Real-Time NMR Monitoring of Spatially Segregated Enzymatic Reactions in Multilayered Hydrogel Assemblies*.

Compartmentalized chemical reactions at the microscale are important in biotechnology, yet monitoring the molecular content at these small scales is challenging. To address this challenge, we integrate a compact, reconfigurable reaction cell featuring electrochemical functionality with high-resolution NMR spectroscopy. We demonstrate the operation of this system by monitoring the activity of enzymes immobilized in chemically distinct layers within a multi-layered chitosan hydrogel assembly. As a benchmark, we observed the parallel activities of urease (Urs), catalase (Cat), and glucose oxidase (GOx) by monitoring reagent and product concentrations in real-time. Simultaneous monitoring of an independent enzymatic process (Urs) together with a cooperative process (GOx + Cat) was achieved, with chemical conversion modulation of the GOx + Cat process demonstrated by varying the order in which the hydrogel was assembled.

An NMR-compatible microfluidic platform enabling in situ electrochemistry.

Combining microfluidic devices with nuclear magnetic resonance (NMR) has the potential of unlocking their vast sample handling and processing operation space for use with the powerful analytics provided by NMR. One particularly challenging class of integrated functional elements from the perspective of NMR are conductive structures. Metallic electrodes could be used for electrochemical sample interaction for example, yet they can cause severe NMR spectral and SNR degradation. These issues are more entangled at the micro-scale since the distorted volume occupies a higher ratio of the sample volume. In this study, a combination of simulation and experimental validation was used to identify an electrode geometry that, in terms of NMR spectral parameters, performs as well as for the case when no electrodes are present. By placing the metal tracks in the side-walls of a microfluidic channel, we found that NMR RF excitation performance was actually enhanced, without compromising B0 homogeneity. Monitoring in situ deposition of chitosan in the microfluidic platform is presented as a proof-of-concept demonstration of NMR characterisation of an electrochemical process.

Spatial and Temporal Control Over Multilayer Bio-Polymer Film Assembly and Composition.

Biosensing applications have taken advantage of lab-on-a-chip technologies for sample handling and sensors integration for highly sensitive, specific detection with high throughput. These systems are based on 2.5D fabrication principles with sensing elements restricted to an array format in two dimensions. In this report, a sensing platform that recovers biosensing capabilites in three spatial dimensions is presented. This is achieved by leveraging chitosan, a stimulus responsive polyaminosaccharide that undergoes a sol-gel transition driven by a change of pH. This process can be repeated, resulting in a multilayered hydrogel stack where each layer carries a unique chemical identity. In addition, the functionality of chitosan can be modified prior to or during the assembly process. This is demonstrated by introducing both a carboxylic acid functionality and additional primary amines to the base chitosan polymer. The assembly process is shown to be compatible with microfluidic dimensions.

Parahydrogen based NMR hyperpolarisation goes micro: an alveolus for small molecule chemosensing.

Complex mixtures, commonly encountered in metabolomics and food analytics, are now routinely measured by nuclear magnetic resonance (NMR) spectroscopy. Since many samples must be measured, one-dimensional proton (1D 1H) spectroscopy is the experiment of choice. A common challenge in complex mixture 1H NMR spectroscopy is spectral crowding, which limits the assignment of molecular components to those molecules in relatively high abundance. This limitation is exacerbated when the sample quantity itself is limited and concentrations are reduced even further during sample preparation for routine measurement. To address these challenges, we report a novel microfluidic NMR platform integrating signal enhancement via parahydrogen induced hyperpolarisation. The platform simultaneously addresses the challenges of handling small sample quantities through microfluidics, the associated decrease in signal given the reduced sample quantity by Signal Amplification by Reversible Exchange (SABRE), and overcoming spectral crowding by taking advantage of the chemosensing aspect of the SABRE effect. SABRE at the microscale is enabled by an integrated PDMS membrane alveolus, which provides bubble-free hydrogen gas contact with the sample solution. With this platform, we demonstrate high field NMR chemosensing of microliter sample volumes, nanoliter detection volumes, and micromolar concentrations corresponding to picomole molecular sensitivity.

Comparison of spin dynamics and magnetic properties in antiferromagnetic closed and open molecular Cr-based rings.

We present magnetization and (1)H nuclear magnetic resonance (NMR) measurements performed in both closed Cr8 and open Cr8Zn antiferromagnetic molecular rings in the temperature range 1.65 < T < 300 K at different external magnetic fields. The magnetization measurements on Cr8Zn are consistent with a small decrease of the exchange constant J(Cr-Cr) and a much smaller gap between the singlet ground state and the first magnetic excited state when compared with the same properties of the closed ring Cr8, in agreement with previous inelastic neutron scattering results. The temperature dependence of the (1)H NMR nuclear spin lattice relaxation rate (NSLR), 1/T1(T), was found to be similar in both open and closed rings with a magnetic field dependent peak centered at a temperature of the order of the corresponding exchange constant J(Cr-Cr). Such main peak in the NSLR could be fitted with a single correlation frequency ω(c1) as in most molecular magnets. At low temperature T < 4 K, a new feature not observed in previous NMR measurements on antiferromagnetic rings and consisting in a smaller peak of 1/T1(T) which is well resolved only in Cr8Zn, was singled out. This low-T peak indicates the presence of a second correlation frequency ω(c2) of the magnetization, found to be quite different between the two rings and thus possibly reflecting the different low temperature level structure associated with the different spin topology. The presence of ω(c2) is confirmed by the NMR spin-spin relaxation rate enhancement, which generates a two-steps wipe-out effect of the NMR signal intensity.

