Full optimization of dynamic nuclear polarization on a 1 tesla benchtop polarizer with hyperpolarizing solids

Abstract

Hyperpolarization by dissolution dynamic nuclear polarization (dDNP) provides the opportunity to dramatically increase the weak nuclear magnetic resonance (NMR) signal of liquid molecular targets using the high polarization of electron radicals. Unfortunately, the solution-state hyperpolarization can only be accessed once since freezing and melting of the hyperpolarized sample happen in an irreversible fashion. A way to expand the application horizon of dDNP can therefore be to find a recyclable DNP alternative. To pursue this ambitious goal, we recently introduced the concept of recyclable hyperpolarized flow (HypFlow) DNP where hyperpolarization happens in porous hyperpolarizing solids placed in a compact benchtop DNP polarizer at a magnetic field of 1 T and a temperature of 77 K. Here we aim to optimize the radical concentrations immobilized in hyperpolarizing solids with the objective of generating as much polarization as possible in a timeframe (<1 s) compatible with future recyclable DNP applications. To do so, the solid-state DNP enhancement factors, build-up rates and DNP spectra of different hyperpolarizing solids containing various nitroxide radical loadings (20-74 μmol cm^-3) are compared against the DNP performance of varying nitroxide concentrations (10-100 mM) solvated in a glassy frozen solution. We demonstrate that in <1 s, polarization enhancement goes up to 56 and 102 with surface-bound and solvated radicals, respectively, under the optimized conditions. For the range of nitroxide concentrations used cross effect DNP seems to be the dominant mechanism under benchtop conditions. This was deduced from the electron paramagnetic resonance (EPR) lineshape of TEMPOL investigated using Q-band EPR measurements.

Fig. 1. Overview of solid-state dynamic nuclear polarization (DNP) principles to hyperpolarize analytes using paramagnetic radicals (a) randomly distributed in an amorphous frozen solution or (b) immobilized on the surface of a porous material when placed inside a magnetic field and cryogenic temperatures. In (I) the Lewis structure of the chemical components required for DNP with solvated radicals or hyperpolarizing solids are highlighted. In (II) a graphical representation of the radical and analyte distribution is provided in both cases. In (III) the principle of transferring spin hyperpolarization from spin polarized electrons (orange) to neighbouring nuclear spins (blue) embedded in most commonly a deuterated matrix (grey) is presented.

Fig. 2. Overview of the hardware and experimental design of the benchtop DNP polarizer. (a) Frontal cut of the prototype benchtop 1 T DNP polarizer co-developed by Bruker Biospin that was used for all of the DNP experiments. The polarizer is equipped with a 1 T permanent magnet (1) that is temperature stabilized at 301 K by 6 PID controlled heating mats (2) and a liquid nitrogen cryostat insert (3) that operates at 77 K. The cryostat itself is suited to fit a 4 mm EPR tube. (b) Transverse cut of the DNP NMR probe. A quartz tube (4) is used to hold the ¹³C solenoid radiofrequency (rf) coil (5) and the ¹H saddle rf coil (6) which can be tuned externally to 10.7 MHz and 42.57 MHz respectively. A Kα-band microwave source is used to amplify microwave frequencies from 26.6 to 28.8 GHz up to 5 watts. A rectangular antenna horn (7) is placed as close as possible to the NMR coils and directs the microwaves onto the sample location after the coaxial to waveguide transition (8). (c) The pseudo-3d pulse sequence is shown that enabled us to extract the hyperpolarization build-up rates using small angle rf pulses (1), the DNP enhancement factors (2) together with the DNP spectra (3). More information is provided in the materials and methods section.

