Efficient Parahydrogen-Induced 13C Hyperpolarization on a Microfluidic Device.

We show the direct production and detection of 13C-hyperpolarized fumarate by parahydrogen-induced polarization (PHIP) in a microfluidic lab-on-a-chip (LoC) device and achieve 8.5% 13C polarization. This is the first demonstration of 13C-hyperpolarization of a metabolite by PHIP in a microfluidic device. LoC technology allows the culture of mammalian cells in a highly controlled environment, providing an important tool for the life sciences. In-situ preparation of hyperpolarized metabolites greatly enhances the ability to quantify metabolic processes in such systems by microfluidic NMR. PHIP of 1H nuclei has been successfully implemented in microfluidic systems, with mass sensitivities in the range of pmol/s. However, metabolic NMR requires high-yield production of hyperpolarized metabolites with longer spin life times than is possible with 1H. This can be achieved by transfer of the polarization onto 13C nuclei, which exhibit much longer T1 relaxation times. We report an improved microfluidic PHIP device, optimized using a finite element model, that enables the direct and efficient production of 13C-hyperpolarized fumarate.

Fully Automated Characterization of Protein-Peptide Binding by Microfluidic 2D NMR.

We demonstrate an automated microfluidic nuclear magnetic resonance (NMR) system that quantitatively characterizes protein-ligand interactions without user intervention and with minimal sample needs through protein-detected heteronuclear 2D NMR spectroscopy. Quantitation of protein-ligand interactions is of fundamental importance to the understanding of signaling and other life processes. As is well-known, NMR provides rich information both on the thermodynamics of binding and on the binding site. However, the required titrations are laborious and tend to require large amounts of sample, which are not always available. The present work shows how the analytical power of NMR detection can be brought in line with the trend of miniaturization and automation in life science workflows.

Imported Toxigenic Corynebacterium Diphtheriae in Refugees with Polymicrobial Skin Infections, Germany, 2022.

During August-December 2022, toxigenic Corynebacterium diphtheriae was isolated from 25 refugees with skin infections and 2 refugees with asymptomatic throat colonization at a refugee reception center in Germany. None had systemic toxin-mediated illness. Of erosive/ulcerative skin infections, 96% were polymicrobial. Erosive/ulcerative wounds in refugees should undergo testing to rule out cutaneous diphtheria.

Enzymatic Reactions Observed with Zero- and Low-Field Nuclear Magnetic Resonance.

We demonstrate that enzyme-catalyzed reactions can be observed in zero- and low-field NMR experiments by combining recent advances in parahydrogen-based hyperpolarization methods with state-of-the-art magnetometry. Specifically, we investigated two model biological processes: the conversion of fumarate into malate, which is used in vivo as a marker of cell necrosis, and the conversion of pyruvate into lactate, which is the most widely studied metabolic process in hyperpolarization-enhanced imaging. In addition to this, we constructed a microfluidic zero-field NMR setup to perform experiments on microliter-scale samples of [1-13C]fumarate in a lab-on-a-chip device. Zero- to ultralow-field (ZULF) NMR has two key advantages over high-field NMR: the signals can pass through conductive materials (e.g., metals), and line broadening from sample heterogeneity is negligible. To date, the use of ZULF NMR for process monitoring has been limited to studying hydrogenation reactions. In this work, we demonstrate this emerging analytical technique for more general reaction monitoring and compare zero- vs low-field detection.

Development of Low-Magnetic Susceptibility Microcoils via 5-Axis Machining for Analysis of Biological and Environmental Samples.

In environmental research, it is critical to understand how toxins impact invertebrate eggs and egg banks, which, due to their tiny size, are very challenging to study by conventional nuclear magnetic resonance (NMR) spectroscopy. Microcoil technology has been extensively utilized to enhance the mass-sensitivity of NMR. In a previous study, 5-axis computer numerical control (CNC) micromilling (shown to be a viable alternative to traditional microcoil production methods) was used to create a prototype copper slotted-tube resonator (STR). Despite the excellent limit of detection (LOD) of the resonator, the quality of the line shape was very poor due to the magnetic susceptibility of the copper resonator itself. This is best solved using magnetic susceptibility-matched materials. In this study, approaches are investigated that improve the susceptibility while retaining the versatility of coil milling. One method involves machining STRs from various copper/aluminum alloys, while the other involves machining ones from an aluminum 2011 alloy and electroplating them with copper. In all cases, combining copper and aluminum to produce resonators resulted in improved line shape and SNR compared to pure copper resonators due to their reduced magnetic susceptibility. However, the copper-plated aluminum resonators showed optimal performance from the devices tested. The enhanced LOD of these STRs allowed for the first 1H-13C heteronuclear multiple quantum coherence (HMQC) of a single intact 13C-labeled Daphnia magna egg (∼4 µg total biomass). This is a key step toward future screening programs that aim to elucidate the toxic processes in aquatic eggs.

