Real-time pH microscopy down to the molecular level by combined scanning electrochemical microscopy/single-molecule fluorescence spectroscopy

Analytical Chemistry
Frank-Mario BoldtMichael Börsch

Abstract

A new technique combining scanning electrochemical microscopy (SECM) and single-molecule fluorescence spectroscopy was developed to accomplish locally and temporally defined pH adjustments in buffer solutions and on surfaces monitored by fluorescence alteration of pH-sensitive fluorophores in real time. Local pH gradients were created by electrochemical generation of H(+) or OH(-) during redox reactions at ultramicro- or nanoelectrodes with radii from 5 microm to 35 nm. Ratiometric fluorescence measurements were performed with a confocal laser microscope using two detectors for different spectral regions. Time-resolved pH measurements were carried out with freely diffusing SNARF-1-dextran. For pH measurements on surfaces, total internal reflection fluorescence microscopy was used in combination with a CCD camera. The fluorophore SNAFL-succinimidyl ester was bound to amino-terminated octadecylsilane-coated coverslips. Local pH determinations could be accomplished with an accuracy of 0.2 unit. The measured pH profiles showed a strong dependence on the tip diameter, the buffer/mediator concentration ratio, and the tip-surface distance. As an application for bionanotechnology using SECM-induced pH changes on the molecular level, th...Continue Reading

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Citations

Jul 21, 2006·Analytical and Bioanalytical Chemistry·Shigeru AmemiyaDarrick A Gross
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Dec 1, 2015·Analytical Chemistry·Rui Hao, Bo Zhang
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Jul 9, 2010·Analytical Chemistry·Bikash Kumar JenaBo Zhang

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