Direct digital sensing of protein biomarkers in solution

Abstract

Highly sensitive detection of proteins is of central importance to biomolecular analysis and diagnostics. Conventional protein sensing assays, such as ELISAs, remain reliant on surface-immobilization of target molecules and multi-step washing protocols for the removal of unbound affinity reagents. These features constrain parameter space in assay design, resulting in fundamental limitations due to the underlying thermodynamics and kinetics of the immunoprobe–analyte interaction. Here, we present a new experimental paradigm for the quantitation of protein analytes through the implementation of an immunosensor assay that operates fully in solution and realizes rapid removal of excess probe prior to detection without the need of washing steps. Our single-step optofluidic approach, termed digital immunosensor assay (DigitISA), is based on microfluidic electrophoretic separation combined with single-molecule laser-induced fluorescence microscopy and enables calibration-free in-solution protein detection and quantification within seconds. Crucially, the solution-based nature of our assay and the resultant possibility to use arbitrarily high probe concentrations combined with its fast operation timescale enables quantitative binding of analyte molecules regardless of the capture probe affinity, opening up the possibility to use relatively weak-binding affinity reagents such as aptamers. We establish and validate the DigitISA platform by probing a biomolecular biotin–streptavidin binding complex and demonstrate its applicability to biomedical analysis by quantifying IgE–aptamer binding. We further use DigitISA to detect the presence of α-synuclein fibrils, a biomarker for Parkinson’s disease, using a low-affinity aptamer at high probe concentration. Taken together, DigitISA presents a fundamentally new route to surface-free specificity, increased sensitivity, and reduced complexity in state-of-the-art protein detection and biomedical analysis.

Tie-lines reveal interactions driving heteromolecular condensate formation

“…This pair of points allows us to construct the tie-line by connecting them with gradient k calculated as (figure 3A). Experimentally, we attached florescent tags to FUS and G3BP1 proteins and used a home-built confocal setup [25] to determine the photon count intensity from the dilute phase, which directly relate to the protein concentration (the conversion is not necessary since the value does not enter the tie-line expression); a microfluidic device was used to trap the phase separating systems in water-in-oil droplets so that measurements could be carried out without surface effects (figure 3B, 3C and Appendix B 1).…”

“…Measurement of tie-lines requires a robust method for measuring dilute phase concentrations of proteins in heterogeneous mixtures of condensates and soluble monomer. In order to carry out these measurements, we utilised a droplet microfluidic technique in which we created microdroplets containing the desired concentrations of protein and co-polymer and then measured these droplets on a home-built confocal setup [25]. In brief a 488 laser line is coupled into a 60x-magnification water-immersion objective (CFI Plan Apochromat WI 60x, NA 1.2, Nikon) and emitted photons are directed onto an avalanche photodiode (APD), a motorised XYZ stage is mounted on top of the objective so that a microfluidic device may be monitored throughout the experiment.…”

“…4. Experimental overview. (A) simplified illustration of the home-built confocal measurement setup, described in detail elsewhere [25]; (B) microfluidic device used, with a zoomed-in view of the droplet-generation section. (C) histogram of mean baseline signal for individual droplets, the red dashed line is a gaussian distribution using the mean and standard deviation calculated for the series of droplet.…”

Phase separating RNA binding proteins form heterogeneous distributions of clusters in subsaturated solutions

“…As an independent probe for the presence of such species, we turned to confocal detection under flow by using Microfluidic confocal spectroscopy (MCS). This is a brightness-based method that combines microfluidic mixing and flow with confocal detection (33)(34)(35). MCS measurements are sensitive to the presence of species that span a broad size range from tens to hundreds of nanometers, and due to convective flow, is not reliant on diffusion alone for sampling species of different sizes in solution (SI Appendix, Fig.…”

The Hsc70 Disaggregation Machinery Removes Monomer Units Directly from α-Synuclein Fibril Ends

“…Briefly, the microscope is equipped with a 488-nm laser line (Cobolt 06-MLD, Hübner Photonics, Derby, UK) and a single-photon counting avalanche photo diode (SPCM-14, PerkinElmer, Seer Green, UK) for subsequent detection of emitted fluorescence photons. Further details of the optical unit have been described previously 53 . Diffusion profile recording was done by continuously moving the confocal observation volume through the centre four channels of the microfluidic device.…”