Fig. 3. Solid-state ¹H-DNP performances at 1 T and 77 K with varying concentrations of TEMPOL radicals (a) randomly distributed in an amorphous frozen solution (10–100 mM in a 2 : 2 : 6 H₂O : D₂O : DMSO-d₆ (v : v : v) solution) and (b) under chemically immobilized conditions in the HYPSO-5 silica mesoporous matrix (20–74 μmol cm⁻³ impregnated with a partially protonated 2 : 8 H₂O : D₂O (v : v) solution). A single pulse sequence (Fig. 2c) with the corresponding MATLAB processing pipeline (available in Zenodo†) enabled us to capture the thermal and DNP hyperpolarized NMR spectra (at the optimal f_μw) (I), the enhancement factors (II), the hyperpolarization build-up profiles (III), the corresponding rates τ_dnp⁻¹ (IV) and the DNP spectra (V) in a single NMR experiment. The enhancement factors were calculated according to eqn (2) in the experimental section, while τ_dnp⁻¹ were fitted using the monoexponential function shown in eqn (3) with typically 15 build-up points. The error boundaries correspond to a 95% confidence interval. More information about the processing and experimental parameters can be found in the Materials and methods section.

Fig. 4. (a) ¹H-DNP spectrum measured for the optimal TEMPOL concentration of 50 mM at 1 T and 77 K. (b) ¹H-DNP build-up rates (τ_dnp⁻¹) at each corresponding f_μw of the DNP spectrum. τ_dnp⁻¹ values were extracted using the MATLAB processing pipeline after correction for the background build-up (see Fig. S4 and S5 (ESI‡)). The same methodology was used for each nitroxide concentration in frozen solution or in the hyperpolarizing solids. The results are presented in Fig. S6 and S7 of the ESI.

Fig. 5. (a) The influence of varying the microwave frequency modulation bandwidth Δf_μw (0–185 MHz) on the ¹H-DNP spectrum (from 27.9 GHz to 28.4 GHz) measured on the best performing HYPSO-5 powder with a nitroxide concentration of 43 μmol cm⁻³. In each experiment a microwave modulation frequency (f_mod) of 60 kHz with a triangle shape was used at the maximum output power of 5 watts. (b) A plot of the maximum negative enhancement factor deduced from the ¹H-DNP spectrum plotted against different Δf_μw. An optimum enhancement of 56 is reached at Δf_μw of 40 MHz.

Fig. 6. (a) Phase-sensitive continuous wave (CW)-EPR experiment measured via sweeping the magnetic field with a field modulation amplitude (ma) of 0.5 G and a modulation frequency (mf) of 50 kHz. (b) Q-band CW-EPR spectrum of the optimal 50 mM TEMPOL radical DNP solution (1) measured at 1.2 T and 77 K. The Easyspin MATLAB package was used to extract the anisotropic g-tensor and hyperfine A-tensor values at 1.2 T. (c) A simulated frequency-swept EPR spectrum of 50 mM TEMPOL to rationalize the EPR profile used for performing DNP at a benchtop polarizer with a specific field of 1.0022 T (ω_I = 42.669 MHz). The contribution of the ¹⁴N hyperfine transitions to the overall simulated EPR spectrum is also visualized in dotted, striped, and dotted-striped lines. See the Materials and methods section for more detailed information on the EPR measurements.

Fig. 7. Simulated ¹H DNP spectra assuming (a) cross effect and (b) solid effect type of polarization transfer using 50 mM TEMPOL in a partially protonated frozen glassy solution at 77 K and 1 T. The calculated line shapes assuming the cross effect and solid effect are overlayed with the experimental ¹H DNP spectrum as a comparison. Solid-state EPR simulations of TEMPOL at 1.0022 T were performed using the Easyspin MATLAB package and based on experimental CW-EPR measured at 1.2 T as can be seen in Fig. 6. The ¹H-DNP spectrum simulations are based on a straightforward analytical electron bin model assuming monochromatic microwave hole burning without spectral diffusion. Detailed information on the acquisition and simulation of the DNP spectra can be found in the Materials and methods section.

Fig. 8. A visual representation of the entire benchtop DNP polarizer setup. The location of the main hardware components (the magnet, the cryostat, the microwave source, the DNP-NMR probe) are highlighted using arrows.