Constructing real-time, wash-free, and reiterative sensors for cell surface proteins using binding-induced dynamic DNA assembly

Abstract

Cell surface proteins are an important class of biomarkers for fundamental biological research and for disease diagnostics and treatment. In this communication, we report a universal strategy to construct sensors that can achieve rapid imaging of cell surface proteins without any separation by using binding-induced dynamic DNA assembly. As a proof-of-principle, we developed a real-time and wash-free sensor for an important breast cancer biomarker, human epidermal growth factor receptor-2 (HER2). We then demonstrated that this sensor could be used for imaging and sensing HER2 on both fixed and live breast cancer cells. Additionally, we have also incorporated toehold-mediated DNA strand displacement reactions into the HER2 sensor, which allows for reiterating (switching on/off) fluorescence signals for HER2 from breast cancer cells in real-time.

Fig. 1. (A) Schematic illustrating the principle of the real-time cell surface sensor. (B) Confocal fluorescent microscopy images of SK-BR-3 cells incubated with real-time HER2-specific DNA sensors (25 nM TB, 25 nM B*C, and 50 nM T*C*:C) and 100 nM 4′,6-diamidino-2-phenylindole (DAPI) at RT for 30 min. (C) Confocal fluorescent microscopy images of SK-BR-3 cells incubated with isotype control probes and 100 nM DAPI. Scale bars at x-axis represent 9 μm.

Fig. 2. Real-time imaging of HER2 from fixed SK-BR-3 cells using HER2-specific DNA sensors. Time-lapse confocal fluorescent microscopy images of SK-BR-3 cells were taken at a 30 s interval immediately after mixing cells with HER2-specific DNA sensors that contained 25 nM TB, 25 nM B*C, and 50 nM T*C*:C. Scale bars represent 20 μm.

Fig. 3. Quantitative measurement of fluorescence increase from each single cell (A) and total fluorescence increase (B) as a function of time. All fluorescence measurements were carried out using ImageJ 1.47. For each cell, fluorescence = integrated density – (area of selected cell × mean fluorescence of background readings).

Fig. 4. Flow cytometry analysis of live SK-BR-3 cells that are labeled with HER2 specific DNA sensors. Fluorescent intensities are displayed in a hyperlog scale at x-axis.

Fig. 5. (A) Schematic illustrating the principle of real-time erasing and reiterating fluorescence for HER2 using DNA strand displacement. (B–D) Fluorescent imaging of SK-BR-3 cells before erasing fluorescence (B), after erasing fluorescence (C), and after regenerating fluorescence (D).