Loss of Panx1 function in zebrafish alters motor behavior in a lab-on-chip model of Parkinson's disease.

Pannexin 1 (Panx1) forms ATP-permeable membrane channels that play roles in purinergic signaling in the nervous system. A link between Panx1 activity and neurodegenerative disorders including Parkinson's disease (PD) has been suggested, but experimental evidence is limited. Here, a zebrafish model of PD was produced by exposing panx1a+/+ and panx1a-/- zebrafish larvae to 6-hydroxydopamine (6-OHDA). Electrical stimulation in a microfluidic chip and quantitative real-time-qPCR of zebrafish larvae tested the role of Panx1 in both pathological and normal conditions. After 72-h treatment with 6-OHDA, the electric-induced locomotor activity of 5 days post fertilization (5dpf) panx1a+/+ larvae were reduced, while the stimulus did not affect locomotor activity of age-matched panx1a-/- larvae. A RT-qPCR analysis showed an increase in the expression of genes that are functionally related to dopaminergic signaling, like the tyrosine hydroxylase (th2) and the leucine-rich repeat kinase 2 (lrrk2). Extending the 6-OHDA treatment duration to 120 h caused a significant reduction in the locomotor response of 7dpf panx1a-/- larvae compared to the untreated panx1a-/- group. The RT-qPCR data showed a reduced expression of dopaminergic signaling genes in both genotypes. It was concluded that the absence of Panx1a channels compromised dopaminergic signaling in 6-OHDA-treated zebrafish larvae and that the increase in the expression of dopaminergic genes was transient, most likely due to a compensatory upregulation. We propose that zebrafish Panx1a models offer opportunities to shed light on PD's physiological and molecular basis. Panx1a might play a role on the progression of PD, and therefore deserves further investigation.

Oscillating dispersed-phase co-flow microfluidic droplet generation: jet length reduction effect.

Microdroplet generation methods are assessed by two important criteria of droplet throughput and size dispersity. The widely-used co-flow droplet generation technique is bottlenecked with droplet polydispersity at high throughputs due to transition to an unstable jetting regime at high dispersed-phase (d-phase) flow rates. In this paper, we introduce a novel technique to oscillate the d-phase nozzle inside the continuous phase (c-phase) channel to suppress the jetting effect. The effect of the nozzle oscillation frequency (0-15 Hz) on the jet length was studied at different d-phase (Qd = 1.8, 2.4 and 3.0 ml min-1) and c-phase (Qc = 6, 12 and 18 ml min-1) flow rates and d-phase viscosities (1, 2.5, and 6 mPa s). The jet length was directly proportional to the d-phase flow rate and inversely proportional to the oscillation frequency. Oscillation-induced jet length reduction was more significant at high jet velocities, but a less steep jet length reduction was always observed at oscillation frequencies higher than 10 Hz. A maximum jet length reduction of 70.8% was obtained at the highest d-phase and lowest c-phase flow rates. Increasing the viscosity of the d-phase resulted in diminishing the effect of oscillation on jet length reduction. Moreover, we observed that nozzle oscillation could disintegrate the long jet into droplets of various sizes that were mostly smaller than the stationary-mode droplets. We hypothesize that oscillating the dispersion nozzle at lower flow rates, without the jetting effect, can simultaneously generate multi-size monodisperse droplets. This active technique can also be implemented into aqueous two-phase systems (ATPSs) in which droplet generation is a difficult task.

Microfluidic curved-channel centrifuge for solution exchange of target microparticles and their simultaneous separation from bacteria.

