Brojo Kishor Shachib Dhali‡
a,
Abubakar Abdurraheem‡
b,
Mustapha Abdulmojeeda,
Anna Samoilenkob,
Megan Pike
a,
Rielly J. Harrison
a,
Franziska Theiss
a,
Boyd M. Goodson
c,
Eduard Y. Chekmenev
*b and
Thomas Theis
*a
aDepartment of Chemistry, North Carolina State University, Raleigh, North Carolina 27965-8204, USA. E-mail: bdhali@ncsu.edu; ttheis@ncsu.edu
bDepartment of Chemistry, Karmanos Cancer Institute, Wayne State University, Detroit, MI 8202, USA. E-mail: gq3501@wayne.edu
cSchool of Chemical & Biomolecular Sciences, Southern Illinois University, Carbondale, Illinois 62901, USA
First published on 4th July 2025
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 45700 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.
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+.
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).
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.
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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 pKa 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.
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 ((yb − c) − 3δ)/m, where yb = 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.
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5cc02890f |
‡ Contributed equally to the experiments. |
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