Simultaneous high-throughput particle-bacteria separation and solution exchange via in-plane and out-of-plane parallelization of microfluidic centrifuges.

Inertial microfluidic devices have gained attention for point-of-need (PoN) sample preparation. Yet, devices capable of simultaneous particle-bacteria solution exchange and separation are low in throughput, hindering their applicability to PoN settings. This paper introduces a microfluidic centrifuge for high-throughput solution exchange and separation of microparticles, addressing the need for processing large sample volumes at elevated flow rates. The device integrates Dean flow recirculation and inertial focusing of microparticles within 24 curved microchannels assembled in a three-layer configuration via in-plane and out-of-plane parallelization. We studied solution exchange and particle migration using singleplex and duplex samples across devices with varying curve numbers (2-curve, 8-curve, and 24-curve). Processing 5 and 10 µm microparticles at flow rates up to 16.8 ml/min achieved a solution exchange efficiency of 96.69%. In singleplex solutions, 10 and 5 µm particles selectively migrated to inner and outer outlets, demonstrating separation efficiencies of 99.7% and 90.3%, respectively. With duplex samples, sample purity was measured to be 93.4% and 98.6% for 10 and 5 µm particles collected from the inner and the outer outlets, respectively. Application of our device in biological assays was shown by performing duplex experiments where 10 µm particles were isolated from Salmonella bacterial suspension with purity of 97.8% while increasing the state-of-the-art particle solution exchange and separation throughput by 16 folds. This parallelization enabled desirable combinations of high throughput, low-cost, and scalability, without compromising efficiency and purity, paving the way for sample preparation at the PoN in the future.

An in-vivo microfluidic assay reveals cardiac toxicity of heavy metals and the protective effect of metal responsive transcription factor (MTF-1) in Drosophila model.

Previous toxicity assessments of heavy metals on Drosophila are limited to investigating the survival, development rate, and climbing behaviour by oral administration while cardiac toxicity of these elements have not been investigated. We utilized a microfluidic device to inject known dosages of zinc (Zn) or cadmium (Cd) into the larvae's hemolymph to expose their heart directly and study their heart rate and arrhythmicity. The effect of heart-specific overexpression of metal responsive transcription factor (MTF-1) on different heartbeat parameters and survival of Drosophila larvae was investigated. The heart rate of wild-type larvae decreased by 24.8% or increased by 11.9%, 15 min after injection of 40 nL of 100 mM Zn or 10 mM Cd solution, respectively. The arrhythmicity index of wild-type larvae increased by 58.2% or 76.8%, after injection of Zn or Cd, respectively. MTF-1 heart overexpression ameliorated these effects completely. Moreover, it increased larvae's survival to pupal and adulthood stages and prolonged the longevity of flies injected with Zn and Cd. Our microfluidic-based cardiac toxicity assay illustrated that heart is an acute target of heavy metals toxicity, and MTF-1 overexpression in this tissue can ameliorate cardiac toxicity of Zn and Cd. The method can be used for cardiotoxicity assays with other pollutants in the future. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03336-7.

Open access tool and microfluidic devices for phenotypic quantification of heart function of intact fruit fly and zebrafish larvae.

In this paper, the heartbeat parameters of small model organisms, i.e. Drosophila melanogaster (fruit fly) and Danio rerio (zebrafish), were quantified in-vivo in intact larvae using microfluidics and a novel MATLAB-based software. Among different developmental stages of flies and zebrafish, the larval stage is privileged due to biological maturity, optical accessibility, and the myogenic nature of the heart. Conventional methods for parametric quantification of heart activities are complex and mostly done on dissected, irreversibly immobilized, or anesthetized larvae. Microfluidics has helped with reversible immobilization without the need for anesthesia, but heart monitoring is still done manually due to challenges associated with the movement of floating organs and cardiac interruptions. In our MATLAB software applied to videos recorded in microfluidic-based whole-organism assays, we have used image segmentation to automatically detect the heart and extract the heartbeat signal based on pixel intensity variations of the most contractile region of the heart tube. The smoothness priors approach (SPA) was applied to remove the undesired low-frequency noises caused by environmental light changes or heart movement. Heart rate and arrhythmicity were automatically measured from the detrended heartbeat signal while other parameters including end-diastolic and end-systolic diameters, shortening distance, shortening time, fractional shortening, and shortening velocity were quantified for the first time in intact larvae, using M-mode images under bright field microscopy. The software was able to detect more than 94% of the heartbeats and the cardiac arrests in intact Drosophila larvae. Our user-friendly software enables in-vivo quantification of D. melanogaster and D. rerio larval heart functions in microfluidic devices, with the potential to be applied to other biological models and used for automatic screening of drugs and alleles that affect their heart.

