Category: News

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

James Eills, William Hale, Manvendra Sharma, Matheus Rossetto, Malcolm H. Levitt and Marcel Utz

Journal of the American Chemical Society, 2019

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 1 μM s , 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.

Pharmacy Day, University of Groningen

Mouse precision-cut liver slices as a disease model to predict drug-induced cholestasis

R.E.H. Karsten, N.J.V.W. Krijnen, M. Grajewski, E. Verpoorte, P. Olinga

University of Groningen, Groningen, The Netherlands

Drug-induced cholestasis is a problematic adverse drug reaction, with no adequate model for early detection of cholestatic drugs mainly because the disease mechanism is complex and variable. The toxicity seen in cholestasis is most likely caused by accumulation of substances in the liver, bile ducts, or blood, which are normally excreted into the bile (e.g. bile salts, cholesterol, bilirubin, drug metabolites). This intrahepatic accumulation is thought to cause hepatocellular apoptosis and necrosis. When chronic, this can lead to progressive organ failure.

Aims

The aim of this project is to maintain metabolism and liver function in mouse precision-cut liver slices incubated in a microfluidic device, to ultimately study the onset of drug-induced cholestasis in mouse liver in real time.

Methods

We are developing an ex vivo, organ-on-a-chip model based on precision-cut liver slices (PCLS)1 to study drug-induced cholestasis in mouse liver. We incubate PCLS (5 mm diameter and 250-300 µm thickness) for 48 h in medium (Williams E Medium, with added glucose and gentamicin) in a 12-well plate. To the medium we also add non-, low- or medium-toxic concentrations of one of three different cholestatic drugs (chlorpromazine, glibenclamide, and cyclosporin A) and a humanized bile-acid mix with relevant mouse in vivo concentrations. Viability studies were performed by measuring intracellular ATP content. Moreover, gene expression was measured by real-time PCR. Gene expression was measured for the bile-uptake transporter, sodium-taurocholate co-transporting polypeptide (NTCP); the canalicular bile-export transporters, multidrug resistance-associated protein 2 (MRP2) and bile salt-export pump (BSEP); and basolateral bile-export transporters, MRP3 and MRP4. Furthermore, the gene expression of the nuclear receptor, Farnesoid X Receptor (FXR), which regulates bile homeostasis, was measured. 

Results / Conclusions

Combined administration of cholestatic drug and mixture of bile acids led to changes in the gene expression of bile-export transporters. This was true for both basolateral and canalicular bile-export transporters. This study is the first that relates gene-expression data to early drug-induced cholestasis development in mPCLS. Once optimized, PCLS will be incubated in a microfluidic device to monitor the onset of drug-induced cholestasis in real time. We will use this model to better elucidate disease mechanisms and perform drug-toxicity screening.

References

  1. de Graaf IAM, Olinga P, de Jager MH, Merema MT, de Kanter R, van de Kerkhof EG, et al. Preparation and incubation of precision-cut liver and intestinal slices for application in drug metabolism and toxicity studies. Nat Protoc. 2010; 5(9):1540–51.

Precision-cut tissue slices as a route to reduced animal use in toxicology studies

The usage of animals for laboratory testing annually costs millions of rodents’ lives worldwide. In our project, we significantly reduce the number of animals used by implementation of the precision-cut tissue slice model (PCTS). This model represents an exciting opportunity for the future of in vitro and toxicology research for a number of reasons.

One advantage of using the PCTS model is that dozens of tissue slices can be produced from even the smallest organ, allowing vast numbers of experimental conditions to be tested for each animal. When compared to in vivo studies, where only one experimental condition can be tested, we are drastically reducing the total number of animals needed.

Moreover, every one of the many slices produced using our method maintains the complex internal structure of each organ. PCTS are therefore also responsible for the generation of robust and highly translatable data, allowing for strong prediction of clinical toxicity and development of effective disease biomarkers.

