The main objective of the project is to fabricate different types of magnetic nanostructures (MNS) with novel spin configurations and demonstrate their potential in medical diagnostics and cancer therapy. Thus, the project has accomplished significant progress in these three pillar: fabrication, diagnosis and cell therapy, with the following results.
Fabrication
Several lithography techniques were used for production of MNS with feature sizes from several microns down to sub-100 nm. Direct laser writing lithography for feature sizes from 2 – 4 microns; laser interference lithography for elements from 250 – 900 nm, and porous alumina anodization for smaller nanostructures from 40 – 70 nm. The lithography process was optimized for each technique; fitting dose, exposition times, developing protocols, etc. Both positive and negative tone resist were tested in laser lithography. In case of porous alumina, two different approaches were developed: in-situ anodization of thin aluminum films and anodization of aluminum foils for a further transfer of alumina membranes. As result, two spin configurations were produced for these geometries: vortex state and synthetic antiferromagnets with in-plane anisotropy.
Diagnosis
The detection technique is based on the magneto-electrical signal of giant magneto-resistance (GMR) sensors, which are integrated in cartridges allocating biosensors and microfluidic channels to direct the analyte on the sensor surface. The sensor was microfabricated on a 150 mm Si/SiO2 100 nm wafer, and the spinvalve materials were deposited by ion beam. The sensors were patterned using laser lithography and ion beam milling, and protected with Si3N4 passivation layer, where the gold pads where then defined over the sensors. The chip layout used has 30 U-shaped spin valve sensors with dimensions 46.6 x 2.6 μm2 arranged in series of two sensors, displayed in 6 distinct sensing regions, with each region compromising 5 biological active sensors coated with a gold film. The sensor surface was functionalized to detect cancer biomarker proteins (Carcinoembryonic Antigen – CEA). The results demonstrated successful implementation of the surface chemistry and molecular recognition strategy on GMR sensor, enabling the detection of CEA protein in buffer at concentrations as low as 100 ng/mL
Therapy
To determine the efficiency of the magneto-mechanical action as cell therapy, the MNS behaviour inside cells under low-frequency magnetic field exposure was investigated. Different incubation times, frequencies and magnetic field doses were tested. Cancer cells were seeded in plates at a density of 7.000 cells. Cells were cultured containing different concentrations of MNS. A magnetic field of 100 Oe was applied in all assays, the cell-well without magnetic disks was used as the control group. PrestoBlue proliferation assay was carried out to assess the cell viability induced by magneto-mechanical configuration. Compared the cells under magnetic field with the control group, it was found that around 30% of cells were entering apoptosis. In addition, most of the cells had a disk on the membrane or internalized, suggesting that after the application of the magnetic field, the cell began an apoptosis process due to magneto-mechanical damage. It is worth noting the absence of toxicity if no magnetic fields are applied. Cellular viability assays with MNS were performed using a human monocytic leukaemia cell line, two human metastatic melanoma cell lines, one of mice macrophages and one of human epidermal melanocytes. No cytotoxic effects were observed for periods up to 48 h, which demonstrate the biocompatibility of the fabricated MNS.
