Enzymatic oxygen scavenging for photostability without pH drop in single-molecule experiments

Abstract

Over the past years, bottom-up bionanotechnology has been developed as a promising tool for future technological applications. Many of these biomolecule-based assemblies are characterized using various single-molecule techniques that require strict anaerobic conditions. The most common oxygen scavengers for single-molecule experiments are glucose oxidase and catalase (GOC) or protocatechuate dioxygenase (PCD). One of the pitfalls of these systems, however, is the production of carboxylic acids. These acids can result in a significant pH drop over the course of experiments and must thus be compensated by an increased buffer strength. Here, we present pyranose oxidase and catalase (POC) as a novel enzymatic system to perform single-molecule experiments in pH-stable conditions at arbitrary buffer strength. We show that POC keeps the pH stable over hours, while GOC and PCD cause an increasing acidity of the buffer system. We further verify in single-molecule fluorescence experiments that POC performs as good as the common oxygen-scavenging systems, but offers long-term pH stability and more freedom in buffer conditions. This enhanced stability allows the observation of bionanotechnological assemblies in aqueous environments under well-defined conditions for an extended time.

Figure 1

(a) GOC, PCD, and POC reactions. (b) Time series of fluorescence spectra of SNARF-1 in solution with GOC and 10 mM Tris:HCl-buffer. An emission spectrum was recorded every 2 min (blue to red). (c) pH value evolution for GOC (dashed), PCD (dotted), and POC (solid) with different starting pH. The grayed area indicates saturation of the SNARF-1 sensor.

Figure 2

(a) Setup sketch for single-molecule bleaching experiments and flow cell design. (b) Normalized lifetimes of Cy3 fluorophores at different starting pH for GOC, POC, and PCD. (c) Cy5 fluorophore lifetimes, same conditions as (b). Error bars are standard error; molecule and field of view counts in Supporting Information.

Figure 3

(a) Molecular blinking characterized by the off-time/on-time ratio before bleaching, averaged over Cy3 molecules at starting pH 7.5, after 1, 5, 30, and 120 min of flushing the buffer into the experimental chamber, for GOC (blue), POC (green), and PCD (red). (b) Same as (a) for Cy5. (c, d) Example traces of single-molecule fluorescence intensity with GOC at starting pH 7.5, 1 min after flush-in, for Cy3 and Cy5, respectively. The dashed line represents the limit for considering the molecule in “off” versus “on”-state. (e, f) Example traces under the same conditions 120 min after flush-in with GOC imaging buffer, for Cy3 and Cy5, respectively.