One of the common operations in sample preparation is to separate specific particles (e.g. target cells, embryos or microparticles) from non-target substances (e.g. bacteria) in a fluid and to wash them into clean buffers for further processing like detection (called solution exchange in this paper). For instance, solution exchange is widely needed in preparing fluidic samples for biosensing at the point-of-care and point-of-use, but still conducted via the use of cumbersome and time-consuming off-chip analyte washing and purification techniques. Existing small-scale and handheld active and passive devices for washing particles are often limited to very low throughputs or require external sources of energy. Here, we integrated Dean flow recirculation of two fluids in curved microchannels with selective inertial focusing of target particles to develop a microfluidic centrifuge device that can isolate specific particles (as surrogates for target analytes) from bacteria and wash them into a clean buffer at high throughput and efficiency. We could process micron-size particles at a flow rate of 1 mL min-1 and achieve throughputs higher than 104 particles per second. Our results reveal that the device is capable of singleplex solution exchange of 11 µm and 19 µm particles with efficiencies of 86 ± 2% and 93 ± 0.7%, respectively. A purity of 96 ± 2% was achieved in the duplex experiments where 11 µm particles were isolated from 4 µm particles. Application of our device in biological assays was shown by performing duplex experiments where 11 µm or 19 µm particles were isolated from an Escherichia coli bacterial suspension with purities of 91-98%. We envision that our technique will have applications in point-of-care devices for simultaneous purification and solution exchange of cells and embryos from smaller substances in high-volume suspensions at high throughput and efficiency.

Magneto-Hydrodynamic Fractionation (MHF) for continuous and sheathless sorting of high-concentration paramagnetic microparticles.

Sorting cells, microorganisms and particles from a solution is of paramount importance in many biological applications. An ideal sorting device should work at high throughput, involve simple design, avoid energy consumption, operate without a diluting sheath flow and perform separation with high purity. However, currently available sorting methods such as pinched flow fractionation, hydrodynamic filtration, magnetophoresis and deterministic lateral displacement meet only a few of the above-mentioned characteristics. In this paper, we report a hybrid technique combining magnetic focusing of particles in a thin microchannel and their hydrodynamic fractionation at a downstream expansion region, to devise a sheathless and high-throughput Magneto-Hydrodynamic Fractionation (MHF) method. First, sheathless magnetic focusing of 11 µm microparticles against the wall of the thin microchannel was investigated over a wide range of flow rates (0.5-5 mL h-1). Then, a mixture of 5 µm and 11 µm paramagnetic particles was injected into the device at a flow rate of 5 mL h-1 to demonstrate their sorting. Both of these magnetic particles were aligned along the wall of the channel and hence focused in the device, however their centers were lying on different streamlines due to their different sizes. Therefore, they were separated into distinct streamlines upon entering into the expansion region. Using this device, we achieved a high throughput sorting of more than 104 particles per second with an approximate on-chip fractionation purity of 98%. This technique has a great potential for separation of more than two magnetic particles for application in immunomagnetic affinity-based sorting of multiple biological substances.

A microfluidic device for quantitative investigation of zebrafish larvae's rheotaxis.

Zebrafish is a model organism for various sensory-motor biological studies. Rheotaxis, or the ability of zebrafish to orient and swim against the water stream, is a common behavior that involves multiple sensory-motor processes such as their lateral line and visual systems. Due to the lack of a controllable and easy-to-use assay, zebrafish rheotaxis at larval stages is not well-understood. In this paper, we report a microfluidic device that can be used to apply the flow stimulus precisely and repeatedly along the longitudinal axis of individual zebrafish larvae to study their coaxial rheotaxis. We quantified rheotaxis in terms of the response rate and location along the channel at various flow velocities (9.5-38 mm.sec-1). The larvae effectively exhibited a similarly high rheotactic response at low and medium velocities (9.5 and 19 mm.sec-1); however, at high velocity of 38 mm.sec-1, despite sensing the flow, their rheotactic response decreased significantly. The flow velocity also affected the response location along the channel. At 9.5 mm.sec-1, responses were distributed evenly along the channel length while, at 19 and 38 mm.sec-1, the larvae demonstrated higher rheotaxis responses at the anterior and posterior ends of the channel, respectively. This result shows that although the response is similarly high at low and medium flow velocities, zebrafish larvae become more sensitive to the flow at medium velocity, demonstrating a modulated rheotactic behavior. Employing our device, further investigations can be conducted to study the sensory-motor systems involved in rheotaxis of zebrafish larvae and other fish species.

High-throughput light sheet imaging of adult and larval C. elegans Parkinson's disease model using a low-cost optofluidic device and a fluorescent microscope.