Direct Production of a Hyperpolarized Metabolite on a Microfluidic Chip.

Microfluidic systems hold great potential for the study of live microscopic cultures of cells, tissue samples, and small organisms. Integration of hyperpolarization would enable quantitative studies of metabolism in such volume limited systems by high-resolution NMR spectroscopy. We demonstrate, for the first time, the integrated generation and detection of a hyperpolarized metabolite on a microfluidic chip. The metabolite [1-13C]fumarate is produced in a nuclear hyperpolarized form by (i) introducing para-enriched hydrogen into the solution by diffusion through a polymer membrane, (ii) reaction with a substrate in the presence of a ruthenium-based catalyst, and (iii) conversion of the singlet-polarized reaction product into a magnetized form by the application of a radiofrequency pulse sequence, all on the same microfluidic chip. The microfluidic device delivers a continuous flow of hyperpolarized material at the 2.5 µL/min scale, with a polarization level of 4%. We demonstrate two methods for mitigating singlet-triplet mixing effects which otherwise reduce the achieved polarization level.

Quantification and Prediction of Imine Formation Kinetics in Aqueous Solution by Microfluidic NMR Spectroscopy.

Quantitatively predicting the reactivity of dynamic covalent reaction is essential to understand and rationally design complex structures and reaction networks. Herein, the reactivity of aldehydes and amines in various rapid imine formation in aqueous solution by microfluidic NMR spectroscopy was quantified. Investigation of reaction kinetics allowed to quantify the forward rate constants k+ by an empirical equation, of which three independent parameters were introduced as reactivity parameters of aldehydes (SE , E) and amines (N). Furthermore, these reactivity parameters were successfully used to predict the unknown forward rate constants of imine formation. Finally, two competitive reaction networks were rationally designed based on the proposed reactivity parameters. Our work has demonstrated the capability of microfluidic NMR spectroscopy in quantifying the kinetics of label-free chemical reactions, especially rapid reactions that are complete in minutes.

Spatially Resolved Kinetic Model of Parahydrogen Induced Polarisation (PHIP) in a Microfluidic Chip.

We report a spatially resolved kinetic finite element model of parahydrogen-induced polarisation (PHIP) in a microfluidic chip that was calibrated using on-chip and off-chip NMR data. NMR spectroscopy has great potential as a read-out technique for lab-on-a-chip (LoC) devices, but is often limited by sensitivity. By integrating PHIP with a LoC device, a continuous stream of hyperpolarised material can be produced, and mass sensitivities of pmols have been achieved. However, the yield and polarisation levels have so far been quite low, and can still be optimised. To facilitate this, a kinetic model of the reaction has been developed, and its rate constants have been calibrated using macroscopic kinetic measurements. The kinetic model was then coupled with a finite element model of the microfluidic chip. The model predicts the concentration of species involved in the reaction as a function of flow rate and position in the device. The results are in quantitative agreement with published experimental data.

The Cu chaperone CopZ is required for Cu homeostasis in Rhodobacter capsulatus and influences cytochrome cbb3 oxidase assembly.

