(a) After passing through a mixing region, the protein stream and two buffer streams (fed from three separate inlets) are directed to three outlets, which are connected to separate reservoirs, whose heights are adjusted to tune mixing and dilution (see Supplementary Note). (b) Diagram of the device showing inlets and outlets. (c) Micrograph of the functional region with a fluorescent solution fed to the protein inlet. Arrows indicate buffer flow (blue), protein flow before mixing (yellow), and protein flow in mixing and detection regions (red). (d) Flow velocity (hollow circles), as measured with fluorescence correlation spectroscopy (FCS), and time after mixing or dilution (solid circles), both as functions of the position along the channel in the deceleration region; dashed line is from 3D flow simulations (COMSOL). (e) Flow velocity (as in (d)) in the focusing, mixing and deceleration regions. (f) Flow diagram: the protein stream is squeezed horizontally by two buffer streams for medium exchange and then directed to smFRET detection region. (g) Simulations of flow velocities and streamlines in two device regions highlighted in insets.

(a)α-synuclein in its intrinsically disordered U-state is mixed with a buffer containing SDS, triggering folding of α-synuclein into F-state, and smFRET is measured as the folding progresses in time. Histograms (~50,000 events) of smFRET efficiency, E_FRET, obtained at different time points are used to generate a 3D histogram in coordinates of time and E_FRET, with the percentage of total events color-coded from blue (0) to red (>5%). (b)Similarly plotted3D histogram for the α-synuclein unfolding reaction. The α-synuclein molecules are initially in the low-E_FRET F-state with SDS bound to the protein; massive dilution of the protein-SDS complexes triggers their dissociation and the unfolding of α-synuclein. (c) Representative E_FRET histograms for various states: intrinsically disordered (U-state, E_FRET ≈0.42, obtained before mixing), collapsed unfolded (U*-state, E_FRET≈0.6, 490 μs after mixing), intermediate (I-state, E_FRET ≈0.8, 1.2 ms after mixing), and extended structures (F-state, E_FRET ≈0.1, >10ms after mixing). (d) Model of the α-synuclein conformational transitions; the mirror representation emphasizes the asymmetry between the folding and unfolding pathways; the structures are color-coded (brown: disordered protein, turquoise: α-helix); green (donor) and purple (acceptor) spheres represent dye molecules.