Recent advancements at the interface of microfluidics technology and light sheet fluorescence microscopy have opened the door for high-throughput and high-content investigation of C. elegans disease models. In this paper, we report on the development of a simple, miniaturized, and low-cost optofluidic platform that can be added to a conventional inverted fluorescent microscope for continuous light sheet imaging of transgenic worm populations with high lateral and axial resolutions of 1.1 µm and 2.4 µm, respectively. The optofluidic device is made entirely of PDMS with integrated optics for light sheet generation. Laser excitation is delivered to the device via a low-cost free space laser, and cross-sections of worm populations are imaged as they pass continuously through a channel. Results show the platform can image NW1229 whole worms with pan-neural fluorescent expression at a throughput of >20 worms per minute at L3 and young adult (YA) stages. As a benchmark test, we show that the low-cost device can quantify the reduced neuronal expressions of L3 and YA NW1229 worms when exposed to 500 µM 6-OHDA neurodegenerative agent. Following the benchmark validation, we utilized the platform in a novel application for imaging human alpha-synuclein reporter in populations of Parkinson's transgenic model (ERS100). Results show the ability of the low-cost platform to reliably detect and quantify the anomalous neural phenotypic changes in ERS100 populations at L3 and YA stages with high spatial resolution. The findings of this study show the potential of our low-cost optofluidic add-on platform to equip conventional fluorescent microscopes with light sheet capability for quantitative phenotypic studies of transgenic C. elegans at high resolution and throughput.

Microfluidic Droplet-Generation Device with Flexible Walls.

Controlling droplet sizes is one of the most important aspects of droplet generators used in biomedical research, drug discovery, high-throughput screening, and emulsion manufacturing applications. This is usually achieved by using multiple devices that are restricted in their range of generated droplet sizes. In this paper, a co-flow microfluidic droplet-generation device with flexible walls was developed such that the width of the continuous (C)-phase channel around the dispersed (D)-phase droplet-generating needle can be adjusted on demand. This actuation mechanism allowed for the adjustment of the C-phase flow velocity, hence providing modulated viscous forces to manipulate droplet sizes in a single device. Two distinct droplet-generation regimes were observed at low D-phase Weber numbers, i.e., a dripping regime at high- and medium-channel widths and a plug regime at low-channel widths. The effect of channel width on droplet size was investigated in the dripping regime under three modes of constant C-phase flow rate, velocity, and Capillary number. Reducing the channel width at a constant C-phase flow rate had the most pronounced effect on producing smaller droplets. This effect can be attributed to the combined influences of the wall effect and increased C-phase velocity, leading to a greater impact on droplet size due to the intensified viscous force. Droplet sizes in the range of 175-913 µm were generated; this range was ~2.5 times wider than the state of the art, notably using a single microfluidic device. Lastly, an empirical model based on Buckingham's Pi theorem was developed to predict the size of droplets based on channel width and height as well as the C-phase Capillary and Reynolds numbers.

Recent advances in biosensors for real time monitoring of pH, temperature, and oxygen in chronic wounds.

Chronic wounds are among the major healthcare issues affecting millions of people worldwide with high rates of morbidity, losses of limbs and mortality. Microbial infection in wounds is a severe problem that can impede healing of chronic wounds. Accurate, timely and early detection of infections, and real time monitoring of various wound healing biomarkers related to infection can be significantly helpful in the treatment and care of chronic wounds. However, clinical methodologies of periodic assessment and care of wounds require physical visit to wound care clinics or hospitals and time-consuming frequent replacement of wound dressing patches, which also often adversely affect the healing process. Besides, frequent replacements of wound dressings are highly expensive, causing a huge amount of burden on the national health care systems. Smart bandages have emerged to provide in situ physiochemical surveillance in real time at the wound site. These bandages integrate smart sensors to detect the condition of wound infection based on various parameters, such as pH, temperature and oxygen level in the wound which reduces the frequency of changing the wound dressings and its associated complications. These devices can continually monitor the healing process, paving the way for tailored therapy and improved quality of patient's life. In this review, we present an overview of recent advances in biosensors for real time monitoring of pH, temperature, and oxygen in chronic wounds in order to assess infection status. We have elaborated the recent progress in quantitative monitoring of several biomarkers important for assessing wounds infection status and its detection using smart biosensors. The review shows that real-time monitoring of wound status by quantifying specific biomarkers, such as pH, temperature and tissue oxygenation to significantly aid the treatment and care of chronic infected wounds.

