Rational design of DNA nanostructures for single molecule biosensing

Abstract

The ability to detect low concentrations of biomarkers in patient samples is one of the cornerstones of modern healthcare. In general, biosensing approaches are based on measuring signals resulting from the interaction of a large ensemble of molecules with the sensor. Here, we report a biosensor platform using DNA origami featuring a central cavity with a target-specific DNA aptamer coupled with a nanopore read-out to enable individual biomarker detection. We show that the modulation of the ion current through the nanopore upon the DNA origami translocation strongly depends on the presence of the biomarker in the cavity. We exploit this to generate a biosensing platform with a limit of detection of 3 nM and capable of the detection of human C-reactive protein (CRP) in clinically relevant fluids. Future development of this approach may enable multiplexed biomarker detection by using ribbons of DNA origami with integrated barcoding.

Fig. 1. Concentric square DNA origami nanostructures.

a Colour-coded schematic representation of DNA nanostructure designs – ConA is a solid tile and ConB and ConC are frame-like with different central cavities. All three structures feature similar external dimensions but varying internal cavity. b AFM micrographs of the respective nanostructures and c their typical translocation ion current signatures. d Scatter plot of individual translocation events with peak amplitude plotted versus dwell time for ConA (blue circles), ConB (green circles) and ConC (black circles), overlaid with the respective 95% confidence ellipses. Source data are in the Source Data file.

Fig. 2. DNA origami carrier-based biosensors.

a Schematic representation of the DNA nanostructure-based biosensing concept exploiting translocation through nanopipettes as the sensing mechanism. b Schematic representation of the design and representative AFM micrographs of the unoccupied and occupied DNA origami carriers. The frame DNA nanostructure is ~95 nm × 95 nm in dimension with a 35 nm × 35 nm inner cavity. The DNA origami comprises small nucleotide ‘anchors’ that protrude into the cavity which facilitate the incorporation of the DNA aptamer via hybridisation. The DNA carrier also includes a polarity marker. c Peak amplitude and dwell time histograms of DNA nanostructure carriers (9 nM) and carriers incubated with CRP at 9 nM concentration, both measured at a final carrier concentration of 500 pM. d Typical ion current signatures upon translocation of CRP molecules, CRP–DNA aptamer complexes, unoccupied carriers and CRP-bound occupied carriers. The scale bars represent 20 pA and 1 ms, respectively. Source data are in the Source Data file.

Fig. 3. CRP binding study.

a Representative selection of ion current signatures for different CRP concentrations. The peak traces are stitched together from individual peaks of a longer trace to remove regions with no events for the purpose of illustration. b Translocation events of carriers (9 nM) incubated with different concentrations of CRP with their peak amplitude plotted against dwell time. The single peaks in the ion current data are coloured blue while the double peaks are coloured orange, and the unclassified events discarded from the quantitative analysis are represented as triangles. The plots are overlaid with the 95% confidence ellipses from Fig. S6a. c Normalised single peak count, i.e., ratio of single peaks vs total classified peaks against CRP concentration (Supplementary Tables 1 and 2). The data were fitted with a Langmuir isotherm (solid line) and revealed a K_d of 11 ± 2 nM. The dashed lines represent the confidence boundaries of the fit. The error bars denote the standard deviation of translocation experiments conducted on different days using three different nanopipettes (n = 3). The sampling time was 2 min for all concentrations. Source data are in the Source Data file.

Fig. 4. CRP detection in diluted human plasma.

Typical ion current traces, and peak amplitude and dwell time histograms for a unoccupied carriers and b CRP-occupied carriers incubated in different concentrations of CRP in 5% plasma. c Normalised single peak count, i.e., ratio of single peaks vs total classified peaks against CRP concentration (Supplementary Tables 3 and 4). The solid line is a guide to the eye. The error bars are standard deviation calculated from three different traces (n = 3). The sampling time was 2 min for all samples. Source data are in the Source Data file.