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

Abstract

Multiplexed protein detection is critical for improving the drug and biomarker screening efficiency. Here, we show that multiplexed protein detection and parallel protein interaction analysis can be realized by evanescent scattering microscopy (ESM). ESM enables binding kinetics measurement with label-free digital single-molecule counting. We implemented an automatic single-molecule counting strategy with high temporal resolution to precisely determine the binding time, which improves the counting efficiency and accuracy. We show that digital single-molecule counting can recognize proteins with different molecular weights, thus making it possible to monitor the protein binding processes in the solution by real-time tracking of the numbers of free and bound proteins landing on the sensor surface. Furthermore, we show that this strategy can simultaneously analyze the kinetics of two different protein interaction processes on the surface and in the solution. This work may pave a way to investigate complicated protein interactions, such as the competition of biomarker–antibody binding in biofluids with biomarker–protein binding on the cellular membrane.

Label-Free Evanescent Imaging of Cellular Heterogeneity in Membrane Protein Binding Kinetics

“…The ESM image intensities of the cells increase along with the WGA binding (Supplementary Video 1). This is expected because the WGA molecules are biological macromolecules, and evanescent waves scattered by WGA molecules can interfere with those scattered by the cell adhesion sites, resulting in increasing image intensity along with the WGA binding 17,18,[24][25][26][27][28][29][30] . After averaging the image intensities of all adhesion sites of the cells and plotting the mean values against the time, the ensemble binding curve can be achieved as shown in Fig.…”

Label-Free Optical Imaging of Nanoscale Single Entities

“…The applications of ESM for single entity imaging were demonstrated with several examples. Similar to PSM and iSCAT, ESM can measure protein binding kinetics by digitally counting individual binding events to the surface antibodies . ESM were also used to visualize DNA conformational changes by monitoring the fluctuation of tethered AuNP (Figure b).…”

“…Similar to PSM and iSCAT, ESM can measure protein binding kinetics by digitally counting individual binding events to the surface antibodies. 72 ESM were also used to visualize DNA conformational changes by monitoring the fluctuation of tethered AuNP (Figure 5b). Hybridization with complementary RNA reduces the AuNP's fluctuation, and the change is quantified by measuring the axial displacement of the AuNP and calculating the spring constant of the single stranded DNA (0.19 N/m) and hybridized duplex (0.36 N/m).…”

Label-free analysis of membrane protein binding kinetics and cell adhesions using evanescent scattering microscopy

“…The ESM still employs the evanescent waves as an illumination field, so it maintains high surface sensitivity and sub-nanometer tracking resolution for analyzing the object movements in the axial direction. [26][27][28][29] At the same time, the ESM only detects the evanescent waves scattered by the objects, where the non-scattered evanescent waves do not contribute to the image contrast, and thus the ESM images can achieve high spatial resolution as PSM without interferences resulting from long decaying length of evanescent waves along the surface, which usually create micrometer scale parabolic tails following the objects in images provided by the traditional evanescent detection systems (Fig. S2 †).…”