Transient isotachophoresis focusing of DNA and DNA-protein complexes is essentially enhanced by spontaneously dissolved aerial carbon dioxide in electrolytes.

ABSTRACT

The formation of a highly adapted high-E zone is critical to isotachophoresis separation and focusing. Recently, we discovered that the high-E zone is present only in a small portion of electrophoresis channel in the presence of EOF (Liu, S. Q. et al. J. Am. Chem. Soc. 2013, 135, 4644-4647). Accordingly, a much narrower high-E zone is presumably present in t-ITP. If so, it is hard to achieve efficient t-ITP focusing. Indeed, by online coupling t-ITP with CE-LIF immunoassay, the immunocomplexes of carcinogenic BPDE-dG adducts are not efficiently focused using a freshly prepared background electrolyte. Intriguingly, we observed that 20-day stored background electrolyte displays a 10-fold better focusing efficiency. We hypothesize that the unexpected phenomenon is associated with the dissolution of aerial carbon dioxide, which is mainly converted to ionic HCO3(-) in the weak alkaline background electrolyte. Consequently, HCO3(-) of high electrophoretic mobility will be continuously injected into the capillary along with the background electrolyte and act as an alternative leading ion to improve the focusing. By addition of dry ice (without causing significant pH decrease, ΔpH < 0.4) to freshly prepared background electrolytes, we immediately observed the enhanced focusing of immunocomplexes of the DNA adducts. NH4HCO3 and Na2CO3, included in the background electrolyte, also improve the focusing efficiency and reproducibility. All these consistently support our hypothesis. To understand the underlying mechanism, an advanced CE-SMFI was exploited to monitor in real time the motion of single DNA molecules and the E change throughout t-ITP. We uncovered that t-ITP can induce a local high-E zone, but the presence of HCO3(-) in the background electrolyte could greatly increase the E value in the high-E zone, which allows more DNA molecules to rapidly move backward and to be efficiently stacked at LE/TE boundary. This study provides new insight into nonuniform electric field-induced electrophoresis focusing.