Microfluidic Sensor Based on Cell-Imprinted Polymer-Coated Microwires for Conductometric Detection of Bacteria in Water.

The rapid, inexpensive, and on-site detection of bacterial contaminants using highly sensitive and specific microfluidic sensors is attracting substantial attention in water quality monitoring applications. Cell-imprinted polymers (CIPs) have emerged as robust, cost-effective, and versatile recognition materials with selective binding sites for capturing whole bacteria. However, electrochemical transduction of the binding event to a measurable signal within a microfluidic device to develop easy-to-use, compact, portable, durable, and affordable sensors remains a challenge. For this paper, we employed CIP-functionalized microwires (CIP-MWs) with an affinity towards E. coli and integrated them into a low-cost microfluidic sensor to measure the conductometric transduction of CIP-bacteria binding events. The sensor comprised two CIP-MWs suspended perpendicularly to a PDMS microchannel. The inter-wire electrical resistance of the microchannel was measured before, during, and after exposure of CIP-MWs to bacteria. A decline in the inter-wire resistance of the sensor after 30 min of incubation with bacteria was detected. Resistance change normalization and the subsequent analysis of the sensor's dose-response curve between 0 to 109 CFU/mL bacteria revealed the limits of detection and quantification of 2.1 × 105 CFU/mL and 7.3 × 105 CFU/mL, respectively. The dynamic range of the sensor was 104 to 107 CFU/mL where the bacteria counts were statistically distinguishable from each other. A linear fit in this range resulted in a sensitivity of 7.35 µS per CFU/mL. Experiments using competing Sarcina or Listeria cells showed specificity of the sensor towards the imprinted E. coli cells. The reported CIP-MW-based conductometric microfluidic sensor can provide a cost-effective, durable, portable, and real-time solution for the detection of pathogens in water.

Low-Cost Resistive Microfluidic Salinity Sensor for High-Precision Detection of Drinking Water Salt Levels.

Rapid, inexpensive, and precise water salinity testing remains indispensable in water quality monitoring applications. Despite many sensors and commercialized devices to monitor seawater salinity, salt detection and quantification at very low levels of drinking water (below 120 ppm) have been overlooked. In this paper, we report on optimization of a low-cost microfluidic sensor to measure water salinity in the range of 1-120 ppm. The proposed design employs two copper microbridge wires suspended orthogonally in a PDMS microchannel to measure salinity based on the electrical resistance between the wires. The preliminary design of the sensor microchannel with a rectangular cross-section width (w) of 900 µm and height (h) of 500 µm could measure the water salinity in the range of 1-20 ppm in less than 1 min with detection sensitivity, limit of detection (LOD), and limit of quantification (LOQ) of 17.1 ohm/ohm·cm, 0.31 ppm, and 0.37 ppm, respectively. Data from the preliminary design was used for developing and validating a numerical model which was subsequently used for parametric studies and optimization to improve the sensor's performance. The optimized design demonstrated an order of magnitude increase in sensitivity (385 ohm/ohm·cm), a 6-fold wider detection range (1-120 ppm), and a 15-fold enhancement in miniaturization of the microfluidic channel (w = 200 µm and h = 150 µm) with LOD and LOQ of 0.39 and 0.44 ppm, respectively. In the future, the sensor can be integrated into a hand-held device to remove present impediments for low-cost and ubiquitous salinity surveillance of drinking water.

Dopaminergic signaling regulates zebrafish larvae's response to electricity.

Electrical stimulation of brain or muscle activities has gained attention for studying the molecular and cellular mechanisms involved in electric-induced responses. We recently showed zebrafish's response to electricity. Here, we hypothesized that this response is affected by the dopaminergic signaling pathways. The effects of multiple dopamine agonists and antagonists on the electric response of 6 days-postfertilization zebrafish larvae were investigated using a microfluidic device with enhanced control of experimentation and throughput. All dopamine antagonists decreased locomotor activities, while dopamine agonists did not induce similar behaviors. The D2-selective dopamine agonist quinpirole enhanced the movement. Exposure to nonselective and D1-selective dopamine agonists apomorphine and SKF-81297 caused no significant change in the electric response. Exposing larvae that were pretreated with nonselective and D2-selective dopamine antagonists butaclamol and haloperidol to apomorphine and quinpirole, respectively, restored the electric locomotion. These results reveal a correlation between electric response and dopamine signaling pathway. Furthermore, they demonstrate that electric-induced zebrafish larvae locomotion can be conditioned by modulating dopamine receptor functions. Our electrofluidic assay has profound application potential for fundamental electric-induced response research and brain disorder studies especially those related to the dopamine imbalance and as a chemical screening method when investigating biological pathways and behaviors.

