Advanced Correlation Spectroscopy and Nanofluidics in Single-Molecule Biophysics

by Damir Sakhapov
Doctoral thesis
Date of Examination:2025-03-28
Date of issue:2025-10-28
Advisor:Prof. Dr. Jörg Enderlein
Referee:Prof. Dr. Helmut Grubmüller
Referee:Prof. Dr. Marina Rodnina
Persistent Address: http://dx.doi.org/10.53846/goediss-11453

 

 

Files in this item

Name:PhD_Thesis_Sakhapov.pdf
Size:13.2Mb
Format:PDF

The following license files are associated with this item:

Abstract

English

Fluorescence Correlation Spectroscopy (FCS) is a powerful and widelyused technique in biophysics for studying biomolecules and their interactionsat the single-molecule level. However, traditional FCS hasnotable limitations. First, it is highly sensitive to imperfections in theoptical system, which are often unavoidable in practical applications.In this work, we extend the use and uncover new applications of advancedFCS techniques—including antibunching FCS, PhotoinducedElectron Transfer (PET) FCS, Dual-Color FCS (2fFCS), and RotationalDiffusion FCS—to investigate a range of phenomena relevant to the lifesciences. These include the photophysics of fluorescent molecules, theintramolecular dynamics of Intrinsically Disordered Proteins (IDPs),and the docking-undocking dynamics of ribosomes with translocons.Secondly, while FCS techniques provide valuable insights, they arefundamentally ensemble-averaging approaches. True single-moleculetechniques, such as the Anti-Brownian Electrokinetic (ABEL) trap andsingle-molecule burst analysis, have emerged over the past decadesbut remain limited in widespread use due to the complexity of therequired instrumentation. Recent advancements in nanofluidics, fueledby knowledge transfer from the field of microfabrication to the lifesciences, offer promising solutions. To harness this potential, we havedeveloped a nanofluidic-based fabrication process from the groundup, aiming to simplify instrumentation and make single-molecule biophysicsmore accessible. Our ultimate objectives include the trappingof single biomolecules within nanochannels and measuring electriccharges of bimolecules under physiological conditions.
Keywords: Biophysics; Nanofluidics; FCS; Nanofabrication
 
Statistik