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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.

Microfluidic devices in combination with fluorescent microscopy offer high-resolution and high-content platforms to study single-cell morphology, behavior and dynamic process in replicative aging of budding yeast, Saccharomyces cerevisiae. However, a huge mass of recorded images makes the data processing labor-intensive and time-consuming to determine yeast replicative lifespan (RLS), a primary criterion in yeast aging. To address this limitation and pursue label-free RLS assays, electrical impedance spectroscopy (EIS) that can be easily functionalized through microelectrodes in microfluidic devices, was introduced to monitor cell growth and division of budding yeast. Herein, a microfluidic device integrated with EIS biosensor was proposed to perform in-situ impedance measurement of yeast proliferation in single-cell resolution so as to identify the momentary events of daughter dissection from its mother. Single yeast cells were reliably immobilized at the bottleneck-like traps for continuous culturing, during which daughter cells were effectively detached from their mother cells by hydraulic shear forces. Time-lapse impedance measurement was performed every 2 min to monitor the cellular process including budding, division and dissection. By using the K-means clustering algorithm to analyze a self-defined parameter “Dissection Indicator,” to our knowledge for the first time, the momentary event of a daughter removing from its mother cell was accurately extracted from EIS signals. Thus, the identification of daughter dissection events based on impedance sensing technology has been validated. With further development, this microfluidic device integrated with electrical impedance biosensor holds promising applications in high-throughput, real-time and label-free analysis of budding yeast aging and RLS.

To facilitate in situ comparative culturing of budding yeast cells in a precisely controlled microenvironment, we developed a microfluidic single-cell array (MiSCA) with 96 traps (16 rows × 6 columns) for single-cell immobilization. Through optimization of the distances between neighboring traps and the applied flow rates by using a hydraulic equivalent circuit of the fluidic network, yeast cells were delivered to each column of the array by laminar focused flows and reliably captured at the traps by hydrodynamic forces with about 90% efficiency of cell immobilization. Immobilized cells in different columns within the same device can then be cultured in parallel while being exposed to different media and compounds delivered by laminar flows. For biological validation of the comparative cell-culturing device, we used budding yeast that can express yellow fluorescent protein upon the addition of β-estradiol in cell-culturing medium. Experimental results show successful induction of fluorescence in cells immobilized in desired columns that have been dosed with β-estradiol. The MiSCA system allows for performing sets of experiments and control experiments in parallel in the same device, or for executing comparative experiments under well-defined laminar-perfusion conditions with different media, as well as in situ monitoring of dynamic cellular responses upon different analytical compounds or reagents for single-cell analysis.

Microfluidic methodologies allow for automatic and high-throughput replicative lifespan (RLS) determination of single budding yeast cells. However, the resulted RLS is highly impacted by the robustness of experimental conditions, especially the microfluidic yeast-trapping structures, which are designed for cell retention, growth, budding, and daughter cell dissection. In this work, four microfluidic yeast-trapping structures, which were commonly used to immobilize mother cells and remove daughter cells for entire lifespan of budding yeast, were systematically investigated by means of finite element modeling (FEM). The results from this analysis led us to propose an optimized design, the yeast rotation (YRot) trap, which is a “leaky bowl”–shaped structure composed of two mirrored microcolumns facing each other. The YRot trap enables stable retention of mother cells in its “bowl” and hydrodynamic rotation of buds into its “leaky orifice” such that matured progenies can be dissected in a coincident direction. We validated the functions of the YRot trap in terms of cell rotation and daughter dissection by both FEM simulations and experiments. With the integration of denser YRot traps in microchannels, the microfluidic platform with stable single-yeast immobilization, long-term cell culturing, and coincident daughter dissection could potentially improve the robustness of experimental conditions for precise RLS determination in yeast aging studies.

This chapter describes an electrode-integrated microfluidic system with multiple functions of manipulating and monitoring single S. cerevisiae cells. In this system, hydrodynamic trapping and negative dielectrophoretic (nDEP) releasing of S. cerevisiae cells are implemented, providing a flexible method for single-cell manipulation. The multiplexing microelectrodes also enable sensitive electrical impedance spectroscopy (EIS) to discern the number of immobilized cells, classify different orientations of captured cells, as well as detect potential movements of immobilized single yeast cells during the overall recording duration by using principal component analysis (PCA) in data mining. The multifrequency EIS measurements can, therefore, obtain sufficient information of S. cerevisiae cells at single-cell level.

This chapter describes an electrode-integrated microfluidic system with multiple functions of manipulating and monitoring single S. cerevisiae cells. In this system, hydrodynamic trapping and negative dielectrophoretic (nDEP) releasing of S. cerevisiae cells are implemented, providing a flexible method for single-cell manipulation. The multiplexing microelectrodes also enable sensitive electrical impedance spectroscopy (EIS) to discern the number of immobilized cells, classify different orientations of captured cells, as well as detect potential movements of immobilized single yeast cells during the overall recording duration by using principal component analysis (PCA) in data mining. The multifrequency EIS measurements can, therefore, obtain sufficient information of S. cerevisiae cells at single-cell level.

This paper presents a microfluidic device for the capture and culture of zebrafish embryos and real-time monitoring of early-stage embryonic development. Innovative claims include: (i) rapid-prototyping and low-cost fabrication and easy-to-operate device; (ii) the use of a degassing chamber to remove air bubbles during live imaging; and (iii) the optimal embryo-trapping regime to reduce potential shear stress on zebrafish embryos. Long-term monitoring of zebrafish embryogenesis at early stages verifies that the microfluidic device enables the reliable capture and culturing of zebrafish embryos under continuous medium perfusion and high-quality screening of their 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.

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/&radic, Hz, a programmable-gain amplifier with a gain of 52 dB, and an anti-aliasing, fully differential low-pass filter with &minus, 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&deg, 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.

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., Micromachines, 10 (3), ISSN:2072-666X