Cu homeostasis depends on a tightly regulated network of proteins that transport or sequester Cu, preventing the accumulation of this toxic metal while sustaining Cu supply for cuproproteins. In Rhodobacter capsulatus, Cu-detoxification and Cu delivery for cytochrome c oxidase (cbb3 -Cox) assembly depend on two distinct Cu-exporting P1B -type ATPases. The low-affinity CopA is suggested to export excess Cu and the high-affinity CcoI feeds Cu into a periplasmic Cu relay system required for cbb3 -Cox biogenesis. In most organisms, CopA-like ATPases receive Cu for export from small Cu chaperones like CopZ. However, whether these chaperones are also involved in Cu export via CcoI-like ATPases is unknown. Here we identified a CopZ-like chaperone in R. capsulatus, determined its cellular concentration and its Cu binding activity. Our data demonstrate that CopZ has a strong propensity to form redox-sensitive dimers via two conserved cysteine residues. A ΔcopZ strain, like a ΔcopA strain, is Cu-sensitive and accumulates intracellular Cu. In the absence of CopZ, cbb3 -Cox activity is reduced, suggesting that CopZ not only supplies Cu to P1B -type ATPases for detoxification but also for cuproprotein assembly via CcoI. This finding was further supported by the identification of a ~150 kDa CcoI-CopZ protein complex in native R. capsulatus membranes.

5-Axis CNC Micromilling for Rapid, Cheap, and Background-Free NMR Microcoils.

The superior mass sensitivity of microcoil technology in nuclear magnetic resonance (NMR) spectroscopy provides potential for the analysis of extremely small-mass-limited samples such as eggs, cells, and tiny organisms. For optimal performance and efficiency, the size of the microcoil should be tailored to the size of the mass-limited sample of interest, which can be costly as mass-limited samples come in many shapes and sizes. Therefore, rapid and economic microcoil production methods are needed. One method with great potential is 5-axis computer numerical control (CNC) micromilling, commonly used in the jewelry industry. Most CNC milling machines are designed to process larger objects and commonly have a precision of >25 µm (making the machining of common spiral microcoils, for example, impossible). Here, a 5-axis MiRA6 CNC milling machine, specifically designed for the jewelry industry, with a 0.3 µm precision was used to produce working planar microcoils, microstrips, and novel microsensor designs, with some tested on the NMR in less than 24 h after the start of the design process. Sample wells could be built into the microsensor and could be machined at the same time as the sensors themselves, in some cases leaving a sheet of Teflon as thin as 10 µm between the sample and the sensor. This provides the freedom to produce a wide array of designs and demonstrates 5-axis CNC micromilling as a versatile tool for the rapid prototyping of NMR microsensors. This approach allowed the experimental optimization of a prototype microstrip for the analysis of two intact adult Daphnia magna organisms. In addition, a 3D volume slotted-tube resonator was produced that allowed for 2D 1H-13C NMR of D. magna neonates and exhibited 1H sensitivity (nLODω600 = 1.49 nmol s1/2) close to that of double strip lines, which themselves offer the best compromise between concentration and mass sensitivity published to date.

High-Resolution Nuclear Magnetic Resonance Spectroscopy with Picomole Sensitivity by Hyperpolarization on a Chip.

We show that high-resolution NMR can reach picomole sensitivity for micromolar concentrations of analyte by combining parahydrogen-induced hyperpolarization (PHIP) with a high-sensitivity transmission line microdetector. The para-enriched hydrogen gas is introduced into solution by diffusion through a membrane integrated into a microfluidic chip. NMR microdetectors, operating with sample volumes of a few µL or less, benefit from a favorable scaling of mass sensitivity. However, the small volumes make it very difficult to detect species present at less than millimolar concentrations in microfluidic NMR systems. In view of overcoming this limitation, we implement PHIP on a microfluidic device with a 2.5 µL detection volume. Integrating the hydrogenation reaction into the chip minimizes polarization losses to spin-lattice relaxation, allowing the detection of picomoles of substance. This corresponds to a concentration limit of detection of better than 1µMs , unprecedented at this sample volume. The stability and sensitivity of the system allow quantitative characterization of the signal dependence on flow rates and other reaction parameters and permit homo- (1H-1H) and heteronuclear (1H-13C) 2D NMR experiments at natural 13C abundance.

Hyperpolarization of Amino Acids in Water Utilizing Parahydrogen on a Rhodium Nanocatalyst.

