Background

Proteins play essential roles in many biological processes and serve as drug targets and biomarkers. Analysis of proteins rely on technologies including electrophoresis, Western Blot, and mass spectrometry, which separate and identify proteins based on the size and charge of protein molecules. Although powerful in detecting proteins, these technologies are not sensitive to single molecules, which is required for elucidating molecular heterogeneity and precision diagnosis. Recently, several single-molecule technologies have been developed to measure the size or charge of single biomolecules. Examples include interferometric scattering microscopy (iSCAT) (Young et al., 2018) and plasmonic scattering microscopy (PSM) (Zhang et al., 2020), which quantify the size by measuring the scattered light from the molecules. Single-molecule electrometry (Ruggeri et al., 2017) and anti-Brownian electrokinetic (ABEL) trap (Wang et al., 2012) measure the charge of single molecules by monitoring single molecule motion in a potential trap. However, simultaneously measuring the size and charge on a single platform remains challenging.

Recently, we have developed a single-molecule technique to solve this problem (Ma et al., 2020). We tether proteins to an indium tin oxide (ITO) surface by polyethylene glycol (PEG) linkers and drive them into oscillation in vertical direction by applying an alternating electric field (Figure 1A). To track the protein oscillation, we place the ITO on an inverted optical microscope and generate evanescent field on the ITO surface. The oscillating proteins scatter the evanescent field, which is collected by the microscope and detected by a CMOS camera (Figure 1B). Using this method, the oscillation can be tracked with nanometer precision. By analyzing the oscillation and electromechanical response of the proteins to the field, the size and charge of individual proteins can be determined. The current protocol describes the assembly of protein oscillators and the optical detection system. Applications including protein size, charge, and binding measurements will also be presented. This protocol is also applicable to other single-molecule detection platforms, such as PSM and iSCAT, to expand the capability in simultaneous charge measurements.

Figure 1. Detection principle and schematic of the detection system. A. The protein is tethered to the ITO surface by a PEG tether. An electric field is applied to the surface which drives the protein into oscillation. An evanescent field is generated on the surface to probe the oscillation of the protein. B. The detection system is based on an inverted microscope. A p-polarized incident light is directed onto the ITO surface via a high-numeric aperture objective with an incident angle slightly lower than the total internal reflection angle. The electric field is applied by a three-electrode electrochemical system, where the WE, RE, and CE represent working electrode, reference electrode, and counter electrode, respectively.