Real‐Time Chiral Metabolic Monitoring of Single Cell Using Microchip Electrophoresis Coupled with Electrospray Ionization Mass Spectrometry

Abstract

Cells are extremely complex and dynamic biochemical entities. Significant biochemical heterogeneity can exist among cells of the same type. This heterogeneity may arise via many mechanisms, including localized damage, mutations, stages in the cell cycle, and differential exposure to external signals among others. Unfortunately, when working with measurement approaches that report average values for larger numbers of cells, critical information is often not obtained. To understanding the chemical differences between cell types, the techniques that can examine only one cell at a time (namely single cell analysis) are necessary. Single cell analysis can provide an insight to the detailed chemistry information of a cell on individual, which allows exploration of the cells and the relationship to their overall pathological mechanism. However, single cell analysis is an emerging field requiring a high level interdisciplinary collaboration to provide detailed insights into the complex organisation, function and heterogeneity of life. And the cells are generally on the order of 5–20 μm in diameter. Many of the macromolecules of interest are present in low copy numbers, so the techniques which can handle such small volumes without significant dilution and which can be directly integrated with ultrasensitive detection schemes are required. Single‐cell methods for genome sequence, transcrip‐tomes, small‐volume analysis of cell‐cell signaling molecules, metabolites, cell fates, DNA methyla‐tion, yeast proteome dynamics, mass spectrometry (MS) imaging, and chromosome conformation have been reported. However, the technologies for real‐time chiral metabolic monitoring in single cell are still a great challenge.

Recent advances in microchip enantioseparation and analysis

“…Other patterns for channel layouts have also been employed, such as specific designs for 2D separation [48], double-T layouts [39], and extended separation channels [53]. In addition, chips with multiple layers have also been designed to integrate other functionalities, for example, automated peak collection/injection for performing consecutive achiral and chiral separations [32], and the integration of cell transport and separation channels with a microchamber for hypoxia stimulation [34]. Side channels have been added to avoid interference with the detection system [31,33,34], and to introduce makeup flows that generate electrosprays and stabilize ionization through nanoemitters for dead-volume free coupling to MS [31,33,34].…”

“…In addition, chips with multiple layers have also been designed to integrate other functionalities, for example, automated peak collection/injection for performing consecutive achiral and chiral separations [32], and the integration of cell transport and separation channels with a microchamber for hypoxia stimulation [34]. Side channels have been added to avoid interference with the detection system [31,33,34], and to introduce makeup flows that generate electrosprays and stabilize ionization through nanoemitters for dead-volume free coupling to MS [31,33,34]. The connecting channel has also been used to enable solvent compatibility, from an organic solvent-based Mannich reaction media to an aqueous electrophoretic buffer without disturbance [53].…”

“…The connecting channel has also been used to enable solvent compatibility, from an organic solvent-based Mannich reaction media to an aqueous electrophoretic buffer without disturbance [53]. Additional microchannels/microchambers used for various functions, for example, for cell culture [31,33,34] and for chemical reactions [53], have also been explored. To provide a near comprehensive overview of enantiomeric separations using microchip-based electro-driven methods from recent years, the main aspects of the cited references are collected in Table 1.…”

“…The chosen modes of separation and types of CSs are critical factors that contribute to developing a reliable enantioselective microchip electroseparation-based analytical platform. To date, a wide variety of chiral compounds and samples have been investigated by microchip enantioseparation, such as therapeutic drugs [22][23][24][25][26][27][28][29][30], neurotransmitters [31][32][33][34], biomolecules in general [22,26,27,29,30,[35][36][37][38][39][40][41][42][43][44][45][46][47][48][49], other small molecules [30,[50][51][52], and chemical reaction products [53,54].…”

Mass Spectrometry for Single-Cell Analysis

“…ESI-MS technique is widely used for the detection and identification of molecules through MS/MS or MS n analysis (Sirikatitham et al 2007). ESI-based MS techniques are explored for the analysis of single-cell metabolome during the last decade (Abed et al 2017;Li et al 2016;Lombard-Banek et al 2016;Pan et al 2014;Yu et al 2014). For the detection of positive ions, the capillary of the instrument is kept at the positive charge, or a counter electrode can also be applied for the electrospray ionization process.…”