Colloidal assemblies of oriented maghemite nanocrystals and their NMR relaxometric properties.

An elevated-temperature polyol-based colloidal-chemistry approach allows for the development of size-tunable (50 and 86 nm) assemblies of maghemite iso-oriented nanocrystals, with enhanced magnetization. (1)H-nuclear magnetic resonance (NMR) relaxometric experiments show that the ferrimagnetic cluster-like colloidal entities exhibit a remarkable enhancement (4-5 times) in transverse relaxivity when compared to that of the superparamagnetic contrast agent Endorem®, over an extended frequency range (1-60 MHz). The marked increase in the transverse relaxivity r2 at a clinical magnetic field strength (∼1.41 T), which is 405.1 and 508.3 mM(-1) s(-1) for small and large assemblies, respectively, makes it possible to relate the observed response to the raised intra-aggregate magnetic material volume fraction. Furthermore, cell tests with a murine fibroblast culture medium confirmed cell viability in the presence of the clusters. We discuss the NMR dispersion profiles on the basis of relaxivity models to highlight the magneto-structural characteristics of the materials for improved T2-weighted magnetic resonance images.

Protein corona affects the relaxivity and MRI contrast efficiency of magnetic nanoparticles.

Magnetic nanoparticles (NPs) are increasingly being considered for use in biomedical applications such as biosensors, imaging contrast agents and drug delivery vehicles. In a biological fluid, proteins associate in a preferential manner with NPs. The small sizes and high curvature angles of NPs influence the types and amounts of proteins present on their surfaces. This differential display of proteins bound to the surface of NPs can influence the tissue distribution, cellular uptake and biological effects of NPs. To date, the effects of adsorption of a protein corona (PC) on the magnetic properties of NPs have not been considered, despite the fact that some of their potential applications require their use in human blood. Here, to investigate the effects of a PC (using fetal bovine serum) on the MRI contrast efficiency of superparamagnetic iron oxide NPs (SPIONs), we have synthesized two series of SPIONs with variation in the thickness and functional groups (i.e. surface charges) of the dextran surface coating. We have observed that different physico-chemical characteristics of the dextran coatings on the SPIONs lead to the formation of PCs of different compositions. (1)H relaxometry was used to obtain the longitudinal, r1, and transverse, r2, relaxivities of the SPIONs without and with a PC, as a function of the Larmor frequency. The transverse relaxivity, which determines the efficiency of negative contrast agents (CAs), is very much dependent on the functional group and the surface charge of the SPIONs' coating. The presence of the PC did not alter the relaxivity of plain SPIONs, while it slightly increased the relaxivity of the negatively charged SPIONs and dramatically decreased the relaxivity of the positively charged ones, which was coupled with particle agglomeration in the presence of the proteins. To confirm the effect of the PC on the MRI contrast efficiency, in vitro MRI experiments at ν = 8.5 MHz were performed using a low-field MRI scanner. The MRI contrasts, produced by different samples, were fully in agreement with the relaxometry findings.

Hybrid iron oxide-copolymer micelles and vesicles as contrast agents for MRI: impact of the nanostructure on the relaxometric properties.

Magnetic resonance imaging (MRI) is at the forefront of non-invasive medical imaging techniques. It provides good spatial and temporal resolution that can be further improved by the use of contrast agents (CAs), providing a valuable tool for diagnostic purposes. Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are attractive MRI contrast agents due to their negative (T2) contrast enhancement capability and biocompatibility. Clusters of USPIOs with polymer material are of particular interest since they can sustain additional functionalities like drug delivery and targeting. Aiming to establish a relationship between the morphology of the clusters and their efficacy as MRI contrast agents (relaxometric properties), we prepared - using three different maghemite (γ-Fe2O3) USPIO diameters - a series of hybrid copolymer/iron oxide CAs presenting two different geometries (micellar or vesicular). The NMR relaxometry profiles confirmed the nature of the physical mechanisms inducing the increase of nuclear relaxation rates at low (magnetic anisotropy) and high (Curie relaxation) magnetic fields. A heuristic model, first proposed by Roch, Muller, Gillis, and Brooks, allowed the fitting of the whole longitudinal relaxivity r1(ν) profile, for samples with different magnetic core sizes. We show that both types of clusters exhibit transverse relaxivity (r2) values comparable to or higher than those of common contrast agents, over the whole tested frequency range. Moreover, in-depth analysis revealed substantially a linear relationship between r2 and the number of encapsulated USPIOs divided by the diameter of the clusters (NUSPIO/DH), for each USPIO size. The cluster structure (i.e. micelle or vesicle) appeared to have a mild influence on the transverse relaxivity value. Indeed, the r2 value was mainly governed by the individual size of the USPIOs, correlated with both the cluster external diameter and the magnetic material volume fraction.