loop regimens. Miniaturized video cameras were utilized to monitor the amphipods movement patterns during the experiments. Furthermore innovative video analysis algorithms was applied for detection of sub-lethal endpoints such as changes in swimming activity that would otherwise go unnoticed. A key advantage of this flow-through system as compared to conventional approach is the automation of analysis and emphasis on sub-lethal behavioral parameters.
We present preliminary data validating the technology and compared to a gold standard method for testing organisms from the order Amphipoda This work provides a foundation to enable automation of ecotoxicity biotests performed on marine test organisms.
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