Proteins govern life. They carry oxygen, catalyse reactions, transmit signals, and form the structural fabric of every cell. Understanding how individual proteins behave, how they fold, interact, and function is fundamental to medicine, diagnostics, and drug discovery. Yet most tools available today require attaching a synthetic fluorescent dye to a protein before it can be detected. This labelling step, while effective, is not neutral: it can alter the protein's shape, disrupt its binding interactions, and distort the very behaviour it was designed to reveal.

Single-molecule fluorescence techniques like FCS, FRET, FLIM, and STED have transformed our understanding of molecular biology. However, they almost universally depend on extrinsic fluorescent labels. The field has long sought an alternative rooted in a simple observation: proteins are not dark. Aromatic amino acids like tryptophan, tyrosine, and phenylalanine emit an autofluorescence under ultraviolet (UV) illumination. More than 90% of all proteins contain at least one such residue.

Exploiting this intrinsic UV autofluorescence for single-molecule detection has been a long-standing challenge. Proteins are orders of magnitude dimmer than conventional dyes, and UV microscopy introduces specific difficulties: rapid photobleaching, high background from buffer and optics, and the absence of nanophotonic platforms optimised for the UV spectral range. Early efforts were limited to large proteins carrying dozens of tryptophan residues, a restriction incompatible with most biologically relevant targets.

Recent years have seen decisive progress. The Wenger group at Institut Fresnel demonstrated that UV-optimised plasmonic nanoantennas, including aluminium nanoapertures, optical horn antennas, and rhodium nanocube dimers, can enhance UV autofluorescence sufficiently to resolve single proteins containing as few as one tryptophan residue (Roy et al., Nano Letters 2023; Barulin et al., Nature Communications 2022). Zero-mode waveguides (ZMWs), nanoscale apertures that confine light below the diffraction limit, have also been shown to boost brightness and enable operation at physiologically relevant micromolar concentrations (Barulin et al., Nano Letters 2019). In parallel, plasmonic nanopores have emerged as powerful single-molecule sensors capable of monitoring the translocation of individual biomolecules with optical readout, moving beyond the limitations of conventional ionic current detection (Verschueren et al., ACS Nano 2019; Zhao et al., ACS Photonics 2022).

Despite these advances, combining UV autofluorescence with nanophotonic enhancement and nanopore-based translocation in a unified, label-free platform remains an open frontier and is precisely what this project addresses.

My project under DYNAMO is centred on UV autofluorescence correlation spectroscopy (UV-FCS), a technique that measures the natural UV emission of individual proteins as they diffuse through a tiny illuminated volume, revealing information about their concentration, size, and dynamics. The intrinsic signal is weak, so two complementary strategies are deployed: reducing noise and amplifying the signal.

First is suppressing the background with FLCS. Fluorescence Lifetime Correlation Spectroscopy (FLCS) adds a time dimension to the measurement and can mathematically separate genuine signal from scattered laser light and other parasitic contributions. This lifetime-gating approach substantially improves the signal-to-background ratio without any modification to the protein or sample.

The second strategy is amplifying the signal with nanophotonics. Zero-mode waveguides (ZMWs) are nanoscale apertures fabricated in metal films confining light to volumes below the diffraction limit. When a protein enters this ultrasmall hotspot, its fluorescence is dramatically enhanced. This makes it possible to detect individual proteins that would otherwise be invisible, and to work at the physiologically relevant concentrations found in living systems.

Further, we plan to extend the detection platform to plasmonic nanopores: nanoscale channels that force proteins to pass through one at a time, driven by an electric field, and give electrical readouts. But our focus is on the optical readout strategy. Each translocation event produces a distinct optical burst, carrying information not only about the protein's presence but also about its dynamics and identity. Combined with the UV autofluorescence readout, this approach opens the possibility of characterising proteins with single-molecule resolution, label-free, in real time.

Together, these tools form a coherent label-free platform for single-protein analysis. The integration of UV autofluorescence with nanopore translocation could enable a new class of biosensor, one capable of identifying proteins by their intrinsic optical fingerprint as they thread through a nanoaperture. This would have direct implications for medical diagnostics, proteomics, and the study of protein misfolding diseases such as Alzheimer's and Parkinson's.