We show that high-resolution NMR can reach picomole sensitivity for micromolar concentrations of analyte by combining parahydrogen induced hyperpolarisation (PHIP) with a high-sensitivity transmission line micro-detector. 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 favourable 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 parahydrogen-induced polarisation (PHIP) on a microfluidic device with 2.5 µL detection volume. Integrating the hydrogenation reaction into the chip minimises polarisation 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 allows quantitative characterisation of the signal dependence on flow rates and other reaction parameters and permits homo-( 1 H-1 H) and heteronuclear( 1 H-13 C) 2D NMR experiments at natural 13 C abundance.
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 B 1 homogeneity. We have successfully acquired 1 H-13 C and 1 H-15 N heteronuclear correlation spectra (HSQC), including a 1 H-15 N HSQC spectrum of 1 mM 15 N labeled ubiquitin in 2.5 µl of sample volume.
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 $$\upmu$$
μ
m to 300 $$\upmu$$
μ
m in diameter in 2.5 $$\upmu$$
μ
L 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 $$\le$$
≤
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 ($$p=3.5\times 10^{-3}$$
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.
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