Single Molecule Discrimination of Heteropolytungstates and Their Isomers in Solution with a Nanometer-Scale Pore

We report a new method to identify metallic nanoclusters (polyoxometalate structures) in solution at the single molecule limit using a nanometer-scale pore. The technique allows the measurement of polyoxometalates with over 2 orders of magnitude lower analyte concentration than conventional analytical chemistry tools. Furthermore, pH-dependent structural changes in phosphotungstic acid are measured with protein nanopores and validated with NMR. We further demonstrate that the method can also discriminate [PW₉O₃₄]^9– structural isomers. The results suggest this technique can serve as a complementary approach to traditional methods

Catalyst-Free Aqueous Hyperpolarized [1-¹³C]Pyruvate Obtained by Re-Dissolution Signal Amplification by Reversible Exchange

Despite great successes in oncology, patient outcomes are often still discouraging, and hence the diagnostic imaging paradigm is increasingly shifting toward functional imaging of the pathology to better understand individual disease biology and to personalize therapies. The dissolution Dynamic Nuclear Polarization (d-DNP) hyperpolarization method has enabled unprecedented real-time MRI sensing of metabolism and tissue pH using hyperpolarized [1-13C]pyruvate as a biosensor with great potential for diagnosis and monitoring of cancer patients. However, current d-DNP is expensive and suffers from long hyperpolarization times, posing a substantial translational roadblock. Here, we report the development of Re-Dissolution Signal Amplification By Reversible Exchange (Re-D SABRE), which relies on fast and low-cost hyperpolarization of [1-13C]pyruvate by chemical exchange with parahydrogen at microtesla magnetic fields. [1-13C]pyruvate is precipitated from catalyst-containing methanol using ethyl acetate and rapidly reconstituted in aqueous media. 13C polarization of 9 ± 1% is demonstrated after redissolution in water with residual iridium mass fraction of 8.5 ± 1.5 ppm; further improvement is anticipated via process automation. Re-D SABRE makes hyperpolarized [1-13C]pyruvate biosensor available at a fraction of the cost (<$10,000) and production time (≈1 min) of currently used techniques and makes aqueous hyperpolarized [1-13C]pyruvate “ready” for in vivo applications

MOSAIC: A Modular Single-Molecule Analysis Interface for Decoding Multistate Nanopore Data

Biological and solid-state nanometer-scale pores are the basis for numerous emerging analytical technologies for use in precision medicine. We developed Modular Single-Molecule Analysis Interface (MOSAIC), an open source analysis software that improves the accuracy and throughput of nanopore-based measurements. Two key algorithms are implemented: ADEPT, which uses a physical model of the nanopore system to characterize short-lived events that do not reach their steady-state current, and CUSUM+, a version of the cumulative sum statistical method optimized for longer events that do. We show that ADEPT detects previously unreported conductance states that occur as double-stranded DNA translocates through a 2.4 nm solid-state nanopore and reveals new interactions between short single-stranded DNA and the vestibule of a biological pore. These findings demonstrate the utility of MOSAIC and the ADEPT algorithm, and offer a new tool that can improve the analysis of nanopore-based measurements

Rapid ¹³C Hyperpolarization of the TCA Cycle Intermediate α‑Ketoglutarate via SABRE-SHEATH

α-Ketoglutarate is a key biomolecule involved in a number of metabolic pathwaysmost notably the TCA cycle. Abnormal α-ketoglutarate metabolism has also been linked with cancer. Here, isotopic labeling was employed to synthesize [1-13C,5-12C,D4]α-ketoglutarate with the future goal of utilizing its [1-13C]-hyperpolarized state for real-time metabolic imaging of α-ketoglutarate analytes and its downstream metabolites in vivo. The signal amplification by reversible exchange in shield enables alignment transfer to heteronuclei (SABRE-SHEATH) hyperpolarization technique was used to create 9.7% [1-13C] polarization in 1 minute in this isotopologue. The efficient 13C hyperpolarization, which utilizes parahydrogen as the source of nuclear spin order, is also supported by favorable relaxation dynamics at 0.4 μT field (the optimal polarization transfer field): the exponential 13C polarization buildup constant Tb is 11.0 ± 0.4 s whereas the 13C polarization decay constant T1 is 18.5 ± 0.7 s. An even higher 13C polarization value of 17.3% was achieved using natural-abundance α-ketoglutarate disodium salt, with overall similar relaxation dynamics at 0.4 μT field, indicating that substrate deuteration leads only to a slight increase (∼1.2-fold) in the relaxation rates for 13C nuclei separated by three chemical bonds. Instead, the gain in polarization (natural abundance versus [1-13C]-labeled) is rationalized through the smaller heat capacity of the “spin bath” comprising available 13C spins that must be hyperpolarized by the same number of parahydrogen present in each sample, in line with previous 15N SABRE-SHEATH studies. Remarkably, the C-2 carbon was not hyperpolarized in both α-ketoglutarate isotopologues studied; this observation is in sharp contrast with previously reported SABRE-SHEATH pyruvate studies, indicating that the catalyst-binding dynamics of C-2 in α-ketoglutarate differ from that in pyruvate. We also demonstrate that 13C spectroscopic characterization of α-ketoglutarate and pyruvate analytes can be performed at natural 13C abundance with an estimated detection limit of 80 micromolar concentration × *%P13C. All in all, the fundamental studies reported here enable a wide range of research communities with a new hyperpolarized contrast agent potentially useful for metabolic imaging of brain function, cancer, and other metabolically challenging diseases