SABRE-SHEATH hyperpolarized ¹⁵N₂-imidazole for Zn²⁺ sensing

Abstract

Zinc ions are essential for numerous biological functions and activities. Accordingly, Zn²⁺ sensors are crucial in biomedical research to understand the role of Zn²⁺ in health and disease. Here, we demonstrated the viability of SABRE-SHEATH hyperpolarized ¹⁵N₂-imidazole, providing an NMR signal enhancement of 45 700 fold (p = 2.15%), as a probe for Zn²⁺ sensing by monitoring the Zn-imidazole interaction using NMR and extracted a LOD of 1.3 mM. This study is one of the first demonstrations of SABRE-SHEATH hyperpolarized ¹⁵N as a sensor of other non-hyperpolarized species, which promises chemical sensing without penetration-depth limitations.

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Nuclear Magnetic Resonance (NMR) is a powerful technique for non-destructive structural elucidation and monitoring of molecular interactions. However, NMR is notoriously insensitive, especially for heteronuclei, such as ¹³C and ¹⁵N, and requires large sample volumes or high concentrations. Therefore, the study of low-concentration targets often remains out of reach.^1,2 In this work, we employed Signal Amplification By Reversible Exchange in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH), to enhance the ¹⁵N NMR signal of ¹⁵N₂-imidazole up to ∼45 700 fold at 1.4 T (p = 2.15%). The hyperpolarized ¹⁵N₂-imidazole is subsequently used to quantify the Zn²⁺ content in a test solution via changes of the ¹⁵N chemical shift upon ¹⁵N₂-imidazole binding to Zn²⁺.

SABRE-SHEATH is a cost-effective and non-destructive hyperpolarization technique. SABRE-SHEATH creates nuclear spin polarization of a target molecule by simultaneous chemical exchange of parahydrogen (p-H₂) and the target on an iridium catalyst.^3–15 The employed pre-catalyst is [IrCl(COD)(IMes)] (where, IMes = 1,3-bis(2,4,6-trimethyl-phenyl)imidazole-2-ylidene), which forms the active polarization transfer catalyst as illustrated in Fig. 1. The afforded signal enhancement is subsequently used to monitor the interaction between the ¹⁵N nuclei of ¹⁵N₂-imidazole and Zn²⁺.

Fig. 1 Illustration of the reversible exchange of p-H₂ and ¹⁵N₂-imidazole on the iridium catalyst promoting polarization transfer through a J-coupling network. Initially, p-H₂ transfers its spin order to catalyst-bound ¹⁵N₂-imidazole, which subsequently exchanges off the catalyst to form free, hyperpolarized ¹⁵N₂-imidazole. Finally, the hyperpolarized ¹⁵N₂-imidazole binds to Zn²⁺. The hyperpolarized ¹⁵N₂-imidazole NMR signal directly reports on the binding event.

Zinc is a crucial trace element with different biological activities, including enzyme catalysis, gene expression, cell signaling, and immune response.^16–23 Zinc ions also regulate neuronal activity and synaptic plasticity in the brain.^24–28 Accordingly, Zn²⁺ sensors play vital roles in biomedical research used to detect and monitor Zn²⁺ ions in biological systems. Understanding the physiological and pathological roles of Zn²⁺ ions in various biological functions and diseases is critical to the development of new diagnostic and therapeutic strategies.^29–33 Literature demonstrates that Zn²⁺ ions interact with the nitrogen atoms of imidazole to form coordination complexes with coordination numbers of one, two, three, or four—predominantly four.^34–38 The present study showcases the ability of hyperpolarized ¹⁵N₂-imidazole to detect Zn²⁺. We selected this system because of imidazole's ability to coordinate with Zn²⁺, and the diamagnetic nature of Zn²⁺, which avoids paramagnetic relaxation of the hyperpolarized ¹⁵N nuclear spins.^34,39

Previous work showed that ¹⁵N₂-imidazole hyperpolarizes more effectively under basic conditions.⁴⁰ Therefore, a sample containing 100 mM ¹⁵N₂-imidazole, 6 mM Ir-IMes pre-catalyst and 1 mM NaOH in methanol was prepared, and a 0.8 mL aliquot of the solution was carefully transferred into an NMR tube, as shown in Fig. 2. The hyperpolarization pre-catalyst in the sample was then activated by flowing p-H₂ at a rate of 100 sccm and a pressure of 100 psi at 52 °C for 10 minutes. Once fully activated, the sample changed color from pale yellow to colorless. The activated sample was transferred to a mu-metal shield, where p-H₂ was bubbled for 40 seconds at an optimized polarization transfer field of 0.4 μT and a temperature of 37 °C to hyperpolarize the ¹⁵N nuclei of ¹⁵N₂-imidazole (see ESI,† Section S8 for the optimization studies of polarization build-up, polarization transfer field and temperature). After hyperpolarization, the sample was transferred to a 1.4 T Magritek benchtop NMR spectrometer and depressurized by disconnecting the NMR sample tube from the bubbling setup. Immediately, a 30-degree pulse was applied to acquire a proton-decoupled ¹⁵N NMR signal of neutral ¹⁵N₂-imidazole. Finally, the analyte of interest (Zn²⁺ solution, or HCl solution, or methanol for the control experiment) was injected into the NMR tube using a syringe. The solutions were mixed by shaking the PTFE catheter, and a 30-degree pulse was applied to acquire the proton-decoupled ¹⁵N NMR signal to detect the chemical interactions (see ESI,† Section S2 for detailed experimental procedure and data processing).

