Electron Paramagnetic Resonance of a Single NV Nanodiamond Attached to an Individual Biomolecule

Abstract

Electron paramagnetic resonance (EPR), an established and powerful methodology for studying atomic-scale biomolecular structure and dynamics, typically requires in excess of 10(12) labeled biomolecules. Single-molecule measurements provide improved insights into heterogeneous behaviors that can be masked in ensemble measurements and are often essential for illuminating the molecular mechanisms behind the function of a biomolecule. Here, we report EPR measurements of a single labeled biomolecule. We selectively label an individual double-stranded DNA molecule with a single nanodiamond containing nitrogen-vacancy centers, and optically detect the paramagnetic resonance of nitrogen-vacancy spins in the nanodiamond probe. Analysis of the spectrum reveals that the nanodiamond probe has complete rotational freedom and that the characteristic timescale for reorientation of the nanodiamond probe is slow compared with the transverse spin relaxation time. This demonstration of EPR spectroscopy of a single nanodiamond-labeled DNA provides the foundation for the development of single-molecule magnetic resonance studies of complex biomolecular systems.

Figure 1

(a) Schematic of the NV center atomic structure (left), composed of a nitrogen impurity adjacent to a vacant site in the diamond tetrahedral lattice. The NV center axis can adopt any of four allowed orientations in the diamond lattice (middle). Our NV nanodiamonds are single-crystalline and range from 10 to 200 nm in diameter (right). (b) Simplified schematic of the electronic structure of the NV center. (c) Optically detected magnetic resonance spectrum of a collection of NV centers in a diamond crystal, exhibiting the Zeeman splitting of the magnetic resonance peaks by an applied magnetic field (shown by dark blue arrows) and the dependence of the splitting on the orientation of the field relative to the NV axis. To see this figure in color, go online.

Figure 2

Workflow of the NVND biotinylation chemistry protocol. To see this figure in color, go online.

Figure 3

Attachment of a single nanodiamond to a single 16.5-μm-long DNA molecule. (a) Schematic representation showing the binding between streptavidin, antidigoxigenin antibodies, individual dual-labeled (digoxigenin and biotin) λ DNA molecules, and biotinylated fluorescent nanodiamonds. The assembly occurs on the surface of the glass coverslip. (b) Epifluorescence microscope image of the attached nanodiamond. On the top is an image of the surface through a 570 nm bandpass filter. On the bottom is the same surface as seen through a 670 nm bandpass filter. The NV diamond fluorescence will pass through the 670 nm filter and the SYBR Gold-dyed DNA molecules will emit through the 570 nm bandpass filter only. To see this figure in color, go online.

Figure 4

(a) Simplified schematic of the custom-built confocal microscope. The optical components (in gray) are tightly integrated with the microwave circuit (components in purple) and flow-cell circuit (in blue). (b) Zoomed-in view of the integrated microwave and fluid circuits at the sample, illustrating eight flow channels for housing the in vitro single-molecule experiment, running perpendicular across the microwave coplanar waveguide. The microwave circuit path is highlighted in purple and the fluid circuit is in blue. To see this figure in color, go online.

Figure 5

ODMR spectra. (a) ODMR spectrum of a single static nanodiamond. The eight distinct peaks arise from NV centers oriented in four directions allowed by the nanodiamond crystal lattice. (b) NV diamond powder dispersion, collected from a large population of randomly oriented nanodiamond crystals (top inset) under three different applied external fields: 0 G (red), 18.7 G (blue), and 32.6 G (green). The uniform intensity of this spectrum reflects the uniform probability of finding an NV center with any particular orientation relative to the applied field. The field values are extracted from the fit for each spectrum (black) based on our model and are in agreement with our field calibration. (c) smEPR spectrum of a nanodiamond crystal attached to a single DNA molecule (bottom inset) under three similar applied external fields (0 G (red), 19.0 G (blue), and 32.1 G (green)), again extracted from the fit (black) for each spectrum, also based on our model. The close similarity to the powder spectrum confirms our expectation that the nanodiamond probe will rotate isotropically, and hence freely, through all possible orientations on a timescale that is slow compared with the characteristic transverse spin relaxation time $T_2$ for NV centers. Each spectrum is the average of five frequency sweeps and each sweep takes 300 s. To see this figure in color, go online.