Next, we monitored the pH value evolution in a flow cell typical for single-molecule experiments (see Figure S3) by using the ratiometric, dual-emission dye SNARF-1 (seminaphtharhodafluor), (14) which changes its emission by protonation or deprotonation of side groups with a p K a ≈ 7.6. Therefore, efficient oxygen scavenging at constant and well-defined pH conditions, with arbitrary buffer strength and pH, is of high priority. (9) The need for high buffer concentrations, however, prevents the use of low ionic strength conditions, yet increased salt concentrations have been associated with the precipitation of proteins, (10) thereby interfering with some single-molecule experiments. The activities of many biomolecules and cells studied in single-molecule experiments are both pH and salt sensitive, while fluorophores may show unfavorable pH-dependent blinking behavior. If not buffered sufficiently, this acid production will lead to a continuous pH drop over time, hence changing the experimental conditions. However, in both cases, the substrate oxidation produces carboxylic acids ( d-gluconic acid and 3,3-carboxy- cis, cis-muconic acid, respectively, Figure 1a, reactions 1 and 2).
![oxygen scavengers oxygen scavengers](http://www.boilerchemicals.com/v/vspfiles/photos/CW-O2-2T.jpg)
![oxygen scavengers oxygen scavengers](https://5.imimg.com/data5/UC/LL/MY-45749351/oxygen-scavenger-chemical-500x500.jpg)
The two most common oxygen scavenger systems are glucose oxidase in combination with catalase (GOC) and the recently introduced protocatechuate-dioxygenase (PCD), (7, 8) with β- d-glucose and 3,4-protocatechuic acid (PCA) as substrate, respectively. This enhanced stability allows the observation of bionanotechnological assemblies in aqueous environments under well-defined conditions for an extended time. 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. We show that POC keeps the pH stable over hours, while GOC and PCD cause an increasing acidity of the buffer system. 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.
![oxygen scavengers oxygen scavengers](http://3.imimg.com/data3/HY/WU/MY-3561838/oxygen-scavengers-250x250.jpg)
These acids can result in a significant pH drop over the course of experiments and must thus be compensated by an increased buffer strength. One of the pitfalls of these systems, however, is the production of carboxylic acids. The most common oxygen scavengers for single-molecule experiments are glucose oxidase and catalase (GOC) or protocatechuate dioxygenase (PCD). Many of these biomolecule-based assemblies are characterized using various single-molecule techniques that require strict anaerobic conditions. Over the past years, bottom-up bionanotechnology has been developed as a promising tool for future technological applications.