Nuclear magnetic resonance at low field strength is an insensitive spectroscopic technique, precluding portable applications with small sample volumes, such as needed for biomarker detection in body fluids. Here we report a compact double resonant chip stack system that implements in situ dynamic nuclear polarisation of a 130 nL sample volume, achieving signal enhancements of up to − 60 w.r.t. the thermal equilibrium level at a microwave power level of 0.5 W. This work overcomes instrumental barriers to the use of NMR detection for point-of-care applications.
Computer aided design (CAD) views of the ODNP probe inside a palm-sized permanent magnet. The microfluidic chip features a sample reservoir and a MW resonator. A stacked figure-8 type RF transceive coil accepts the sample container and is sandwiched between a set of bi-planar electrical shim coils. A B0-field modulation coil is part of phase sensitive EPR detection. Figure used with permission.
We present a microfluidic platform that allows in operando nuclear magnetic resonance (NMR) observation of serial mixing experiments. Gradually adding one reagent to another is a fundamental experimental modality, widely used to quantify equilibrium constants, for titrations, and in chemical kinetics studies. NMR provides a non-invasive means to quantify concentrations and to follow structural changes at the molecular level as a function of exchanged volume. Using active pneumatic valving on the microfluidic device directly inside an NMR spectrometer equipped with a transmission-line NMR microprobe, the system allows injection of aliquots and in situ mixing in a sample volume of less than 10 μL.
It is challenging to investigate fast supramolecular processes due to the lack of appropriate characterization methods with high structural resolution. In this study, microfluidic NMR spectroscopy as employed to monitor the kinetics of threading and dethreading of a pseudorotaxane system. By employing the high-time resolution of microfluidic NMR, 1H and 2D-ROESY NMR spectra were recorded at any time point 1.5 s after the onset of the process. By employing this technique, kinetic intermediates and rate-determining steps were successfully identified, which is not possible by other spectroscopic techniques. Our study demonstrates the capability of microfluidic NMR in investigating the mechanism of complex supramolecular systems.
We present a quantitative study of the metabolic activity of a single spheroid culture of human cancer cells. NMR (nuclear magnetic resonance) spectroscopy is an ideal tool for observation of live systems due to its non-invasive nature. However, limited sensitivity has so far hindered its application in microfluidic culture systems. We have used an optimised micro-NMR platform to observe metabolic changes from a single spheroid. NMR spectra were obtained by directly inserting microfluidic devices containing spheroids ranging from 150 μm to 300 μm in diameter in 2.5 μL of culture medium into a dedicated NMR probe. Metabolite concentrations were found to change linearly with time, with rates approximately proportional to the number of cells in the spheroid. The results demonstrate that quantitative monitoring of a single spheroid with ≤ 2500 cells is possible. A change in spheroid size by 600 cells leads to a clearly detectable change in the l-Lactic acid production rate (p=3.5×10−3). The consumption of d-Glucose and production of l-Lactic acid were approximately 2.5 times slower in spheroids compared to monolayer culture of the same number of cells. Moreover, while cells in monolayer culture were found to produce l-Alanine and l-Glutamine, spheroids showed slight consumption in both cases.
Post-doc Maciej Grajewski has left the TISUMR project to join SG Papertronics B.V., a start up company founded by himself and Gert Salentijn.
Maciej has recently recieved seed investement for commercialization of sample concentration technology, that is technology allowing accumulation of all the reacted samples in a pre-selected area of a porous membrane, ensuring the completeness of the signal accumulation that in return allows for a full quantification of the tested molecules in the sample. His new company will focus on this commercialization.
We are grateful to Maciej for the work he has done on the TISuMR project and wish him all the best for the future.
Marek Plata, Manvendra Sharma, William Hale, Jörn Werner and Marcel Utz
University of Southampton
In the field of protein analytics NMR possesses nearly unparalleled capabilities. However, low sensitivity and resulting need for significant amounts of protein poses continuing challenge in its application. With introduction of miniaturised high-sensitivity detectors it has become possible to scale down sample use  to only few micrograms. Additionally, this has opened the doors for coupling of detection with microfluidic sample delivery into complex Lab-on-a-Chip systems. Here, we present a novel design for sequential volume exchange experiments, with an in operando detection by NMR, suited for qualitative and quantitative analysis of protein-ligand interactions.
KEYWORDS: Microfluidics, Micromixers, NMR, Protein
NMR is uniquely placed at providing information of protein 3D structure, dynamics and interactions with a wide range of substrates with atomic resolution. Quantification of binding constants is typically based on repeated exchange of small quantities of fluid between two samples: one at zero ligand concentration, the other containing ligand concentration sufficient to ensure protein saturation. Binding is then monitored by exchanging fractions of the two solutions and recording NMR spectra of both samples. Chemical shift, linewidth or intensity changes of the nuclei involved in the interaction provide a read-out for the fraction bound. This process can ideally be implemente on a Lab-on-a-Chip device, eliminating the need for extensive, costly, and error-prone manipulations of the samples.
All microfluidic devices are produced from PMMA layers, bonded under heat and pressure. After initial sample loading, all experimental steps are carried out in situ inside the NMR spectrometer. Sample solutions (A & B, see Fig. 1) are contained within the microfluidic chip and the connecting reservoir capillary. Overall 40 ml of sample, equally divided into two solutions, is used. At the initial stage, only solution A is present inside the active sample circuit (Vs). A stepper-motor micropump (LabSmith) controls the injection volume (Vi) of sample B into the Vs. Mixing of the two volumes is achieved using a pneumatic microvalve  system to create a peristaltic flow  inside the Vs. NMR data are obtained from a 2.5 ml volume of the detection chamber by the double resonance Transmission Line Probe (TLP) , allowing for acquisition of proton-detected heteronuclear correlation spectra of proteins.