Fig. 2 Schematic diagram depicting the process of Zn²⁺ sensing using hyperpolarized ¹⁵N₂-imidazole. (a) The sample was activated by bubbling p-H₂ while it was in a heated water bath. (b) The activated sample was hyperpolarized in the mu-metal shield by bubbling p-H₂ at an optimized polarization transfer field. (c) The hyperpolarized solution was transferred to the benchtop NMR, depressurized, and the target (Zn²⁺) was injected.

The primary objective of this study was to quantify the Zn²⁺ content in a test solution via changes of the ¹⁵N chemical shift upon ¹⁵N₂-imidazole binding to Zn²⁺. Previous work showed that hyperpolarized ¹⁵N₂-imidazole can be used as a pH probe.⁴⁰ Because the analyte of interest, ZnCl₂, is slightly acidic,^40–42 the ¹⁵N chemical shift of imidazole may not only respond to zinc binding but also to changes in pH. Therefore, it was necessary to distinguish pH-induced shifts from binding-induced shifts. To test the pH effect in isolation, we titrated the hyperpolarized ¹⁵N₂-imidazole with a strong acid (HCl) to identify a range where ¹⁵N₂-imidazole remains in its original form (neutral structure) and its peak position remains unchanged. As illustrated in Fig. 3, the neutral structure of ¹⁵N₂-imidazole displayed identical chemical shifts (203.4 ppm) for both ¹⁵N nuclei as a result of rapid proton hopping between the two nitrogen sites. Imidazole reacts as a weak base, and adopts a protonated structure in an acidic environment, resulting in a decrease in ¹⁵N NMR frequency below pH ≈ 6.2. Note that in these ¹⁵N spectra, no ¹H splitting was observed because the spectra were proton-decoupled (see ESI,† Fig. S1, for a spectrum over a larger bandwidth). The additional minor peaks in the ¹⁵N spectra correspond to the Ir-bound ¹⁵N₂-imidazole species at various positions within the ¹⁵N₂-imidazole hexacoordinate Ir complex. The exact spectral assignment of the bound ¹⁵N peaks remains ambiguous at this time (see ESI,† Section S3 for further discussion). In the titration study with HCl, we found that the main (free) ¹⁵N peak position remained stable up to a concentration of 20 mM HCl (pH ≈ 6.2) as shown in Fig. 3. Since HCl is highly acidic compared to ZnCl₂, this finding gave us confidence to add ZnCl₂ up to 20 mM in our sample and attribute any chemical shift changes to binding between Zn²⁺ ions and ¹⁵N₂-imidazole.

Fig. 3 Titration of ¹⁵N₂-imidazole with HCl. No shift in peak position was observed up to a concentration of 20 mM HCl, with sensitivity beginning below a pH of ≈6.2. Species “a” and “b” represent unidentified Ir-bound ¹⁵N₂-imidazole at two different positions within the ¹⁵N₂-imidazole hexacoordinate Ir complex. The HCl (mM) column represents the HCl concentration in the mixture, whereas the pH column reports the pH of the final mixture (see ESI,† Section S6 for more information on pH and Fig. S4 for the measurement of the pK_a value of ¹⁵N₂-imidazole in methanol, ESI†).

We then proceeded with Zn²⁺ sensing by adding ZnCl₂ solution up to 15.8 mM into our hyperpolarized ¹⁵N₂-imidazole sample and acquiring ¹⁵N NMR spectra to observe chemical shift changes, as illustrated in Fig. 4. The pH of the sample mixture was recorded as ≈7.3 after adding ZnCl₂ to a final concentration of 15.8 mM and was well within the region where peak position remains unchanged due to pH (see Fig. 3). Therefore, any observed shifts can be attributed exclusively to Zn²⁺ binding. The ¹⁵N NMR spectrum of hyperpolarized ¹⁵N₂-imidazole before and after the addition of ZnCl₂ solution clearly indicated a reduction in chemical shift, confirming the interaction between Zn²⁺ ions and ¹⁵N₂-imidazole, as shown in Fig. 4. In addition to a chemical shift change, line broadening is observed at higher Zn²⁺ concentrations, which can be attributed to ligand exchange on Zn²⁺. Upon Zn²⁺ binding, the chemical equivalence of the two ¹⁵N sites in ¹⁵N₂-imidazole is broken, which also contributes to the observed broadening in addition to exchange. Although the line broadening effect could also be used to quantify Zn²⁺ binding, the change in chemical shift is a more reliable measure to quantify Zn²⁺ concentration, which we quantified with the calibration curve presented in Fig. 5.

