Fig. 1: Working principle of DigitISA and its implementation.

a Schematic illustration of the DigitISA platform and overview of the experimental workflow. DigitISA integrates electrophoretic separation and single-molecule detection in a platform for single-step sensing of target proteins in solution using only a single affinity reagent. The sample including a mixture of the target protein and its fluorescently labeled affinity probe (e.g., aptamer) is injected into a micron-scale electrophoretic separation unit. The application of an electric field allows protein-bound probe molecules to be discriminated from those probe molecules that are not bound to the protein target, owing to a difference in their electrophoretic mobilities. Confocal scanning across the separation chamber is performed and the number of molecules traversing the confocal volume at each of the scanned positions is estimated ‘digitally’ from the recorded photon-count time trace using a combined inter-photon time (IPT) and photon-count threshold burst-search algorithm. From the obtained counts, an electropherogram is created allowing for a discrimination between protein-bound affinity probe and free probe. b Design of the free-flow electrophoresis device. Sample is flown into the microfluidic chip by the central injection port where it is then surrounded by the carrier buffer solution. The electrophoresis chamber is connected to a co-flowing electrolyte solution (3 M KCl) via bridges, which allows for a narrow sheet of electrolyte to flow along the sides of the chamber. An electric field is applied from metal clips at the outlets of the electrolyte channels, which propagates along the electrolyte sheet and enables separation of molecules perpendicular to the flow direction. c Schematic of the confocal microscopy setup used for single-molecule detection. A diode laser is used to excite the sample through an objective, and a single-photon counting avalanche photodiode (APD) is used to register emitted photons from the sample. The confocal volume is moved across the cross-section of the chip in a stepwise manner with the aid of a motorized stage. This allows the flux of the protein-bound probe molecules to be estimated. Details of the setup are described in the Methods section (Data analysis).