The complete cessation of animal tissue use is unlikely to occur soon. Looking forward, however, the use of the PCTS model in combination with body-on-a-chip systems may someday remove the need for whole animal studies altogether. The development and characterization of the PCTS model is therefore most certainly a step in the right direction to reduce the ecological footprint of toxicology research both now and in the future.

Nikolaas Krijnen

Micro-textures inversely designed with overlayed-lithography manufacturability for wetting behavior in Cassie–Baxter status

Yongbo Deng, Zhenyu Liu, Yasi Wang, Huigao Duan, Jan G. Korvink

Aplied Mathematical Modelling, 2019

Robust Cassie–Baxter wettability of a rough solid surface with micro-textures is a key factor for stable hydrophobicity. Overlayed micro-textures are potentially more effective in ensuring the robustness of the surface properties, because of the layer-by-layer increase of the duty ratio and their effective approximation of the full hierarchy. However, a design methodology that includes considering manufacturability is lacking. In this article, we address this deficiency and present a monolithic inverse design approach, composed of a series of topology optimizations, to derive micro-textures with hierarchy approximated by overlayed geometries. The optimization are implemented in a dimensionless manner using a periodic regular-polygon tiling of the plane, in which the corresponding dimensionless Young-Laplace equation is used to describe the physics at the liquid/vapor interface. Two sequential and neighboring optimization tasks are linked through the design domain of the downward layer, determined by a conformal extension of the physical density representing the pattern of the upward layer. This ensures the manufacturability e.g. for an overlayed lithography process. Layer-by-layer robustness enhancement is thereby achieved, and the capability to anchor the three-phase contact line after the collapse of the liquid/vapor interface supported by the upward layer. In generating the overlayed micro-textures, a rigorous scaling factor for the patterns was determined, leading to a recursion inequality based on the depth of the liquid/vapor interfaces at the critical static pressures that determines the extrusion distance of the patterns. The trace height and minimal aspect ratio of the micro-textures are specified by the scaling factor and extrusion distance for a layer. This allows a compromise between performance and manufacturability, and thereby avoid instabilities caused by elasto-capillary collapse of the micro-/nano-structures. We computationally confirm the optimality by comparing the derived micro-textures with previously reported designs.

Broadband and multi-resonant sensors for NMR

Hossein Davoodi, Mazin Jouda, Jan G. Korvink, Neil MacKinnon, Vlad Badilita

Progress in Nuclear Magnetic Resonance Spectroscopy, 2019

It has always been of considerable interest to study the nuclear magnetic resonance response of multiple nuclei simultaneously, whether these signals arise from internuclear couplings within the same molecule, or from uncoupled nuclei within sample mixtures. The literature contains numerous uncorrelated reports on techniques employed to achieve multi-nuclear NMR detection. This paper consolidates the subset of techniques in which single coil detectors are utilized, and highlights the strengths and weaknesses of each approach, at the same time pointing the way towards future developments in the field of multi-nuclear NMR. We compare the different multi-nuclear NMR techniques in terms of performance, and present a guide to NMR probe designers towards application-based optimum design. We also review the applicability of micro-coils in the context of multi-nuclear methods. Micro-coils benefit from compact geometries and exhibit lower impedance, which provide new opportunities and challenges for the NMR probe designer.

Modular transmission line probes for microfluidic nuclear magnetic resonance spectroscopy and imaging

Manvendra Sharma, Marcel Utz

Journal of Magnetic Resonance, 2019

Microfluidic NMR spectroscopy can probe chemical and bio-chemical processes non-invasively in a tightly controlled environment. We present a dual-channel modular probe assembly for high efficiency microfluidic NMR spectroscopy and imaging. It is compatible with a wide range of microfluidic devices, without constraining the fluidic design. It collects NMR signals from a designated sample volume on the device with high sensitivity and resolution. Modular design allows adapting the detector geometry to different experimental conditions with minimal cost, by using the same probe base. The complete probe can be built from easily available parts. The probe body mainly consists of prefabricated aluminium profiles, while the probe circuit and detector are made from printed circuit boards. We demonstrate a double resonance HX probe with a limit of detection of 1.4 nmol s−1/2 for protons at 600 MHz, resolution of 3.35 Hz, and excellent B1homogeneity. We have successfully acquired 1H-13C and 1H-15N heteronuclear correlation spectra (HSQC), including a 1H-15N HSQC spectrum of 1 mM 15N labeled ubiquitin in 2.5 μl of sample volume.

