Quantum relaxometry for detecting biomolecular interactions with single NV centers

Abstract

The investigation of biomolecular interactions at the single-molecule level has emerged as a pivotal research area in life science, particularly through optical, mechanical, and electrochemical approaches. Spins existing widely in biological systems, offer a unique degree of freedom for detecting such interactions. However, most previous studies have been largely confined to ensemble-level detection in the spin degree. Here, we developed a molecular interaction analysis method approaching single-molecule level based on relaxometry using the quantum sensor, nitrogen-vacancy (NV) center in diamond. Experiments utilized an optimized diamond surface functionalized with a polyethylenimine nanogel layer, achieving $\sim$10 nm average protein distance and mitigating interfacial steric hindrance. Then we measured the strong interaction between streptavidin and spin-labeled biotin complexes, as well as the weak interaction between bovine serum albumin and biotin complexes, at both the micrometer scale and nanoscale. For the micrometer-scale measurements using ensemble NV centers, we re-examined the often-neglected fast relaxation component and proposed a relaxation rate evaluation method, substantially enhancing the measurement sensitivity. Furthermore, we achieved nanoscale detection approaching single-molecule level using single NV centers. This methodology holds promise for applications in molecular screening, identification and kinetic studies at the single-molecule level, offering critical insights into molecular function and activity mechanisms.

Figures (5)

