NMR on small cell ensembles
Investigating the metabolism of small cell ensembles via micro-NMR
Nuclear magnetic resonance (NMR) spectroscopy is a very information rich analytical technique. The inherently low sensitivity of NMR impedes, however, many interesting scientific investigations. This holds in particular, when the mass and the volume of a sample is limited. One approach to overcome such limitations is the development of µ-scale, highly sensitive waveguide RF detectors. The helical coil geometry commonly used for this purpose imposes restrictions on the sample shape and impedes measurements in microfluidic chips. The planar design of the ISAS microslot waveguide NMR detector, however, suits to any sample geometry and especially microfluidic chips. With this design, microfluidic chips can be used as samples without implementing a RF coil or detector in the chip. This feature helps to reduce the technical effort and the costs of manufacturing the chip. Biochemical networks show high variability due to stochastic fluctuations and heterogeneity of the cell ensemble. Therefore, studies of the information flow in biological systems benefit from metabolic studies on synchronized cell ensembles rather than on cellular population averages, as genetically identical populations may well belong to different phenotypes. Aim of the project is to keep an ensemble of cells within a microfluidic cage for approximately five hours and to study the metabolism using microslot NMR. For the investigations, the colon adenocarcinoma cell line HT-29 was chosen. A microfluidic chip, which serves both as a cage for the cells to be investigated as well as a reservoir for metabolite monitoring, was manufactured (Figure 1) and is currently tested at the new high field NMRspectrometer. The chip is employed with an inlet channel of 0.5 mm width and an outlet channel of 0.2 mm width. The reservoir for the cells has a volume of 0.5 µL and is to be positioned onto the sensitive area of the NMR probe. An anti-Helmholtz gradient coil for the generation of pulsed field gradients for the suppression of the water signal in the 1H NMR spectra is currently designed and manufactured. In order to exploit the high sensitivity of the new NMR cryoprobe, a second microfluidic chip is developed that fits in a standard 5mm NMR tube. The above mentioned cryoprobe allows also for 31P NMR measurements and hence for investigations of the energy metabolism of the HT-29 cells.