Single-Cell Point Constrictions for Reagent-Free High-Throughput Mechanical Lysis and Intact Nuclei Isolation.

Xiaomin Huang,Levent Yobas,Chun Ning Ng,Xiaoxing Xing

doi:10.3390/mi10070488

Abstract

Highly localized (point) constrictions featuring a round geometry with ultra-sharp edges in silicon have been demonstrated for the reagent-free continuous-flow rapid mechanical lysis of mammalian cells on a single-cell basis. Silicon point constrictions, robust structures formed by a single-step dry etching process, are arranged in a cascade along microfluidic channels and can effectively rupture cells delivered in a pressure-driven flow. The influence of the constriction size and count on the lysis performance is presented for fibroblasts in reference to total protein, DNA, and intact nuclei levels in the lysates evaluated by biochemical and fluoremetric assays and flow-cytometric analyses. Protein and DNA levels obtained from an eight-constriction treatment match or surpass those from a chemical method. More importantly, many intact nuclei are found in the lysates with a relatively high nuclei-isolation efficiency from a four-constriction treatment. Point constrictions and their role in rapid reagent-free disruption of the plasma membrane could have implications for integrated sample preparation in future lab-on-a-chip systems.

Highlights

Cell lysis—the process of breaking open cells by disrupting the cytoplasmic membrane to release the cellular content—is the first step in “sample preparation”, whether it be for the extraction and purification of nucleic acids or proteins or the fractionation of subcellular organelles
We have demonstrated continuous-flow mechanical cell lysis and intact nuclei isolation using silicon point constrictions
Single-cell constrictions are conducive to isolating intact nuclei because of their characteristic geometry

Summary

Introduction

Cell lysis—the process of breaking open cells by disrupting the cytoplasmic membrane to release the cellular content—is the first step in “sample preparation”, whether it be for the extraction and purification of nucleic acids or proteins or the fractionation of subcellular organelles (e.g., mitochondria). Automating the “sample preparation” without compromising the quantity (yield), as well as the quality (undenatured state), is desired to address the bottleneck issue in accessing subcellular targets To this end, researchers have turned to microfluidic systems and investigated various forms of on-chip cell lysis for obtaining a fully integrated lab-on-a-chip sample preparation. Collisions with particles (bead beating) on a spinning platform [17,18] and compression under a deforming diaphragm [19] were shown to be effective for mechanically rupturing cells While each of these approaches has its own merits, limitations do exist, including the denaturation of proteins due to excessive heat or pH, a low throughput, a tight operating window, and the requirement of reagents or special hardware operated by a skilled user

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Conclusion