• Figure 1: Quantum relaxometry for detecting biomolecular interactions using NV centers. (A) Schematic of the experimental setup. Protein A (yellow) is immobilized on the diamond surface to capture spin-labeled Protein B (blue). The NV center detects magnetic noise from the spin label via acceleration of its spin relaxation rate. (B) $T_1$ measurement sequence. The spin state of the NV center is initialized and read out using 532 nm laser pulses (green). A microwave $\pi$-pulse (blue) is applied to flip the spin state between $|0\rangle \leftrightarrow |\pm1\rangle$. Varying the dark evolution time $\tau$ yields the $T_1$ relaxation profile. (C) $T_1$ curves measured with only protein A (yellow) and with captured spin-labeled protein B (blue). Two relaxation curves were measured using the upper and lower pulse sequences shown in (B), corresponding to initial spin states $|0\rangle$, $|\pm1\rangle$. The difference of the curves was normalized to the reference point at $\tau=0$, yielding the normalized intensity defined as: $(I_{0,\tau}-I_{\pm1,\tau})/(I_{0,0\ \upmu\text{s}}-I_{\pm1,0\ \upmu\text{s}})$.
• Figure 2: Micrometer-scale biomolecular interaction detection using ensemble NV centers. (A) Schematic of biomolecular interaction detection with ensemble NV centers in bulk diamond. The diamond surface is functionalized with a polyethylenimine (PEI) nanogel to achieve surface amination. Then the immobilized proteins, such as streptavidin (SA) or bovine serum albumin (BSA), are sequentially bonded to the surface. Finally, spin-labeled molecules are introduced for detection. (B) Topography image of the diamond surface coated with SA and biotin-Ub(Mn) using atomic force microscope (AFM). Proteins in the square depression area were removed using AFM contact mode. The inset shows a total thickness of 5.8$\pm$1.3 nm. (C-E) Relaxation results of SA+biotin-Ub(Mn), as shown in the inset in (C). (C) $T_1$ curves measured in regions with (27 red lines)/without (21 blue lines) proteins shown in (B). The dashed lines are experimental curves and the hollow dots are averaged data. These dashed lines were fitted using a biexponential decay function to obtain weighted relaxation rates (details in Materials and Methods). (D) Comparison of the averaged relaxation rate derived from the dashed lines in (C). The error bar is the standard deviation. The difference in relaxation rates between the two regions is (4.8$\pm$0.5)$\times10^3$ s$^{-1}$. (E) 2D map of the relaxation signal. By fixing the waiting time of the $T_1$ measurement sequence at $\tau_\text{fix}=350\ \upmu s$, the fluorescence intensity normalized to $\tau=0\ \upmu s$ was obtained around the depression area shown in (B). Inset: Profile of the black line in the 2D map. (F-H) Relaxation results of BSA+biotin-Ub(Mn), as shown in the inset in (F). (F) $T_1$ curves measured in regions with (29 red lines)/without (31 blue lines) proteins. (G) Comparison of the averaged relaxation rate derived from the dashed lines in (F). The relaxation rate difference between the two regions is (0.8$\pm$0.3)$\times10^3$ s$^{-1}$. (H) 2D map of the relaxation signal at $\tau_\text{fix}=350\ \upmu s$. The proteins in the square depression area have been removed.
• Figure 3: Nanoscale biomolecular interaction detection using single NV centers. (A) Schematic of the interaction detection with single NV centers in nano-pillar. The expanded view shows the side profile of a nano-pillar coated with PEI, pre-immobilized proteins (e.g., SA or BSA), and spin-labeled protein (e.g., biotin-Ub(Mn)). (B) $T_1$ curves of a typical single NV center under four conditions. The dots are experimental data points and the solid lines are fitting curves to the single exponential decay function. (C) Relaxation rates of 195 single NV centers under these four surface conditions. The mean rates for each condition are presented as mean $\pm$ standard deviation, which are $(0.92\pm0.04)\times10^3\ s^{-1}$, $(0.9\pm0.1)\times10^3\ s^{-1}$, $(1.0\pm0.2)\times10^3\ s^{-1}$, $(6.5\pm0.7)\times10^3\ s^{-1}$. (D) Histogram distributions of relaxation accelerations for 195 single NV centers relative to the bare diamond. The dashed lines represent Gaussian fits. The fitting results presented as the peak $\pm$ standard deviation are $(-0.2\pm0.3)\times10^3\ s^{-1}$ (green line, SA) and $(-0.2\pm0.4)\times10^3\ s^{-1}$ (purple line, BSA+biotin-Ub). The solid red line is the theoretical curve obtained from the Monte Carlo simulation, assuming an average distance of 12 nm between SA proteins (details in Figure S21).
• Figure 4: Simulation of the magnetic noise signal measured with single NV centers. (A) Schematic of the Monte Carlo simulation. Single NV centers are modeled with depths ($d$) following a Gaussian distribution ($\mu=5.5\ nm$, $\sigma=2.8\ nm$) derived from SRIM ion implantation simulations. SA is approximated as a cubic structure with 4 biotin-binding sites distributed on the diamond surface at an average spacing of 12 nm. (B) Correlation between the NV center's background relaxation rate $\Gamma_{1,\text{BG}}$ and the detected magnetic signal intensity $\Delta\Gamma$. Yellow circles are the experimental data from 195 single NV centers. The colormap displays the simulated probability density of obtaining $\Delta\Gamma$ at given $\Gamma_{1,\text{BG}}$. Outliers (low $\Gamma_{1,\text{BG}}$, high $\Delta\Gamma$) indicate localized protein aggregates or depth estimation deviations. (C) Single-molecule detection probability. The background illustrates the simulated single-molecule detection probability $P_\text{single}$, depicted by the color scale. $P_\text{single}$ quantifies the probability that the observed $\Delta\Gamma$ originates from individual SA-biotin-Ub(Mn) complexes at the given $\Gamma_{1,\text{BG}}$. The blue curve marks the contour of $P_\text{single}=0.5$.
• Figure 5: New evaluation method for relaxation rate of ensemble NV center. (A) $T_1$ curve of an ensemble NV center. The solid (dashed) line is the (bi)exponential decay fitting curve. (B) $T_1$ curves of 3 single NV centers. Solid lines are fitting curves to the single exponential decay function. (C) Histogram of background relaxation rates of 471 single NV centers. The solid line is a fitting curve based on the surface magnetic noise model (SI Appendix Sec. 9.2, Figure S20). (D) Averaged $T_1$ data of 471 single NV centers in (C). The pink (green) line is a fitting curve to the (bi-)exponential decay function. (E) Schematic of surface magnetic noise model for shallow NV center. Bulk magnetic noise and surface paramagnetic impurities jointly influence background relaxation rates. (F) Simulated distribution of $\Gamma_{1,\text{BG}}$ for shallow single NV centers as $\sigma_\text{surf}=0.40\ nm^{-2}$ (SI Appendix Sec. 9.3). (G) Comparison of the simulated $T_1$ curve (purple line) and the experimental $T_1$ data (black dots) for the ensemble NV center. The simulated $T_1$ curve is the average data of single exponential decay curves from the distribution in (F). (H) Comparison of simulated (solid lines) and experimental $T_1$ data (hollow dots). The diamond surface is only bonded with Ub(Mn) and the derived bonding density is $\sigma_\text{Ub}=(1/9)^2\ nm^{-2}$. (I) Dependence of the ensemble NV relaxation rate change $\Delta \Gamma$ on the Ub(Mn) bonding density $\sigma_\text{Ub}$ with relaxation rate evaluation methods: (1) characteristic rate $1/T_{1,\text{stre}}$ from stretched exponential fits, (2) slower characteristic rate $1/T_{1,\text{long}}$ from biexponential fits, (3) weighted rate $\Gamma_{1,\text{w}}$ (our method). Black points are the arithmetic average of relaxation rate accelerations for single NV centers constituting an ensemble NV center. The solid lines are linear fitting curves of data corresponding to the bonding spacing from 20 nm to 7 nm.