Real-Time pH Microscopy down to the Molecular Level by Combined Scanning Electrochemical Microscopy/Single-Molecule Fluorescence Spectroscopy

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, the proton-driven ATP synthesis by single membrane-bound F(0)F(1)-ATP synthases was investigated. ATP synthesis resulted in stepwise subunit rotation within the enzyme that was monitored by single-molecule fluorescence resonance energy transfer.

Elektrochemische Rastermikroskopie zur direkten Abbildung von Reaktionsgeschwindigkeiten

“…[450] Sehr interessant ist auch die Kombination von SECM und konfokaler Mikroskopie, weil die konfokale Mikroskopie eine dreidimensionale Vermessung von Diffusionschichten ermöglicht, [451] die mit der lateralen Auflösung des SECM ohne weitere Modifikationen kompatibel ist. [451,452] Darüber hinaus können photochemische Anregungsprozesse elektrochemisch verfolgt oder lokale Variationen der Lösungs-zusammensetzung als Triebkraft für Grenzflächenreaktionen genutzt und dann optisch charakterisiert werden. [452] So erzeugten Boldt et al an UMEs eine lokale pH-Variation, die das Enzym ATP-Synthase an der Probenoberfläche antrieb.…”

“…[451,452] Darüber hinaus können photochemische Anregungsprozesse elektrochemisch verfolgt oder lokale Variationen der Lösungs-zusammensetzung als Triebkraft für Grenzflächenreaktionen genutzt und dann optisch charakterisiert werden. [452] So erzeugten Boldt et al an UMEs eine lokale pH-Variation, die das Enzym ATP-Synthase an der Probenoberfläche antrieb. [452] Die Bewegung der ATP-Synthase konnte durch Messungen des resonanten Fluoreszenzenergietransfers (FRET) simultan verfolgt werden.…”

Evidence for major structural changes in subunit C of the vacuolar ATPase due to nucleotide binding

“…FCS data were recorded in 50 mM Tris/HCl, pH 8.5 and 100 mM NaCl with the fluorophore concentration of approximately 100 pM, 3.8 mM CaCl 2 , and protein concentrations between 7 and 410 lM, respectively. The fluorescence of single molecules was measured with a confocal setup of local design based on an inverted microscope (IX71; Olympus) similar to [20,21]. Fluorescence excitation with epi-illumination was performed with an argon ion laser (Spectra Physics, Germany) at 488 nm for BODIPY-FL-ATP, or 514,5 nm for Atto-532-ADP, respectively.…”

Electrochemical Biosensors - Sensor Principles and Architectures

“…Reproduced with permission. SECM) [159,160,161], fluorescence spectroscopy (FS-SECM) [162], photoelectrochemical microscopy (PEM) or near-field scanning optical microscopy (NSOM-SECM) [163], electrogenerated chemiluminescence (ECL-SECM) [164] and atomic force microscopy (AFM-SECM) [153,154,165]. The most widely used of these hybrid techniques would be AFM-SECM.…”