NMR offers many possibilities in chemical analysis, structural investigations, and medical diagnostics. Although it is broadly used, one of NMR spectroscopies main drawbacks is low sensitivity. Hyperpolarization techniques enhance NMR signals by more than four orders of magnitude allowing the design of new contrast agents. Parahydrogen induced polarization that utilizes the para-hydrogen's singlet state to create enhanced signals is of particular interest since it allows to produce molecular imaging agents within seconds. Herein, we present a strategy for signal enhancement of the carbonyl 13 C in amino acids by using parahydrogen, as demonstrated for glycine and alanine. Importantly, the hyperpolarization step is carried out in water and chemically unmodified canonical amino acids are obtained. Our approach thus offers a high degree of biocompatibility, which is crucial for further application. The rapid sample hyperpolarization (within seconds) may enable the continuous production of biologically useful probes, such as metabolic contrast agents or probes for structural biology.

Cooperation between two periplasmic copper chaperones is required for full activity of the cbb3 -type cytochrome c oxidase and copper homeostasis in Rhodobacter capsulatus.

Copper (Cu) is an essential micronutrient that functions as a cofactor in several important enzymes, such as respiratory heme-copper oxygen reductases. Yet, Cu is also toxic and therefore cells engage a highly coordinated Cu uptake and delivery system to prevent the accumulation of toxic Cu concentrations. In this study, we analyzed Cu delivery to the cbb3 -type cytochrome c oxidase (cbb3 -Cox) of Rhodobacter capsulatus. We identified the PCuA C-like periplasmic chaperone PccA and analyzed its contribution to cbb3 -Cox assembly. Our data demonstrate that PccA is a Cu-binding protein with a preference for Cu(I), which is required for efficient cbb3 -Cox assembly, in particular, at low Cu concentrations. By using in vivo and in vitro cross-linking, we show that PccA forms a complex with the Sco1-homologue SenC. This complex is stabilized in the absence of the cbb3 -Cox-specific assembly factors CcoGHIS. In cells lacking SenC, the cytoplasmic Cu content is significantly increased, but the simultaneous absence of PccA prevents this Cu accumulation. These data demonstrate that the interplay between PccA and SenC not only is required for Cu delivery during cbb3 -Cox assembly but also regulates Cu homeostasis in R. capsulatus.

Visualisation of quantum evolution in the Stern-Gerlach and Rabi experiments.

The Stern-Gerlach experiment is a seminal experiment in quantum physics, involving the interaction between a particle with spin and an applied magnetic field gradient. A recent article [Wennerström et al., Phys. Chem. Chem. Phys., 2012, 14, 1677-1684] claimed that a full understanding of the Stern-Gerlach experiment can only be attained if transverse spin relaxation is taken into account, generated by fluctuating magnetic fields originating in the magnetic materials which generate the field gradient. This interpretation is contrary to the standard quantum description of the Stern-Gerlach experiment, which requires no dissipative effects. We present simulations of conventional quantum dynamics in the Stern-Gerlach experiment, using extended Wigner functions to describe the propagation of the quantum state in space and time. No relaxation effects are required to reproduce the qualitative experimental behaviour. We also present simulations of quantum dynamics in the Rabi experiment, in which an applied radiofrequency field induces spin transitions in the particle wave.

Power and signal transmission protocol for a contactless subdural spinal cord stimulation device.

Wireless signal transmission will play a critical role in developing reliable subdural spinal cord stimulation systems. We have developed an approach to inductively coupling signals across the epidural spacing between the pial and epidural surfaces. The major design constraints include tolerance of coil misalignments from spinal cord geometries in addition to reasonable power efficiencies within the expected range of movement. The design of the primary side as a driving circuit is simplified by several turns of commercial magnetic wire, whereas the implanted secondary side is implemented in a micro-planar spiral coil tuned to a resonant frequency of 1.6 MHz. We present the results of wireless inductive coupling experiments that demonstrate the ability to transmit and receive a frequency modulated 1.6 MHz carrier signal between primary and secondary coil antennae scaled to 10 mm. Power delivery is in the range of 400 mW at a link efficiency of 32 % for strong coupling (coil separations of 0.5 mm ) and in the range of 70 mW at 4 % efficiency for weak coupling (coil separations of 10 mm).

Operando NMR metabolomics of a microfluidic cell culture.