RESULTS AND DISCUSSION
Fig. 2 demonstrates the successful volume exchange of fumaric acid and DSS solutions inside the microfluidic device, as monitored by the intensity changes of the corresponding signals (Fig. 3). The concentrations were determined by calibration against a signal from sodium acetate, present in both solutions. The probe capacity for protein NMR is exemplified by the heteronuclear 15N and 13C spectra obtained for ubiquitin and the SH3 domain of human protein FYN (fynSH3) (Fig. 4). Experiments were carried out at 14.1T on 1 mM (ubiquitin) and 2 mM (fynSH3) samples with overall acquisition time of 400 min in each spectrum.
In this work we present a novel microfluidic platform for NMR detected protein-ligand studies. This system operates inside the NMR spectrometer, and allows for detailed control over sample injection and mixing, operating with a mixing volume of approx. 10 ml, and essentially zero dead volume.
This project is jointly funded by the EU Horizon 2020 TISuMR project, and Institute for Life Sciences, University of Southampton, UK
Saraí M. Torres Delgado (35) obtained her B. Eng. degree in electronic engineering with a specialisation on power electronics from the Morelia Institute of Technology, Mexico in 2008. After working in industrial automation for three years she then moved to Germany to continue her studies, where in 2014 she received the M. Sc. degree on microsystems engineering from the University of Freiburg. She completed her doctoral studies in October 2018 under the supervision of Prof. Jan G. Korvink at the Department of Microsystems Engineering (IMTEK) at the same university. Her work focused on the development of an electronic platform for Lab on a Disc diagnostics. Sarai then joined the TisuMR project as a postdoc developing electronic solutions, NMR and lab-on-a-disc compatible. She recently rejoined industry where she works in the R&D-electronics department of an international company. Her main interests remain in the health care, sustainability, renewable energies and E-mobility sectors.
We introduce a low-complexity, low-cost, yet sufficiently accurate automatic tune and match system for NMR and MRI applications. The ArduiTaM builds upon an Arduino Uno embedded system that drives a commercial frequency synthesiser chip to perform a frequency sweep around the Larmor frequency. The generated low-power signal is fed to the NMR coil, after which the reflected waves are detected using a directional coupler and amplified. The signal shape is then extracted by means of an envelope detector and passed on to the Arduino, which performs a dip search while continuously generating actuator control patterns to adjust the tune and match capacitors. The process stops once the signal dip reaches the Larmor frequency. The ArduiTaM works readily with any spectrometer frequency in the range from 1 to 23 T. The speed of the ArduiTaM is mainly limited by the clock of the Arduino and the capacitor actuation mechanism. The Arduino can easily be replaced by a higher-speed microcontroller, and varactors can replace stepper-motor controlled variable capacitors. The ArduiTaM is made available in open source, and so is easily duplicated.
This paper presents the topology optimization of hierarchical microtextures for wetting behavior in the Cassie-Baxter mode, considering a structural unit of the hierarchical microtexture composed of base and secondary structures. The geometrical configuration of the considered structural unit can be described as a fiber bundle composed of an external surface of the base structure and the pattern of the secondary structures. Thus, two design variables are defined, one for the external surface of the base structure, and the other for the pattern of the secondary structures. The Young-Laplace equation, including a term depending on the mean curvature of the external surface, is used to describe the liquid/vapor interface imposed with a surface tension in the Cassie-Baxter mode. To overcome the difficulty of numerically computing the second-order derivative of the external surface, two partial differential equation filters are sequentially applied to the design variable of the base structure to ensure the numerical accuracy and feasibility of using an efficient linear-element-based finite element method to solve the Young-Laplace equation. To improve the performance of the hierarchical microtextures, the volume of the liquid bulges suspended at the liquid/vapor interface in the Cassie-Baxter mode, before the transition into the Wenzel mode, is minimized to optimize the match between the external surface of the base structure and the pattern of the secondary structures. In the topology optimization process, penalization of the material density of the surface tension is achieved by an artificial Marangoni phenomenon. In numerical examples, solid surfaces are tiled into textures with axial symmetry, radial symmetry, chirality, and quasiperiodicity; and structural units are derived consisting of base structures with peak shapes and dense secondary structures surrounding the crests of the peaks. The optimized performance of the derived structural units has been confirmed by comparisons.
This paper presents topology optimization on general two-dimensional
manifolds for phenomena described by second-order partial differential
equations, where the material interpolation is implemented by using the
material distribution method. When a physical field is defined on a
two-dimensional manifold, the material interpolation is implemented on a
material parameter in the partial differential equation used to
describe the distribution of the physical field. When the physical field
is defined on a three-dimensional domain with its boundary conditions
defined on a two-dimensional manifold corresponding a surface or an
interface of this three-dimensional domain, the material density is used
to formulate a mixed boundary condition of the partial differential
equation for the physical field and implement the penalization between
two different boundary types. Based on the homeomorphic property of
two-dimensional manifolds, typical two-dimensional manifolds, e.g.,
sphere, torus, Möbius strip and Klein bottle, are included in the
numerical tests, which are used to demonstrate this topology
optimization approach for the design problems of fluidic mechanics, heat
transfer and electromagnetics.