In-Situ NMR Lab-on-a-Chip system for studying protein ligand interactions

Poster Abstract

Marek Plata, Manvendra Sharma, William Hale, Jörn Werner and Marcel Utz

University of Southampton

MicroTAS 2020

ABSTRACT

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 [1] 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

INTRODUCTION

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.

EXPERIMENTAL

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 [2] system to create a peristaltic flow [3] 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) [4], 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.

CONCLUSION

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.

ACKNOWLEDGMENTS

This project is jointly funded by the EU Horizon 2020 TISuMR project, and Institute for Life Sciences, University of Southampton, UK

CONTACT
Corresponding author: marcel.utz@gux.net

In-Situ NMR lab-on-a-chip system for studying protein-ligand interactions

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