Real-time pH microscopy down to the molecular level by combined scanning electrochemical microscopy/single-molecule fluorescence spectroscopy.
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.
Boldt
Frank-Mario
FM
Freiburger Materialforschungszentrum (FMF), Institut für Physikalische Chemie I, Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany.
Heinze
Jürgen
J
Diez
Manuel
M
Petersen
Jan
J
Börsch
Michael
M
D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D001692:Biological Transport; D002384:Catalysis; D003911:Dextrans / Q000737:chemistry*; D004563:Electrochemistry; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D020527:Microscopy, Scanning Probe / Q000295:instrumentation / Q000379:methods; D017785:Photons; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism; D012996:Solutions / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods; D013499:Surface Properties
D003911:Dextrans; D012996:Solutions; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases