Real-Time Monitoring of Dissection Events of Single Budding Yeast in a Microfluidic Cell-Culturing Device Integrated With Electrical Impedance Biosensor

Abstract

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.

Keywords: dissection event; electrical impedance spectroscopy (EIS); microfluidic biosensor; replicative lifespan; single-cell analysis (SCA); yeast aging.

FIGURE 1

Schematics of EIS-biosensor-integrated microdevice for real-time monitoring of single budding yeast cells. (A) Top view and Cross-sectional view along CC′ in the impedance sensing area of the microfluidic device. Insert shows the impedance sensing unit with an immobilized budding yeast cell. For better illustration, dimensions are not to scale. (B) In-situ sensing principle of the yeast dissection event using the electrical impedance biosensor. The instantaneous detachment of daughter cell results in a drastic increase (from 2 to 3) of EIS signal amplitude.

FIGURE 2

(A) Photograph of an assembled microfluidic device indicating fluidic connections for inlets and outlets. (B) Micrograph of a sensing unit: a bottleneck-like trap immobilized with a single yeast cell. Scale bar is 10 μm.

FIGURE 3

Time-lapse images at 10-min intervals of single budding yeast cells during continuous culturing. (A) Budding and daughter dissection of an immobilized yeast cell in one generation. (B) Budding and daughter dissections of an immobilized yeast cell in two generations with successful reorientation of newborn buds towards the side channel by flowing fluid. Scale bar is 4 μm.

FIGURE 4

Real-time EIS monitoring of budding yeast growth and division with 4 daughter-dissection events in sequence. (A) Raw amplitude curve of EIS signals at 1 MHz along the recording time period. Sampling interval is 2 min (B) “Dissection Index” curve combined with K-means cluster analysis clearly showing 4 sharp spikes corresponding to daughter-dissection events of the recorded mother cell. Insets show micrographs of the yeast mother cell right before and after daughter removal. Scale bar is 4 μm.