Micron-Scale NV-NMR Spectroscopy with Signal Amplification by Reversible Exchange

Optically probed nitrogen-vacancy (NV)) quantum defects in diamond can detect nuclear magnetic resonance (NMR) signals with high-spectral resolution from micron-scale sample volumes of about 10 pl. However, a key challenge for NV-NMR spectroscopy is detecting samples at millimolar concentrations. Here we demonstrate an increase in NV-NMR proton concentration sensitivity by hyperpolarizing sample proton spins to about $0.5\mathrm{\%}$ through signal amplification by reversible exchange (SABRE), enabling micron-scale NMR spectroscopy of small-molecule sample concentrations as low as 1 mM in picoliter volumes. The SABRE-enhanced NV-NMR technique may enable detection and chemical analysis of low-concentration molecules and their dynamics in complex micron-scale systems such as single cells.

Popular Summary

Nuclear magnetic resonance (NMR) spectroscopy is widely used for chemical analysis and molecular structure determination. Because it typically relies on the weak magnetic fields produced by nuclear spins at room temperature, NMR spectroscopy suffers from low sensitivity compared with other analytical techniques. A conventional NMR apparatus typically uses sample volumes of about 1 ml—large enough to contain millions of biological cells. In this work, we demonstrate a quantum sensing technique that allows high-resolution NMR spectroscopy of small molecules at roughly the concentration found in, and volume of, a single cell.

To realize this advance, we integrate an ensemble of nitrogen-vacancy (NV) quantum defects in a diamond chip with a technique to hyperpolarize (i.e., polarization well above thermal equilibrium) the nuclear spins in the sample. NVs serve as a room temperature magnetometer with optical readout capability. Because of their closeness to the sample, NV ensembles can detect NMR signals from micron-scale sample volumes. Signal amplification by reversible exchange (SABRE) is a parahydrogen-based hyperpolarization technique that provides close to 1% proton spin polarization for dilute sample molecules in a liquid solvent. SABRE-enhanced NV magnetometry allows high-resolution NMR spectroscopy of small-molecule samples with concentrations as low as 1 mM and with a sensing volume of about 10 pl.

This technique augments the growing toolbox for sensitive, high-resolution NMR spectroscopy in micron-scale samples using NV quantum defects in diamond. Compared with other signal enhancement methods, SABRE provides significantly higher concentration sensitivity while being applicable to a wide range of small-molecule analytes. By implementation of the SABRE technique at tesla-scale magnetic fields, SABRE-enhanced NV-NMR spectroscopy may become a high-impact tool for biological applications, such as tracking and monitoring of chemical reactions of metabolites in single cells.

Figure 1

NV-NMR sensor integrated with SABRE. (a) Experimental schematic of the ensemble NV-NMR sensor. A 532-nm optical beam illuminates the diamond chip via a total internal reflection configuration. A microwave (MW) antenna on the diamond chip drives the electronic spins of the NV centers. Parahydrogen diffused into the sample through the capillary tube initiates the SABRE reaction, and thereby hyperpolarizes sample proton spins via an iridium-based catalyst in the sample solution. The hyperpolarized NMR signal is sensed with use of the electronic spins of the ensemble of NV centers. (b) Energy level diagram for the NV centers in diamond. The expanded view shows the Zeeman splitting of the ground triplet state ${}^3{A}_2$ in the presence of a magnetic field. (c) SABRE hyperpolarization process. The SABRE catalyst is in reversible exchange (indicated by red arrows) with parahydrogen (left side) and a small-molecule substrate (e.g., pyridine; right side). In the transient (millisecond) bound state of the catalyst-substrate complex (center), spin order flows from the hydrides to the substrate, leading to the buildup of polarization on the free substrate in solution, which is the sample to be probed with NV-NMR spectroscopy. (d) Pulse sequence for SABRE hyperpolarization and NV-NMR detection. The parahydrogen, bubbled into the sample solution, activates the catalyst and hyperpolarizes the small-molecule substrate (the sample). A $\pi /2$ pulse induces a FNP signal from the hyperpolarized sample, which is detected by NV sensor spins via a CASR pulse sequence. DAQ, data acquisition.

