Ultraviolet Photostability Improvement for Autofluorescence Correlation Spectroscopy on Label-Free Proteins

TL;DR

This paper presents a novel method to enhance the photostability of label-free proteins for UV fluorescence correlation spectroscopy by combining enzymatic oxygen scavengers, antioxidants, and triplet state quenchers, enabling single-molecule detection.

Contribution

The study introduces a general photostability enhancement strategy for protein autofluorescence, allowing detection of proteins with fewer tryptophan residues without external labels.

Findings

Achieved UV fluorescence correlation spectroscopy on proteins with only 24 tryptophan residues.

Significantly increased protein photostability and autofluorescence detection rate.

Demonstrated the generality of photochemical concepts from organic dyes to proteins.

Abstract

The poor photostability and low brightness of protein autofluorescence have been major limitations preventing the detection of label-free proteins at the single molecule level. Overcoming these issues, we report here a strategy to promote the photostability of proteins and use their natural tryptophan autofluorescence in the ultraviolet (UV) for fluorescence correlation spectroscopy (FCS). Combining enzymatic oxygen scavengers with antioxidants and triplet state quenchers greatly promotes the protein photostability, reduces the photobleaching probability and improves the net autofluorescence detection rate. Our results show that the underlying photochemical concepts initially derived for organic visible fluorescent dyes are quite general. Using this approach, we achieved UV fluorescence correlation spectroscopy on label-free streptavidin proteins containing only 24 tryptophan residues, 6.5x less than the current state-of-the-art. This strategy greatly extends the possibility to detect single label-free proteins with the versatility of single molecule fluorescence without requiring the presence of a potentially disturbing external fluorescent marker. It also opens new perspectives to improve the UV durability of organic devices.