Experimental Nuclear Magnetic Resonance Conference, Pacific Grove, California

Integrated Impedance Spectroscopy for Automated High Throughput MR Measurements on Fluidic Plugs

Seminar Abstract

Omar Nassar, Mazin Jouda, Nan Wang, Michael Rapp, Jan Korvink, Dario Mager, and Neil MacKinnon

Institute of Microstructure Technology, Karlsruhe Institute of Technology (KIT), Germany

Microfluidic technologies combined with micro-NMR have become increasingly popular given their promise of increased sample throughput and mass-sensitivity. A common challenge is the relatively large dead volume in the tubing feeding the micro-NMR detector. To circumvent this issue, concentrated sample plugs may be dispersed within an immiscible fluid. This two-phase flow eliminates the dead volume, but requires continuous online measurement to track the plug positions and ensure that the desired sample completely fills the NMR detection volume. In this contribution, a flow system featuring i) integrated impedance spectroscopy sensors for sample position and flow rate measurement, and ii) a micro saddle coil for NMR spectroscopy is demonstrated. By loading a tube with several samples separated by an immiscible fluid, this flow system enables fully automated NMR spectroscopy while simultaneously taking advantage of the improved experimental time per sample due to continuous sample flow.

Interdigitated electrode (IDE) sensors and a micro saddle coil were fabricated on a Kapton foil using an established process (Wang, N., et al., J Micromech Microeng 28 (2018) 025003). A 25 μm Kapton foil laminated on one side with a 9 μm copper layer was used, which was structured using photolithography. An insert was built (Fig. 1) by rolling the structured Kapton foil around a 2 mm glass tube with a 1.6 inner channel. The two IDE sensors were positioned 1 cm before and after the saddle coil to enable flow sensing in both directions and flow rate measurement, permitting real-time calculation of the appropriate delay before triggering NMR acquisition. Each IDE sensor covered a length of 5 mm and the saddle coil had a detection region of 5 mm. A Teflon tube carried the samples and fit inside the glass capillary to avoid fluidic junctions and ensure smooth sample flow. The inlet and outlet of the tubing were connected to a syringe pump and flow was controlled from outside of the magnet. A Zurich Instruments UHFLI amplifier was used to drive the IDE sensors (frequency = 540 MHz) and measure the impedance to sense the position and the speed of the water plug and trigger NMR acquisition. NMR spectra were acquired at a 1H Larmor frequency of 500 MHz using a Bruker AVANCE III spectrometer.

Two experiments were performed. In the first experiment , the tube was filled with a single aqueous plug (20 cm long) bordered by FC-43, and the flow was controlled by the syringe pump at a flow rate of 1.0 cm / s running continuously until the aqueous plug left the NMR probe. NMR acquisition was done with and without the triggering system. In the second experiment, the tube was filled with nine different 5 cm aqueous plugs each separated by 10 cm of FC-43. The first plug was used as a dummy sample to calculate the trigger delay and the flow was running continuously until all the sample plugs left the NMR probe.

Impedance measurements could robustly distinguish the water and oil phases. Importantly, driving the electrodes had no visible deleterious effect on the 1 H NMR spectrum. It was observed that the SNR was improved by 75% when implementing the automated triggered data acquisition (Fig. 2), even with a relatively long sample plug. Micro-coil integration with impedance-based sensing of flow has been demonstrated. With this technology we believe high-throughput, automated sample measurement (Fig. 3) will be possible in a dual-phase, droplet-based approach where each droplet could be a different sample, thereby reducing the required sample volume significantly.