Figure 2

SABRE-enhanced NV-NMR spectra of pyridine. Comparison of measured NV-NMR spectra of 100 mM pyridine sample with (red circles, one acquisition) and without (blue circles, ${10}^4$ acquisitions) SABRE hyperpolarization for a CASR pulse sequence duration of 2 s. The solid red line is a Lorentzian fit to the SABRE-enhanced NV-NMR spectrum, giving a linewidth of 2.3(5) Hz, a signal enhancement of about $2.22(3)\times {10}^5$, and a proton spin polarization of 0.503(7)$\mathrm{\%}$, with a proton number sensitivity of 66.48(15) $\mathrm{fmol}/\sqrt{\mathrm{Hz}}$ for a SNR of 3. Small side peaks in the CASR spectra and deviations from the Lorentzian fits may arise from technical artifacts in the experiment [10]. The inset shows the CASR resonance frequency $F_{\mathrm{res}}$ of hyperpolarized pyridine (green squares) obtained by our varying the bias magnetic field $B_0$. A linear fit (green line) of $F_{\mathrm{res}}$ versus $B_0$ gives ${\gamma }_p=42.5355(40)$ MHz/T, consistent with the proton gyromagnetic ratio.

Figure 3

SABRE-enhanced NV-NMR measurement for various pyridine concentrations. (a) CASR-detected NV-NMR spectra (blue circles) for hyperpolarized pyridine samples and the associated Lorentzian fits (solid red line) at concentrations of 100, 30, 10, 5, and 1 mM in methanol. (b) CASR-detected NV-NMR signal amplitude (red dots) of hyperpolarized pyridine at various concentrations. The solid blue curve is a power function model of the form $a{x}^b+c$ with fit parameters $a=0.042$, $b=0.6855$, and $c=0.012$.

Figure 4

SABRE-enhanced molecular NV-NMR spectra. (a) Single-shot CASR spectrum of hyperpolarized ${}^{15}\mathrm{N}$-labeled pyridine (blue circles) for a sensing duration of 3 s at a concentration of 100 mM. The double Lorentzian fit (solid red line) indicates a splitting of $\mathrm{\Delta }{f}_J\approx 10(1)$ Hz (indicated by the vertical dashed lines) due to J coupling between the ${}^{15}\mathrm{N}$ nucleus and the protons. The inset shows the chemical structure of ${}^{15}\mathrm{N}$-labeled pyridine. (b) Single-shot CASR spectrum of hyperpolarized nicotinamide (blue circles) for a sensing duration of 2 s at a concentration of 100 mM. The Lorentzian fit (solid red line) gives a spectral linewidth of approximately $4(1)$ Hz. The inset shows the chemical structure of nicotinamide. Small side peaks in the CASR spectra and deviations from the Lorentzian fits may arise from technical artifacts in the experiment [10].

Figure 5

Experimental apparatus for parahydrogen production and delivery to the NV-NMR setup (“experimental cuvette”). Parahydrogen is produced by our flowing hydrogen gas through an iron oxide catalyst at 77 K. The parahydrogen is then bubbled through the capillary tube located within the experimental cuvette, where it interacts with the SABRE catalyst and hyperpolarization of the small-molecule substrate occurs. Optical NV-NMR detection is performed with a diamond chip integrated with the capillary tube in a manner similar to that previously reported [9, 10]. Other key aspects of the NV-NMR setup are not shown, including the electromagnet, green laser, optics, and microwave antenna.

Figure 6

Chemical activation of the SABRE catalyst precursor to become the SABRE active spin-order transfer catalyst.

Figure 7

Reversible hydrogen exchange that leads to hyperpolarization buildup on the free molecular substrate in solution. Left: free (unbound) hydrogen in solution. Center: SABRE active spin-order transfer catalyst. Right: free (unbound) substrate in solution. Steady-state excess of parahydrogen in the sample solution, maintained by periodic bubbling, leads to steady-state excess of hyperpolarized free substrate (Sub).

Figure 8

A simplified spin system that promotes spin-order transfer on the SABRE catalyst. The J-coupling values are as follows: $J_{HH}\approx -7$ Hz, $J_{HS}^{\mathrm{\prime }}\approx$ 1.5 Hz, $J_{HS}\approx 0$ Hz, and $J_{HS}\approx$ 0 Hz.

Figure 9

Experimental protocol. Experimental process such as sample preparation, parahydrogen generation, and catalyst activation are performed only once. NMR measurement, including parahydrogen bubbling and CASR NV-NMR measurement, can be repeated $N$ times, where $N$ is the number of averages used to increase the SNR.

Figure 10

(a) CASR NV measurements of the ac magnetic test signals from a nearby coil at $f_{\mathrm{coil}}=280$ kHz. The coil drive voltage $V_c$ for the ac signal source of the coil is varied from $V_c=0$ (purple trace) to $V_c=0.6$ V (magenta trace). (b) CASR signal data (blue points) as a function of coil voltage $V_c$ at time $t=0.302$ ms, obtained along the dashed line in (a). The blue line is a sinusoidal fit to the data, which gives the magnetic test signal calibration of 5.7(2) $\mu \mathrm{T}/$V. (c) CASR-detected NV-NMR spectra (peak A) of pyridine and test signal (peak B).