A Three-Dimensional Flow Control Concept for Single-Cell Experiments on a Microchip. 2. Fluorescein Diacetate Metabolism and Calcium Mobilization in a Single Yeast Cell As Stimulated by Glucose and pH Changes

Abstract

Using a three-dimensional flow control concept to manipulate and retain a single yeast cell in a microchip, we were able to study the kinetics of intracellular metabolism and calcium mobilization at the single-cell level, as stimulated by glucose and pH changes. As a model study, the fluorogenic substrate fluorescein diacetate (FDA) was chosen to study how the intracellular carboxylesterase metabolize it. A single yeast cell was first cultured in the microchip. Thereafter, under a constant concentration of FDA, influx of FDA into the yeast cell occurred and FDA was hydrolyzed or metabolized. It was found that changes in both pH and glucose stimulated the FDA metabolism in a yeast cell, and the stimuli can elicit multiple responses from the cell. Since it was carried out within the microchip, the whole experiment on one single yeast cell could last for as long as 10 h. The dormant cell, budding cell, and pretreated budding cell (in low-pH buffer) of yeast resulted in different responses. Experimental data provided details on the FDA metabolism at the single-cell level and revealed strong correlations between FDA metabolism and calcium mobilization. Furthermore, efflux of the FDA metabolite fluorescein could start spontaneously if there was glucose in the medium. The experiments on a single cell were of the "human cell conservation" style because the cell responded to the reagent changes implemented by the human researcher. A mathematical model was also developed to study the influx-hydrolysis-efflux processes of the FDA metabolism using single-cell fluorescent data. These long overdue single-cell experiments are now rendered possible using the three-dimensional flow control in the microchip.

A microfluidic‐based system for analysis of single cells based on Ca2+ flux

“…When ionomycin permeated into CHO cells it caused cells to release Ca 21 from internal stores into the cytosol and the released Ca 21 would quickly combine with the calcium indicator, Fluo-4 AM. The reason for the signal decline following its maximum is unclear, although it is possible that an efflux of Ca 21 and/or the calcium indicator from the cell may occur, which in turn will reduce the fluorescent signal observed in the cell [36].…”

Microfluidic-Based Single-Cell Study: Current Status and Future Perspective

“…Peng et al reported a PDMS-based microfluidic device for the detection of intracellular calcium mobilization and fluorescein diacetate (FDA) at single-cell level. In their work, the variation of PH and glucose and the retaining of single cells within microchannels were all controlled by 3D liquid flow [ 104 , 105 ]. They further developed a CD-like microfluidic device with spiral microchannels for DNA hybridizations under similar principle and manipulation [ 106 ].…”

Development of Yeast Molecular Display Systems Focused on Therapeutic Proteins, Enzymes, and Foods: Functional Analysis of Proteins and its Application to Bioconversion

“…Previously, a single yeast cell cultivation system using a microchamber array had not been reported because the small amount of medium in the microscale cultivation dried up. In a different type of system, another yeast cell cultivation system was also reported [129,130], which involved a liquid flow channel system with a small tank within a modified glass slide for the cocultivation of a number of yeast cells. The purpose of the system was to observe cell cycles and the fluorescence of cells within a small cultivation bath.…”