Following DLS analysis, we also monitored nanogel degradation by a single-molecule detection method called fluorescence correlation spectroscopy (FCS). We previously used FCS for a detailed study of MMP-responsive degradation using another peptide-based nanostructure for diagnostic applications. Herein, FCS was employed to first observe nanogel degradation and subsequently to quantitatively analyse model biomolecule loading and release. In order to study MMP-7 triggered degradation of nanogels by FCS, cross-linked nanogels were labelled with Cy5-azide and incubated with excess MMP-7 (9 µM). Successful labelling of nanogels with Cy5-azide can be seen when calculating the hydrodynamic diameters from the FCS autocorrelation analysis (Figure ) that yielded nanogel size rather than free dye (Figure ). Further, non-crosslinked Cy5-azide labelled copolymers in nonassembled form revealed an intermediate diffusion time between free dye and nanogels (Figure ). FCS curves of control experiments to check for non-specific interactions of dyes with nanogels/copolymers yielded diffusion times similar to free dye (Figure ), which confirms successful specific labelling in the case of Cy5-azide (Figure ).