The development of miniaturized devices for studying zebrafish embryos has been limited due to complicated fabrication and operation processes. Here, we reported on a microfluidic device that enabled the capture and culture of zebrafish embryos and real-time monitoring of dynamic embryonic development. The device was simply fabricated by bonding two layers of polydimethylsiloxane (PDMS) structures replicated from three-dimensional (3D) printed reusable molds onto a flat glass substrate. Embryos were easily loaded into the device with a pipette, docked in traps by gravity, and then retained in traps with hydrodynamic forces for long-term culturing. A degassing chamber bonded on top was used to remove air bubbles from the embryo-culturing channel and traps so that any embryo movement caused by air bubbles was eliminated during live imaging. Computational fluid dynamics simulations suggested this embryo-trapping and -retention regime to exert low shear stress on the immobilized embryos. Monitoring of the zebrafish embryogenesis over 20 h during the early stages successfully verified the performance of the microfluidic device for culturing the immobilized zebrafish embryos. Therefore, this rapid-prototyping, low-cost and easy-to-operate microfluidic device offers a promising platform for the long-term culturing of immobilized zebrafish embryos under continuous medium perfusion and the high-quality screening of the developmental dynamics.

This work presents a microfluidic device, which was patterned with (i) microstructures for hydrodynamic capture of single particles and cells, and (ii) multiplexing microelectrodes for selective release via negative dielectrophoretic (nDEP) forces and electrical impedance measurements of immobilized samples. Computational fluid dynamics (CFD) simulations were performed to investigate the fluidic profiles within the microchannels during the hydrodynamic capture of particles and evaluate the performance of single‐cell immobilization. Results showed uniform distributions of velocities and pressure differences across all eight trapping sites. The hydrodynamic net force and the nDEP force acting on a 6 μm sphere were calculated in a 3D model. Polystyrene beads with difference diameters (6, 8, and 10 μm) and budding yeast cells were employed to verify multiple functions of the microfluidic device, including reliable capture and selective nDEP‐release of particles or cells and sensitive electrical impedance measurements of immobilized samples. The size of immobilized beads and the number of captured yeast cells can be discriminated by analyzing impedance signals at 1 MHz. Results also demonstrated that yeast cells can be immobilized at single‐cell resolution by combining the hydrodynamic capture with impedance measurements and nDEP‐release of unwanted samples. Therefore, the microfluidic device integrated with multiplexing microelectrodes potentially offers a versatile, reliable, and precise platform for single‐cell analysis.

High‐resolution microscopic imaging may cause intensive image processing and potential impact of light irradiation on yeast replicative lifespan (RLS). Electrical impedance spectroscopy (EIS) could be alternatively used to perform high‐throughput and label‐free yeast RLS assays. Prior to fabricating EIS‐integrated microfluidic devices for yeast RLS determination, systematic modeling and theoretical investigation are crucial for device design and optimization. Here, we report three‐dimensional (3D) finite‐element modeling and simulations of EIS measurement in a microfluidic single yeast in situ impedance array (SYIIA), which is designed by patterning an electrode matrix underneath a cell‐trapping array. SYIIA was instantiated and modeled as a 5 × 5 sensing array comprising 25 units for cell immobilization, culturing, and time‐lapse EIS recording. Simulations of yeast growing and budding in a sensing unit demonstrated that EIS signals enable the characterization of cell growth and daughter‐cell dissections. In the 5 × 5 sensing array, simulation results indicated that when monitoring a target cell, daughter dissections in its surrounding traps may induce variations of the recorded EIS signals, which could cause mistakes in identifying target daughter‐cell dissections. To eliminate the mis‐identifications, electrode array pitch was optimized. Therefore, the results could conduct the design and optimization of microfluidic electrode‐array‐integrated devices for high‐throughput and accurate yeast RLS assays.

In this work, we report on the design of a wide-band digital lock-in amplifier (DLIA) of up to 65 MHz and its application for electrical impedance measurements in microfluidic devices. The DLIA is comprised of several dedicated technologies. First, it features a fully differential analog circuit, which includes a preamplifier with a low input noise of 4.4 nV/√Hz, a programmable-gain amplifier with a gain of 52 dB, and an anti-aliasing, fully differential low-pass filter with −76 dB stop-band attenuation. Second, the DLIA has an all-digital phase lock loop, which features a phase deviation of less than 0.02° throughout the frequency range. The phase lock loop utilizes an equally accurate period-frequency measurement, with a sub-ppm precision of frequency detection. Third, a modified clock link is implemented in the DLIA to improve the signal-to-noise ratio of the analog-to-digital converter affected by clock jitter of up to 20 dBc. A series of measurements were performed to characterize the DLIA, and the results showed an accurate performance. Additionally, impedance measurements of standard-size microparticles were performed by frequency sweep from 300 kHz to 30 MHz, using the DLIA in a microfluidic device. Different diameters of microparticle could be accurately distinguished according to the relative impedance at 2.5 MHz. The results confirm the promising applications of the DLIA in microfluidic electrical impedance measurements.

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Background Budding yeast, Saccharomyces cerevisiae, has been extensively favored as a model organism in aging and age-related studies, thanks to versatile microfluidic chips for cell dynamics assay and replicative lifespan (RLS) determination at single-cell resolution. However, previous microfluidic structures aiming to immobilize haploid yeast may impose excessive spatial constraint and mechanical stress on cells, especially for larger diploid cells that sprout in a bipolar pattern. Results We developed a high-throughput microfluidic chip for diploid yeast long-term culturing (DYLC), optical inspection and cell-aging analysis. The DYLC chip features 1100 “leaky bowl”-shaped traps formatted in an array to dock single cells under laminar-perfused medium and effectively remove daughter cells by hydraulic shear forces. The delicate microstructures of cell traps enable hydrodynamic rotation of newborn buds, so as to ensure bud reorientation towards downstream and concerted daughter dissection thereafter. The traps provide sufficient space for cell-volume enlargement during aging, and thus properly alleviate structural compression and external stress on budding yeast. Trapping efficiency and long-term maintenance of single cells were optimized according to computational fluid dynamics simulations and experimental characterization in terms of critical parameters of the trap and array geometries. Owing to the self-filling of daughter cells dissected from traps upstream, an initial trapping efficiency of about 70% can rapidly reach a high value of over 92% after 4-hour cell culturing. During yeast proliferation and aging, cellular processes of growth, budding and daughter dissection were continuously tracked for over 60 h by time-lapse imaging. Yeast RLS and budding time interval (BTI) were directly calculated by the sequential two-digit codes indicating the budding status in images. With the employed diploid yeast strain, we obtained an RLS of 24.29 ± 3.65 generations, and verified the extension of BTI in the first couple of generations after birth and the last several generations approaching death, as well as cell de-synchronization along diploid yeast aging. Conclusions The DYLC chip offers a promising platform for reliable capture and culturing of diploid yeast cells and for life-long tracking of cell dynamics and replicative aging processes so that grasping comprehensive insights of aging mechanism in complex eukaryotic cells. Graphical Abstract

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