Simultaneous screening of zebrafish larvae cardiac and respiratory functions: a microfluidic multi-phenotypic approach.

Multi-phenotypic screening of multiple zebrafish larvae plays an important role in enhancing the quality and speed of biological assays. Many microfluidic platforms have been presented for zebrafish phenotypic assays, but multi-organ screening of multiple larvae, from different needed orientations, in a single device that can enable rapid and large-sample testing is yet to be achieved. Here, we propose a multi-phenotypic quadruple-fish microfluidic chip for simultaneous monitoring of heart activity and fin movement of 5-7-day postfertilization zebrafish larvae trapped in the chip. In each experiment, fin movements of four larvae were quantified in the dorsal view in terms of fin beat frequency (FBF). Positioning of four optical prisms next to the traps provided the lateral views of the four larvae and enabled heart rate (HR) monitoring. The device's functionality in chemical testing was validated by assessing the impacts of ethanol on heart and fin activities. Larvae treated with 3% ethanol displayed a significant drop of 13.2 and 35.8% in HR and FBF, respectively. Subsequent tests with cadmium chloride highlighted the novel application of our device for screening the effect of heavy metals on cardiac and respiratory function at the same time. Exposure to 5 $\mu$g/l cadmium chloride revealed a significant increase of 8.2% and 39.2% in HR and FBF, respectively. The device can be employed to monitor multi-phenotypic behavioral responses of zebrafish larvae induced by chemical stimuli in various chemical screening assays, in applications such as ecotoxicology and drug discovery.

Designing microfluidic devices for behavioral screening of multiple zebrafish larvae.

BACKGROUND: Microfluidic devices are being used for phenotypic screening of zebrafish larvae in fundamental and pre-clinical research. A challenge for the broad use of these microfluidic devices is their low throughput, especially in behavioral assays. Previously, we introduced the tail locomotion of a semi-mobile zebrafish larva evoked on-demand with electric signal in a microfluidic device. Here, we report the lessons learned for increasing the number of specimens from one to four larvae in this device. METHODS AND RESULTS: Multiple parameters including loading and testing time per fish and loading and orientation efficiencies were refined to optimize the performance of modified designs. Flow and electric field simulations within the final device provided insight into the flow behavior and functionality of traps when compared to previous single-larva devices. Outcomes led to a new design which decreased the testing time per larva by ≈60%. Further, loading and orientation efficiencies increased by more than 80%. Critical behavioral parameters such as response duration and tail beat frequency were similar in both single and quadruple-fish devices. CONCLUSION: The developed microfluidic device has significant advantages for greater throughput and efficiency when behavioral phenotyping is required in various applications, including chemical testing in toxicology and gene screening.

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.

High-speed label-free confocal microscopy of Caenorhabditis elegans with near infrared spectrally encoded confocal microscopy.

Caenorhabditis elegans (C. elegans) is an optically transparent nematode that shares many gene orthologs and homologs with humans. C. elegans are widely used in large populations for genetic studies relevant to human biology and disease. Success of such studies frequently relies on the ability to image C. elegans structure at high-resolution and high-speed. In this manuscript, we report on the feasibility and suitability of a high-speed variant of reflectance confocal microscopy, known as spectrally encoded confocal microscopy (SECM), for label-free imaging of C. elegans. The developed system utilizes near-infrared illumination in conjunction with refractive and diffractive optics to instantaneously image a confocal image line at a speed of up to 147 kHz with lateral and axial resolutions of 2µm and 10µm, respectively. Our imaging results from wild-type C. elegans and four mutant strains (MT2124, MT1082, CB61, and CB648) demonstrate the ability of SECM in revealing the overall geometry, key internal organs, and mutation-induced structural variations, opening the door for downstream integration of SECM in microfluidic platforms for high throughput structural imaging of C. elegans.

