Methods and microfabrication techniques for subnanoliter magnetic resonance spectroscopy

Nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR) and ferromagnetic resonance (FMR) spectroscopy can open up the possibility of studying many scientifically and biologically relevant samples at the µm and sub-µm scale. Examples of volume-limited systems include numerous species of microorganisms, mammalian zygotes, the majority of cells, proteins limited in growth, micro and nanostructured devices for the analysis of spin dynamics at the sub-µm scale. These volume-limited samples cannot be addressed by commercially available inductive spectrometers due to their constraint in sensitivity. It was previously proposed in our group that CMOS technology can be used to realize miniaturized high sensitivity inductive detection systems, having spin sensitivities at least two orders of magnitude greater than the commercially available spectrometers. During my PhD work, I developed methods and microfabrication techniques to perform NMR, EPR and FMR spectroscopy at the µm and sub-µm scale by using high sensitivity single chip CMOS detectors, previously proposed in our group. Microfluidic systems for the non-invasive handling of liquid samples and biological entities immersed in liquids are realized and integrated with the CMOS single chip NMR and EPR detectors. Microfluidic channels are fabricated via conventional microfabrication techniques and via two-photon polymerization, a 3D printing technique with a lateral resolution of 300 nm. The 3D printing technique is found to be an exceptional solution for NMR applications. Due to the flexibility in the design of the microfluidic systems, it is possible to reduce the magnetic field non-uniformieties with a consequent improval in spectral resolution. Spectral resolutions down to 0.007 ppm are reported for liquids having sample volumes of 100 pL. For the first time, NMR studies on intact biological entities submerged in liquid media of choice are performed, using 3D printed microfluidic systems. A spin sensitivity of 2.5·1013 spins/¿Hz is shown, sufficient to detect highly concentrated endogenous compounds in active volumes down to 100 pL with measurement times down to 3 h. EPR measurements on subnanoliter liquids and frozen solutions are reported, by the combination of commercially available capillaries and EPR single chip detectors. This is a first but important step towards the study of biological samples, whose paramagnetic ions have relaxation times too short to be measured at room temperature. Moreover, a novel method for the sensing of magnetic microbeads is presented, which is based on the detection of the change of susceptibility in FMR condition by the CMOS integrated detector. Due to the frequency and field dependence of the susceptibility, the detected variation is 20 times greater than the change in magnetization measured in static conditions by other approaches. The proposed detection scheme allows for single bead sensitivity over an active area of about 5·104 µm2. Lastly, sub-