RESUMEN
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.
Asunto(s)
Microfluídica , Proteínas , Ligandos , Proteínas/química , Espectroscopía de Resonancia Magnética/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , PéptidosRESUMEN
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.