Conventional and microfluidic methods for airborne virus isolation and detection.

With the COVID-19 pandemic, the threat of infectious diseases to public health and safety has become much more apparent. Viral, bacterial and fungal diseases have led to the loss of millions of lives, especially in the developing world. Diseases caused by airborne viruses like SARS-CoV-2 are difficult to control, as these viruses are easily transmissible and can circulate in the air for hours. To contain outbreaks of viruses such as SARS-CoV-2 and institute targeted precautions, it is important to detect them in air and understand how they infect their targets. Point-of-care (PoC) diagnostics and point-of-need (PoN) detection methods are necessary to rapidly test patient and environmental samples, so precautions can immediately be applied. Traditional benchtop detection methods such as ELISA, PCR and culture are not suitable for PoC and PoN monitoring, because they can take hours to days and require specialized equipment. Microfluidic devices can be made at low cost to perform such assays rapidly and at the PoN. They can also be integrated with air- and liquid-based sampling technologies to capture and analyze viruses from air and body fluids. Here, conventional and microfluidic virus detection methods are reviewed and compared. The use of air sampling devices to capture and concentrate viruses is discussed first, followed by a review of analysis methods such as immunoassays, RT-PCR and isothermal amplification in conventional and microfluidic platforms. This review provides an overview of the capabilities of microfluidics in virus handling and detection, which will be useful to infectious disease researchers, biomedical engineers, and public health agencies.

Low-cost optofluidic add-on enables rapid selective plane illumination microscopy of C. elegans with a conventional wide-field microscope.

SIGNIFICANCE: Selective plane illumination microscopy (SPIM) is an emerging fluorescent imaging technique suitable for noninvasive volumetric imaging of C. elegans. These promising microscopy systems, however, are scarce in academic and research institutions due to their high cost and technical complexities. Simple and low-cost solutions that enable conversion of commonplace wide-field microscopes to rapid SPIM platforms promote widespread adoption of SPIM by biologist for studying neuronal expressions of C. elegans. AIM: We sought to develop a simple and low-cost optofluidic add-on device that enables rapid and immobilization-free volumetric SPIM imaging of C. elegans with conventional fluorescent microscopes. APPROACH: A polydimethylsiloxane (PDMS)-based device with integrated optical and fluidic elements was developed as a low-cost and miniaturized SPIM add-on for the conventional wide-field microscope. The developed optofluidic chip contained an integrated PDMS cylindrical lens for on-chip generation of the light-sheet across a microchannel. Cross-sectional SPIM images of C. elegans were continuously acquired by the native objective of microscope as worms flowed in an L-shape microchannel and through the light sheet. RESULTS: On-chip SPIM imaging of C. elegans strains demonstrated possibility of visualizing the entire neuronal system in few seconds at single-neuron resolution, with high contrast and without worm immobilization. Volumetric visualization of neuronal system from the acquired cross-sectional two-dimensional images is also demonstrated, enabling the standard microscope to acquire three-dimensional fluorescent images of C. elegans. The full-width at half-maximum width of the point spread function was measured as 1.1 and 2.4 µm in the lateral and axial directions, respectively. CONCLUSION: The developed low-cost optofluidic device is capable of continuous SPIM imaging of C. elegans model organism with a conventional fluorescent microscope, at high speed, and with single neuron resolution.

Nanopore sensors for viral particle quantification: current progress and future prospects.