Fig. 4 The response of ¹⁵N₂-imidazole chemical shift to the addition of Zn²⁺. The frequency of ¹⁵N₂-imidazole is reduced as Zn²⁺ is added gradually, indicating the formation of Zn-bound ¹⁵N₂-imidazole complexes. Species “a” and “b” represent unidentified Ir-bound ¹⁵N₂-imidazole at two different positions within the ¹⁵N₂-imidazole hexacoordinate Ir complex. The Zn²⁺ (mM) column represents the final Zn²⁺ concentration in the mixture.

Fig. 5 Calibration curve for Zn²⁺ sensing with hyperpolarized ¹⁵N₂-imidazole. A linear correlation between the ¹⁵N₂-imidazole peak shift and Zn²⁺ concentration is identified (R² = 0.984). LOD estimation is depicted, with the blank measurement (black) separated from the subsequent measurement (brown) by 3δ, yielding a LOD value of 1.3 mM.

The calibration curve shown in Fig. 5 revealed a linear dependence of peak shift with Zn²⁺ concentration. To obtain the depicted calibration curve, peak positions were identified as the center of the Full Width at Half Maximum (FWHM), as opposed to choosing the location of highest intensity in the spectrum. Using the depicted calibration curve, we proceeded to determine the Limit of Detection (LOD) for Zn²⁺ sensing with hyperpolarized ¹⁵N₂-imidazole. LOD is calculated as ((y_b − c) − 3δ)/m, where y_b = blank measurement (peak position of free ¹⁵N₂-imidazole without addition of Zn²⁺), c = intercept of the calibration curve, m = slope of the calibration curve, and δ represents the standard deviation of the blank measurements.^43–45 In this method, the FWHM serves as the most direct measure of uncertainty, as it defines the minimum chemical shift difference required to distinguish two peaks. Therefore, we set δ = FWHM/2 of the NMR line of blank measurement. This concept is illustrated in Fig. 5, showing the blank measurement (black) separated from the subsequent measurement (brown) by 3δ, which allows for distinction of the peaks and thus determines the LOD. As a result, the LOD was determined as 1.3 mM for Zn²⁺ using hyperpolarized ¹⁵N₂-imidazole as the sensor (see ESI,† Section S7 for full calculation).

In conclusion, we introduced ¹⁵N hyperpolarized tracers as biochemical sensors for indirect sensing of a non-hyperpolarized species. Specifically, the interaction between hyperpolarized ¹⁵N₂-imidazole with Zn²⁺ was shown using a 1.4 T Magritek benchtop NMR spectrometer. To this end, we first isolated peak shifts due to pH changes from peak shifts due to Zn²⁺ binding by carefully characterizing the system's behavior in the pH range of 10 to 5.8. Subsequently, we observed the changes in the ¹⁵N NMR spectra as a function of added ZnCl₂ solution and found a significant response of the peak position and the line broadening. The peak position was used to establish a calibration curve for Zn²⁺ sensing with hyperpolarized ¹⁵N₂-imidazole. From the calibration curve, a LOD of 1.3 mM was extracted. This work demonstrates the use of hyperpolarized ¹⁵N probes as molecular sensors, exemplified by ¹⁵N₂-imidazole as a probe for Zn²⁺ sensing. For future physiological applications, it will be crucial to enhance the Zn²⁺ sensor's sensitivity to the micromolar-to-nanomolar range, for example, via decreasing the concentration of hyperpolarized ¹⁵N₂-imidazole in the sample. Furthermore, ensuring biocompatibility by eliminating methanol as a solvent is essential. Possible strategies to achieve this include phase separation or gas stripping.^46,47 The emerging technology is positioned to probe the role of Zn²⁺ or other ions and their biological activities, such as enzyme catalysis, gene expression, or cell signaling.

This material is based upon work supported by the U.S. Department of Energy, Office of Biological and Environmental Research (BER) under award number(s) DE-SC0023334 and DE-SC0025315 (TT & EYC). Funding of this work was also provided by the NIH under R21EB033872 (EYC & BMG), and NSF grants CHE-2404387 (BMG) and CHE-2404388 (EYC). This report was prepared as an account of work sponsored by an agency of the United States Government. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency.

Conflicts of interest

E. Y. C. and B. M. G. are co-founders and equity holders of XeUS Technologies LTD.

Data availability

The data supporting this article have been included as part of the ESI.†

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5cc02890f

‡ Contributed equally to the experiments.

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