Variable gain DNA nanostructure charge amplifiers for biosensing

Abstract

Electronic measurements of engineered nanostructures comprised solely of DNA (DNA origami) enable new signal conditioning modalities for use in biosensing. DNA origami, designed to take on arbitrary shapes and allow programmable motion triggered by conjugated biomolecules, have sufficient mass and charge to generate a large electrochemical signal. Here, we demonstrate the ability to electrostatically control the DNA origami conformation, and thereby the resulting signal amplification, when the structure binds a nucleic acid analyte. Critically, unlike previous studies that employ DNA origami to amplify an electrical signal, we show that the conformation changes under an applied field are reversible. This applied field also simultaneously accelerates structural transitions above the rate determined by thermal motion. We tuned this property of the structures to achieve a response that was ≈2 × 10⁴ times greater (i.e., a gain or amplification) than the value from DNA hybridization under similar conditions. Because this signal amplification is independent of DNA origami-analyte interactions, our approach is agnostic of the end application. Furthermore, since large signal changes are only triggered in response to desirable interactions, we minimize the deleterious effects of non-specific binding. The above benefits of self-assembled DNA origami make them ideally suited for multiplexed biosensing when paired with highly parallel electronic readout.

Fig. 1:

(A) Schematic representation of the DNA origami that shows the two arms of the hinge and the lock strands (yellow) that interact with a complementary DNA analyte. (B) Representative cryogenic electron microscopy (cryo-EM) images of the DNA origami. The scale bar is 50 nm. (C) Cross-sectional schematic and three-dimensional reconstruction of a hinge half from cryo-EM images. The scale bar is 6 nm.

Fig. 2:

Electrochemical impedance spectroscopy (EIS) of (A) normally closed and (B) normally open DNA origami in the absence (blue) and presence of 1 nmol/L (nM) analyte (orange) that has a sequence complementary to the lock strand. Solid lines in each case represent fits of a simplified Randles circuit model. (insets) Representation of the starting and final state for each DNA nanostructure variant upon adding a complementary DNA sequence (orange) that binds the lock strands (blue) A representative measurement is shown here. Full data sets from three independent measurements used to estimate fit parameters are shown in the Supplementary Information (section S2).

Fig. 3:

Measurements and modeling of the capacitance of DNA origami as a function of applied DC bias $\left(V_{DC}-V_{Ag\text{/}AgCl}\right)$ relative to an AgCl reference electrode. (A) Schematic and equivalent circuit model of a DNA origami. (B) Modeled capacitance per origami molecule computed using Eq. 1 and Eq. 2. $C_t$, as a function of hinge angle, $\theta$ inferred by solving the equivalent circuit in panel A. (C) Capacitance measurements vs. $V_{DC}-V_{Ag\text{/}AgCl}$ of normally closed (blue) DNA origami $\left(C_{structure}\right)$ conjugated to a gold surface in the absence of analyte. The capacitance was measured with an applied AC field with a frequency of 100 Hz and amplitude ${\text{V}}_{\text{pk}}=20\text{mV}$ summed with $V_{DC}-V_{Ag\text{/}AgCl}$. The solid line is a fit of the model to the data. (D) Capacitance measurements vs. $V_{DC}-V_{Ag\text{/}AgCl}$ of DNA probe strands $\left(C_{DNA}\right)$, with identical sequence to the DNA nanostructure lock, in the absence (green) and presence of 1 nM (nmol/L) analyte (red). For all plots in the figure, three independent measurements were used to estimate the expanded uncertainties are reported with coverage factor $k\text{=}2$ (95 % confidence interval).

Fig. 4:

Relative change in capacitance $\left(\Delta C_{structure}\text{/}{C}_0\right)$, where $C_0$ is the initial capacitance and gain of DNA origami relative to the change in capacitance of DNA hybridization $\left(\Delta C_{ssDNA}\right)$. (A) and (B) $\Delta C_{structure}\text{/}{C}_0$ of the normally closed and normally open DNA origami in the presence of 1 nM (nmol/L) analyte as a function of the DC bias $\left(V_{DC}-V_{Ag\text{/}AgCl}\right)$ relative to the absence of analyte. The dashed lines in each panel show $\Delta C_{ssDNA}$ in the presence of 1 nM (nmol/L) of DNA analyte. (C) Gain $\left(G\right)$ of the normally closed (blue) and normally open (orange) DNA origami over the DNA hybridization case. (D) Schematic of the forces from the hinge spring constant, $F_H$, and the portion of electric force normal to the hinge, $F_E$, at the relevant angles for the two structures as described in the main text. (E) $\Delta C_{structure}\text{/}{C}_0$ as a function of the analyte concentration in solution. The zero-concentration case represents a control with a non-complementary sequence. For all plots in the figure, three independent measurements were used to estimate expanded uncertainties are reported with coverage factor $k\text{=}2$ (95 % confidence interval).