Localized microinjection of intact Drosophila melanogaster larva to investigate the effect of serotonin on heart rate.

In this paper, we present a novel hybrid microfluidic device for localized microinjection and heart monitoring of intact Drosophila melanogaster larvae at different developmental stages. Drosophila heart at the larval stage has been used as a model for cardiac disorder studies. However, previous pharmacological and toxicological cardiac studies are limited to dissected (semi-intact) Drosophila larvae which cannot be used for post-treatment studies. Challenges associated with microinjection of intact larvae include delicate handling of individual larvae, proper orientation for microneedle penetration, localized microinjection with controlled amount of chemicals into the hemolymph and reversible immobilization for post-injection phenotypic studies, all addressed by our microfluidic device. Larva loading and orientation were achieved by glass capillaries integrated into the PDMS microfluidic device. Side suction channels were used for immobilization prior to heart activity recording. Localized microinjection was achieved with a one degree-of-freedom microneedle and a custom-made pressure driven reagent delivery system, without any adverse effect on heart rate and animal viability. Precision in localized injection into the body cavity close to the heart chamber or the fat body was demonstrated with our microfluidic device. A MATLAB-based heartbeat quantification technique was used to investigate the dose-dependent effect of serotonin (5-hydroxytryptamine), a neurotransmitter, on the heart rate of intact Drosophila larvae, for the first time. Injection of 40 nL serotonin with ≥0.01 mM concentration significantly increased the heart rate of 3rd instar larvae by 21 ± 7% (SEM). Injection of 5 nL serotonin with a concentration of 0.01 mM significantly increased the heart rate of 2nd instar larvae by 12 ± 3% (SEM). The proposed microfluidic injection and heartbeat monitoring technique can be used for dye angiography and hemolymph circulation studies as well as screening intravenous drugs in vivo using the whole-animal Drosophila melanogaster.

Fly-on-a-Chip: Microfluidics for Drosophila melanogaster Studies.

The fruit fly or Drosophila melanogaster has been used as a promising model organism in genetics, developmental and behavioral studies as well as in the fields of neuroscience, pharmacology, and toxicology. Not only all the developmental stages of Drosophila, including embryonic, larval, and adulthood stages, have been used in experimental in vivo biology, but also the organs, tissues, and cells extracted from this model have found applications in in vitro assays. However, the manual manipulation, cellular investigation and behavioral phenotyping techniques utilized in conventional Drosophila-based in vivo and in vitro assays are mostly time-consuming, labor-intensive, and low in throughput. Moreover, stimulation of the organism with external biological, chemical, or physical signals requires precision in signal delivery, while quantification of neural and behavioral phenotypes necessitates optical and physical accessibility to Drosophila. Recently, microfluidic and lab-on-a-chip devices have emerged as powerful tools to overcome these challenges. This review paper demonstrates the role of microfluidic technology in Drosophila studies with a focus on both in vivo and in vitro investigations. The reviewed microfluidic devices are categorized based on their applications to various stages of Drosophila development. We have emphasized technologies that were utilized for tissue- and behavior-based investigations. Furthermore, the challenges and future directions in Drosophila-on-a-chip research, and its integration with other advanced technologies, will be discussed.