Single-Cell Chemical Lysis Method for Analyses of Intracellular Molecules Using an Array of Picoliter-Scale Microwells

Abstract

Analyzing the intracellular contents and enzymatic activities of single cells is important for studying the physiological and pathological activities at the cellular level. For this purpose, we developed a simple single-cell lysis method by using a dense array of microwells of 10-30-pL volume fabricated by poly(dimethylsiloxane) (PDMS) and a commercially available cell lysis reagent. To demonstrate the performance of this single-cell lysis method, we carried out two different assays at the single-cell level: detection of proteins by antibody conjugated microbeads and measurement of protease activity by fluorescent substrates. The results indicated that this method readily enabled us to monitor protein levels and enzymatic activities in a single cell. Because this method required only an array of PDMS microwells and a fluorescence microscope, the simplicity of this platform opens a way to explore the biochemical characteristics of single cells even by those who are not familiar with microfluidic technology.

Review of methods to probe single cell metabolism and bioenergetics

“…In these, a cell suspension is introduced over the microwell array, and cell loading in the microwells commonly occurs by sedimentation (gravity) (Parce et al, 1989). Single cell immobilization is ensured by employing appropriate seeding cell concentration, seeding time and microwell dimensions (Rettig and Folch, 2005; Sasuga et al, 2008) (Fig. 3b).…”

Basic Technologies for Droplet Microfluidics

“…Other advantages of droplet-based [76] microfluidic system include precisely controllable reaction time inside a droplet by adjusting the length of channel and the flow rates of fluids. The availability of a wide range of technologies for flexible generation and manipulation of droplets has enabled the applications of droplet microfluidics in a wide variety of fields, from chemical reactions [9,79] and protein crystallization [10,11] to material synthesis [12,13,[80][81][82][83], single cell analysis [84][85][86][87][88][89][90][91], DNA amplification [16], protein engineering [62,92], and high-throughput screening technologies [17,93]. It is not possible to cover all application areas in this review.…”

Analytical Chemistry on the Femtoliter Scale

“…PDMS arrays of approximately 20 000 wells with pL volumes were used to isolate single pheochromcytoma cells from rats. [70] These cells were lysed individually in the microwells to determine intracellular protein concentrations or enzymatic activities. Cells expressing the recombinant fusion protein FLAG-GST were lysed in the presence of surface-immobilized antibody-labeled beads in the PDMS container.…”