Microfabricated NMR-probes for the analysis of living cells

Microfabricated probes for Nuclear Magnetic Resonance (NMR) spectroscopy of living cells in very small quantities have been developed in this thesis. These microprobes are based on two different sample interfaces: Channel microprobes, which confine a stack of cells in a microfluidic channel, and surface microprobes, where a monolayer of cells is immobilized on the flat surface of the microprobe. Channel microprobes allow integrating advanced 3D-coil structures. Helmholtz and, for the first time, solenoidal coils have been microfabricated for the use in a NMR-probe. Systematic testing with water sample confirms that these coils demonstrate superior performance in NMR experiments compared to planar microcoils. The 1H spectra of 9 nL water taken with a Helmholtz coil of 240 μm inner diameter show a signal-to-noise ratio (SNR) of 620 and a linewidth of 5 Hz at an acquisition frequency of 300 MHz. The spin excitation uniformity, i.e. the ratio of the signal acquired at a 450° spin excitation flip angle over the signal acquired at a 90° angle, is higher than 70 % using Helmholtz or solenoidal coils, which implies a high uniformity of the RF-fields. Surface microprobes are based on planar microcoils. They have been characterized by confining sucrose solution in lipid vesicles that are immobilized on the microprobe surface. The negative effects of the inhomogeneous magnetic field produced by planar microcoils are addressed by patterning the sample on the probe surface. Experiments demonstrate that patterning the vesicles in the center of the coil by aligned microcontact printing improves the spin excitation uniformity to 90 %. However, the sensitivity of this probe type is generally inferior compared to that of channel microprobes, since flat sample layers have low filling factors. A model based on 3D finite element simulation of the coils' magnetic fields and the static magnetic field is used to compute the NMR performance of the microprobes. The model has successfully predicted the performance of the presented microprobes and allows one to derive design rules for improved sensitivity and reduced static magnetic field perturbations. In order to analyze living cells, functional structures are integrated into the microprobes for cell loading, concentration and perfusion. In particular, the microchannel probe allows integration of a wide variety of devices and structures due to the compatibility of the microfluidic channel with the concept of Micro Total Analysis Systems (μTAS). Mechanical filters, microfabricated within the channels, successfully concentrate the cell sample in the sensitive region of the coil. Using surface microprobes, the cell sample is concentrated by mere sedimentation and subsequent immobilization on the surface. By adding a macro channel, which provides an inlet and an outlet to the surface microprobe, living cells that are immobilized on the surface have successfully been perfused during NMR experiments. The 1H spectra of Chinese Hamster Ovaries have been recorded using both types of microprobes. The total detected cell volume is about 4 nL when using the channel microprobe with a Helmholtz coil of 400 μm inner diameter, and about 25 nL employing the surface probe with a planar coil of 1000 μm inner diameter. The cells have been concentrated in order to increase the SNR. The spectra show the peaks of the major compounds found within living cells. To our knowledge, this is the first time that spectra of living cells have been taken using microfabricated NMR-probes. The model results, experimental results and discussions presented in this thesis give an insight into the limits as well as the potential of microfabricated NMR probes. The obtained results and developed modeling tools allow profound evaluation of the feasibility and applicability of NMR microprobes to new applications in the field of biology and biochemistry.


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