Electrochemical DNA-based sensors for measuring cell-generated forces

Abstract

Mechanical forces play an important role in cellular communication and signaling. We developed in this study novel electrochemical DNA-based force sensors for measuring cell-generated adhesion forces. Two types of DNA probes, i.e., tension gauge tether and DNA hairpin, were constructed on the surface of a smartphone-based electrochemical device to detect piconewton-scale cellular forces at tunable levels. Upon experiencing cellular tension, the unfolding of DNA probes induces the separation of redox reporters from the surface of the electrode, which results in detectable electrochemical signals. Using integrin-mediated cell adhesion as an example, our results indicated that these electrochemical sensors can be used for highly sensitive, robust, simple, and portable measurements of cell-generated forces.

Keywords: Cellular forces; DNA probe; Electrochemical sensor; Hairpin DNA; Tension gauge tether.

Fig. 1.

Schematic and photo image of the smartphone-based electrochemical DNA-based force sensors. Thiolated tension gauge tether (TGT) or DNA hairpin probes were immobilized on the surface of a gold screen-printed electrode. Cell adhesion force mediated by a specific receptor–ligand pair could separate the double-stranded TGT probes or unfold the DNA hairpin, and as a result, the attached methylene blue reporter exhibited a decreased electrochemical signal.

Fig. 2.

(a) Nyquist plots of the unmodified Au-SPE (green line), 3’-thiolated anchor strand-modified Au-SPE (red line), 1-hexanethiol-passivated 3’-thiolated anchor strand-modified Au-SPE (blue line), and that after adding 5’-methylene blue-3’-biotin-modified reporter strand (purple line). The electrochemical impedance spectroscopy measurement was performed in a solution containing (v/v) 50% DMEM, 50% phosphate buffer (0.2 M, pH 7.4), and 5 mM ferrocene carboxylate, signals were recorded at an AC potential of 5 mV, a DC potential of 0.17 V, and in the frequency range of 100,000–0.1 Hz. The equivalent electric circuit compatible with the Nyquist diagrams was shown on the top. Here, R_s is the solution resistance, R_ct is the charge transfer resistance, C_dl is double layer capacitance, and Z_W is Warburg impedance. (b) Stability of the cyclic voltammetry signals of Ru(NH₃)₆³⁺ on 1-hexanethiol-passivated thiolated anchor strand-modified Au-SPE during 25 times potential scans at the scan rate of 100 mV·s⁻¹ in a solution containing (v/v) 50% DMEM and 50% phosphate buffer (0.2 M, pH 7.4). (c–e) Square wave voltammetry of the electrochemical c) 12 pN TGT sensor, d) 43 pN TGT sensor, e) 56 pN TGT sensor after adding 100 μL of 1×10⁶ HeLa cells/mL for 0, 15, 30, 45, and 60 min, respectively, followed by washing. The measurement was performed in a solution containing (v/v) 50% DMEM and 50% phosphate buffer (0.2 M, pH 7.4). The step potential was set as 20 mV, the pulse amplitude was at 50 mV, and the frequency was at 20 Hz. (f) Cell incubation time-dependent changes in the peak current value of 12 pN, 43 pN, and 56 pN TGT sensors. Shown are the mean and standard error peak values after subtracting the background signals from four replicated tests.

Fig. 3.

(a) The square wave voltammetry of the 12 pN TGT sensor in the absence of HeLa cell (dotted line) or in the presence of 100 μL of 1×10³, 1×10⁴, 1×10⁵, or 1×10⁶ HeLa cells/mL for 60 min and washing. (b) The relative peak current intensity change (I_%) as a function of adding different concentrations of the HeLa cells. The peak current value obtained from the dotted line, i.e., without adding HeLa cells, was used as the reference, I₀. (c) The square wave voltammetry of the 12 pN TGT sensor in the absence of HeLa cell (dotted line) or after adding 100 μL of 1×10⁶ HeLa cells/mL that have been treated with 0 μM, 20 μM, or 40 μM latrunculin B for 60 min and washing. (d) The relative peak current intensity change (I_%) as a function of latrunculin B treatment. The peak current value obtained from the dotted line, i.e., without adding HeLa cells, was used as the reference, I₀. The measurement was performed in a solution containing (v/v) 50% DMEM and 50% phosphate buffer (0.2 M, pH 7.4). The step potential was set as 20 mV, the pulse amplitude was at 50 mV, and the frequency was at 20 Hz. Shown are representative curves from four replicated tests.

Fig. 4.

(a) Nyquist plots of the unmodified Au-SPE (green line), thiolated DNA hairpin probe-modified Au-SPE (orange line), 1-hexanethiol-passivated thiolated DNA hairpin probe-modified Au-SPE (blue line), 1-hexanethiol-passivated thiolated DNA hairpin probe-modified Au-SPE after adding streptavidin and biotinylated cRGDfK, i.e., DNA hairpin force sensor (red line), and that after adding 100 μL of 1×10⁶ HeLa cells/mL onto the DNA hairpin force sensor (purple line). The electrochemical impedance spectroscopy measurement was performed in a solution containing (v/v) 50% DMEM, 50% phosphate buffer (0.2 M, pH 7.4), and 5 mM ferrocene carboxylate, signals were recorded at an AC potential of 5 mV, a DC potential of 0.17 V, and in the frequency range of 100,000–0.1 Hz. The equivalent electric circuit compatible with the Nyquist diagrams were shown on the right. R_s is the solution resistance, R_ct is the charge transfer resistance, C_dl is double-layer capacitance, and Z_W is Warburg impedance. (b) Stability of the cyclic voltammetry signals of methylene blue on the DNA hairpin force sensor-modified Au-SPE during 25 times potential scans at the scan rate of 400 mV.s⁻¹ in a solution containing (v/v) 50% DMEM and 50% phosphate buffer (0.2 M, pH 7.4). (c) Before (dotted line) and after (solid line) adding HeLa cells, a linear correlation was also observed between both anodic (orange line) and cathodic (blue line) peak currents (I_p) and the changes in the scan rate (ν).

Fig. 5.

(a) The square wave voltammetry of the DNA hairpin sensor in the absence of HeLa cell (dotted line) or in the presence of 100 μL of 1×10³, 1×10⁴, 1×10⁵, or 1×10⁶ HeLa cells/mL for 60 min and washing. (b) The relative peak current intensity change (I_%) as a function of adding different concentrations of the HeLa cells. The peak current value obtained from the dotted line, i.e., without adding HeLa cells, was used as the reference, I₀. (c) The square wave voltammetry of the DNA hairpin sensor in the absence of HeLa cell (dotted line) or after adding 100 μL of 1×10⁶ HeLa cells/mL that have been treated with 0 μM, 20 μM, or 40 μM latrunculin B for 60 min and washing. (d) The relative peak current intensity change (I_%) as a function of latrunculin B treatment. The peak current value obtained from the dotted line, i.e., without adding HeLa cells, was used as the reference, I₀. (e) The square wave voltammetry of the DNA hairpin sensor in the absence of HeLa cell (dotted line) or after adding 1×10⁶ cells/mL HeLa cells for different incubation times. (f) Cell incubation time-dependent changes in the peak current value of the DNA hairpin sensor. Shown are the mean and standard error peak values after subtracting the background signals from four replicated tests. The measurement was performed in a solution containing (v/v) 50% DMEM and 50% phosphate buffer (0.2 M, pH 7.4). The step potential was set as 20 mV, the pulse amplitude was at 50 mV, and the frequency was at 20 Hz. Shown are representative curves from four replicated tests.