A Microfluidic-NMR platform to culture and study live systems

Manvendra Sharma, Bishnubrata Patra, William Hale and Marcel Utz

University of Southampton

ENC 2019

We present a novel microfluidic-NMR platform to combine high sensitivity NMR with microfluidics to perform generic NMR experiments on live systems under tightly controlled environment through microfluidics.
The current setup has already been used for a number of applications including microimaging of mouse liver tissue slices, heteronuclear 2D-NMR correlation experiments on proteins, parahydrogen induced hyperpolarisation, droplet NMR, reaction monitoring, and metabolic studies of mammalian cells.

A Microfluidic-NMR platform to culture and study live systems

Mehrdad Alinaghian

Mehrdad Alinaghian received his Bachelor and Master degrees in Mechanical Engineering from IA University, Iran. His master thesis was on numerical buckling analysis of functionally graded sectorial disks using a generalized differential quadrature (GDQ) method.

Mehrdad has started his PhD in Prof. Jan. Korvink’s group at the Karlsruhe Institute of Technology (KIT). He is working on pulse program customization of magnetic resonance velocimetry (so-called flow MRI) in order to evaluate flow behavior in microfluidic networks. His research interests are Phase Contrast MRI (PC-MRI), Contrast Enhancement MRA (CEMRA), Time-Of-Flight (TOF) Angiography and pulse programming.

Using his expertise in flow MRI, Mehrdad will be supporting the TISuMR project by observing the liquid processes in and around the perfusion chamber containing the liver.

4th Munich Point-of-Care Testing Symposium, Munich, Germany

eLOAD -centrifugal microfluidics with modular embedded systems for easy operation and maintainability at the POC

Poster Abstract

Dario Mager

In resource-limited settings, for example in parts of India and Africa, access to everyday commodities, such as clean water and electricity, is restricted. This makes healthcare in the developing world vastly different from the experiences of high-income countries. In these low-infrastructure settings, exposure to difficult environmental conditions is commonplace, including high levels of humidity, heat and dust. Electricity is often intermittent or non-existent, compounding the harshness of the environment and posing significant challenges for equipment and data connectivity. In addition, lack of trained staff makes it hard to provide a high standard of diagnostic testing and throughput of patients.

The need for medical solutions at the Point-of-Care is an obvious challenge without question and many approaches are made to help solving these issues. Nevertheles many of the proposed solutions orientate themselves at the conditions in rich countries (both with respect to the lab conditions as well as the availability of trained personal). The reality at the PoC is that the permanently available personnel is often purely trained (most others leave to a better job) and service technicians need to fly in from another country. Also the operation of a device which’s cost equivalents the years income of half the village were it is used is morally questionable.

We present an approach – the eLoaD – that is based on the wide available Arduino microcontroller, that allows to automate centrifugal microfluidics (or Lab-on-a-Disc) in many important aspects. It allows the operation of these tests using common and hence cheap microelectronic sensors. The integration of embedded systems allows automation both in diagnostic operations as well as in maintenance, reducing the need for highly trained personnel. The device consists of a generic part containing the energy handling, the Arduino, the SD-card, and the Bluetooth communication. On the other side it has an ApplicationDisc that caries the sensors and actuators [1] and is design for a specific application. Following this approach feedback controlled arbitrary switching of fluids on the spinning disc [2] as well as a chemiluminescence based ELISA test could be performed [3]. The presented system is cheap and highly flexible, yet it is highly sensitive.

  1. Torres et al., Biosens Bioelectron. 2018;117:464–73.
  2. Torres et al. Biosens Bioelectron. 2018;109:214–23
  3. Torres et al., Lab Chip. 2016;16(20):4002–11.