Evanescent scattering imaging of single protein binding kinetics and DNA conformation changes

Abstract

Evanescent illumination has been widely used to detect single biological macromolecules because it can notably enhance light-analyte interaction. However, the current evanescent single-molecule detection system usually requires specially designed microspheres or nanomaterials. Here we show that single protein detection and imaging can be realized on a plain glass surface by imaging the interference between the evanescent lights scattered by the single proteins and by the natural roughness of the cover glass. This allows us to quantify the sizes of single proteins, characterize the protein–antibody interactions at the single-molecule level, and analyze the heterogeneity of single protein binding behaviors. In addition, owing to the exponential distribution of evanescent field intensity, the evanescent imaging system can track the analyte axial movement with high resolution, which can be used to analyze the DNA conformation changes, providing one solution for detecting small molecules, such as microRNA. This work demonstrates a label-free single protein imaging method with ordinary consumables and may pave a road for detecting small biological molecules.

Multiplexed Protein Detection and Parallel Binding Kinetics Analysis with Label-Free Digital Single-Molecule Counting

“…The 450 nm laser is conditioned to illuminate one indium tin oxide (ITO)-coated cover glass with an incident angle of ∼65° through a 60× objective. The incident angle is larger than the critical angle to excite evanescent waves on the surface (Figure a) . A 50× objective is placed on the top of ITO-coated cover glass to collect the evanescent waves scattered by proteins and surface roughness to form the ESM images.…”

“…The incident angle is larger than the critical angle to excite evanescent waves on the surface (Figure 1a). 25 A 50× objective is placed on the top of ITO-coated cover glass to collect the evanescent waves scattered by proteins and surface roughness to form the ESM images.…”

“…In the recent decade, label-free single-molecule imaging approaches have been developed to push beyond ensemble averages and conduct the statistical analysis of intrinsic protein properties such as molecular weight and binding processes. These techniques include interferometric scattering microscopy (iSCAT), − photothermal microscopy, , and recently developed plasmonic scattering microscopy (PSM) − and evanescent scattering microscopy (ESM) . Among these technologies, the ESM presents distinct advantages.…”

“…These techniques include interferometric scattering microscopy (iSCAT), 12−18 photothermal microscopy, 19,20 and recently developed plasmonic scattering microscopy (PSM) 21−24 and evanescent scattering microscopy (ESM). 25 Among these technologies, the ESM presents distinct advantages. In contrast to iSCAT and photothermal microscopy, which employ conventional illuminations, the ESM utilizes evanescent illuminations for enhanced light− analyte interactions and is immune to the interference of molecules and impurities in the bulk solution.…”

“…In our previous work, we measured single-pair protein− protein binding kinetics and a single-molecule effective spring constant to the surface using ESM. 25 To discover the weak single-molecule signal, we remove the strong but static backgrounds with differential processing, which is performed by subtracting or dividing one frame by the previous frame. Averaging of multiple frames over a 100 ms time interval prior to the differential step is required to suppress the shot noise at the same time.…”