In this work we demonstrate the use of microfluidic NMR for in situ culture and quantitative analysis of metabolism in hepatocellular carcinoma (HCC) cell lines. A hydrothermal heating system is used to enable continuous in situ NMR observation of HCC cell culture over a 24 h incubation period. This technique is nondestructive, non-invasive and can measure millimolar concentrations at microlitre volumes, within a few minutes and in precisely controlled culture conditions. This is sufficient to observe changes in primary energy metabolism, using around 500-3500 cells per device, and with a time resolution of 17 min. The ability to observe intracellular responses in a time-resolved manner provides a more detailed view of a biological system and how it reacts to stimuli. This capability will allow detailed metabolomic studies of cell-culture based cancer models, enabling quantification of metabolic reporgramming, the metabolic tumor microenvironment, and the metabolic interplay between cancer- and immune cells.

New detection modality for label-free quantification of DNA in biological samples via superparamagnetic bead aggregation.

Combining DNA and superparamagnetic beads in a rotating magnetic field produces multiparticle aggregates that are visually striking, enabling label-free optical detection and quantification of DNA at levels in the picogram per microliter range. DNA in biological samples can be quantified directly by simple analysis of optical images of microfluidic wells placed on a magnetic stirrer without prior DNA purification. Aggregation results from DNA/bead interactions driven either by the presence of a chaotrope (a nonspecific trigger for aggregation) or by hybridization with oligonucleotides on functionalized beads (sequence-specific). This paper demonstrates quantification of DNA with sensitivity comparable to that of the best currently available fluorometric assays. The robustness and sensitivity of the method enable a wide range of applications, illustrated here by counting eukaryotic cells. Using widely available and inexpensive benchtop hardware, the approach provides a highly accessible low-tech microscale alternative to more expensive DNA detection and cell counting techniques.

Contactless NMR spectroscopy on a chip.

Inductively coupled planar resonators offer convenient integration of high-resolution NMR spectroscopy with microfluidic lab-on-a-chip devices. Planar spiral resonators are fabricated lithographically either by gold electroplating or by etching Cu laminated with polyimide. Their performance is characterized by NMR imaging as well as spectroscopy. A single-scan limit of detection LOD(t) = 0.95 nmol s(1/2) was obtained from sample volumes around 1 µL. The sensitivity of this approach is similar to that obtained by microstripline and microslot probes.

Synergies between Hyperpolarized NMR and Microfluidics: A Review.

Hyperpolarized nuclear magnetic resonance and lab-on-a-chip microfluidics are two dynamic, but until recently quite distinct, fields of research. Recent developments in both areas increased their synergistic overlap. By microfluidic integration, many complex experimental steps can be brought together onto a single platform. Microfluidic devices are therefore increasingly finding applications in medical diagnostics, forensic analysis, and biomedical research. In particular, they provide novel and powerful ways to culture cells, cell aggregates, and even functional models of entire organs. Nuclear magnetic resonance is a non-invasive, high-resolution spectroscopic technique which allows real-time process monitoring with chemical specificity. It is ideally suited for observing metabolic and other biological and chemical processes in microfluidic systems. However, its intrinsically low sensitivity has limited its application. Recent advances in nuclear hyperpolarization techniques may change this: under special circumstances, it is possible to enhance NMR signals by up to 5 orders of magnitude, which dramatically extends the utility of NMR in the context of microfluidic systems. Hyperpolarization requires complex chemical and/or physical manipulations, which in turn may benefit from microfluidic implementation. In fact, many hyperpolarization methodologies rely on processes that are more efficient at the micro-scale, such as molecular diffusion, penetration of electromagnetic radiation into a sample, or restricted molecular mobility on a surface. In this review we examine the confluence between the fields of hyperpolarization-enhanced NMR and microfluidics, and assess how these areas of research have mutually benefited one another, and will continue to do so.

Theory and Simulation Framework for the Relaxation of Nuclear Spin Order in Porous Media.

The theory of nuclear spin relaxation in a liquid permeating a solid structure of irregular geometry is examined. The effects of restricted diffusion and the demagnetizing field generated by an inhomogeneous distribution of magnetic susceptibility in the system are explored. A framework comprising Brownian Dynamics, average Hamiltonian theory, and Liouville-space spin dynamics is proposed for simulating nuclear spin relaxation in 3D models of random structures obtained from CT scans of actual samples. Simulations results are compared with experimental data. An analytical solution valid within approximation is also reported.