Rapid, inexpensive, and laboratory-free diagnostic of viral pathogens is highly critical in controlling viral pandemics. In recent years, nanopore-based sensors have been employed to detect, identify, and classify virus particles. By tracing ionic current containing target molecules across nano-scale pores, nanopore sensors can recognize the target molecules at the single-molecule level. In the case of viruses, they enable discrimination of individual viruses and obtaining important information on the physical and chemical properties of viral particles. Despite classical benchtop virus detection methods, such as amplification techniques (e.g., PCR) or immunological assays (e.g., ELISA), that are mainly laboratory-based, expensive and time-consuming, nanopore-based sensing methods can enable low-cost and real-time point-of-care (PoC) and point-of-need (PoN) monitoring of target viruses. This review discusses the limitations of classical virus detection methods in PoN virus monitoring and then provides a comprehensive overview of nanopore sensing technology and its emerging applications in quantifying virus particles and classifying virus sub-types. Afterward, it discusses the recent progress in the field of nanopore sensing, including integrating nanopore sensors with microfabrication technology, microfluidics and artificial intelligence, which have been demonstrated to be promising in developing the next generation of low-cost and portable biosensors for the sensitive recognition of viruses and emerging pathogens.

Microfluidic electric parallel egg-laying assay and application to in-vivo toxicity screening of microplastics using C. elegans.

Environmental pollutants like microplastics are posing health concerns on aquatic animals and the ecosystem. Microplastic toxicity studies using Caenorhabditis elegans (C. elegans) as a model are evolving but methodologically hindered from obtaining statistically strong data sets, detecting toxicity effects based on microplastics uptake, and correlating physiological and behavioural effects at an individual-worm level. In this paper, we report a novel microfluidic electric egg-laying assay for phenotypical assessment of multiple worms in parallel. The effects of glucose and polystyrene microplastics at two concentrations on the worms' electric egg-laying, length, diameter, and length contraction during exposure to electric signal were studied. The device contained eight parallel worm-dwelling microchannels called electric traps, with equivalent electrical fields, in which the worms were electrically stimulated for egg deposition and fluorescently imaged for assessment of neuronal and microplastic uptake expression. A new bidirectional stimulation technique was developed, and the device design was optimized to achieve a testing efficiency of 91.25%. Exposure of worms to 100 mM glucose resulted in a significant reduction in their egg-laying and size. The effects of 1 µm polystyrene microparticles at concentrations of 100 and 1000 mg/L on the electric egg-laying behaviour, size, and neurodegeneration of N2 and NW1229 (expressing GFP pan-neuronally) worms were also studied. Of the two concentrations, 1000 mg/L caused severe egg-laying deficiency and growth retardation as well as neurodegeneration. Additionally, using single-worm level phenotyping, we noticed intra-population variability in microplastics uptake and correlation with the above physiological and behavioural phenotypes, which was hidden in the population-averaged results. Taken together, these results suggest the appropriateness of our microfluidic assay for toxicological studies and for assessing the phenotypical heterogeneity in response to microplastics.

Integration of microfluidic sample preparation with PCR detection to investigate the effects of simultaneous DNA-Inhibitor separation and DNA solution exchange.

In this paper, we applied a curved-channel microfluidic device to separate DNA from PCR-inhibitor-containing water and simultaneously wash them into clean water for detection using a portable PCR thermocycler. Environmental DNA (eDNA) sampling has become an effective surveying approach for detecting rare organisms. However, low concentration eDNA molecules may be masked by PCR inhibitors during amplification and detection, increasing the risk of false negatives. Therefore, technologies for on-site DNA separation and washing are urgently needed. Our device consisted of a half-circle microchannel with a DNA-inhibitor sample inlet, a clean buffer inlet, and multiple outlets. By using the flow-induced inertial forces, 10 µm DNA-conjugated microparticles were focused at the inner-wall of the curved microchannel while separation from 1 µm inhibitor-conjugated microparticles and DNA washing were achieved simultaneously with the Dean flow. We achieved singleplex focusing, isolation and washing of 10 µm particles at an efficiency of 94.5 ± 2.0%. In duplex experiments with 1 µm and 10 µm particles, larger particles were washed with an efficiency of 92.1 ± 1.6% and a purity of 79 ± 2%. By surface-functionalizing the microparticles with affinity groups against Atlantic salmon DNA and humic acid (HA), and processing samples of various concentrations in our device, we achieved an effective purification and detection of DNA molecules using the portable PCR thermocycler. Our method significantly decreased PCR quantitation cycles from Cq > 38 to Cq = 30.35 ± 0.5, which confirmed enhancement of PCR amplification. The proposed device takes a promising step forward in sample preparation towards an integrated device that can be used for simultaneous purification and solution exchange of DNA in point-of-need environmental monitoring applications.