The project commenced by investigating the various techniques for fabricating nano-patterned substrates. Here, three fabrication techniques were developed, nano-sphere self-assembly, in-cavity hierarchical nano-patterning, and Au over 3D-nanoprinted nano-patterns. The former approach is a multi-stage technique in which Au cavity array was formed by depositing Au electrochemically through a nano-sphere template. Large uniform array (up to 6 × 6 mm2) can be successfully prepared. The second approach aim to produce sub-micron structures within the cavities, which lead to 3 times improvement in detection sensitivity compared to Au cavities without in-cavity sub-structures. The third approach involves duplication of nano-patterns from a 3D-nanoprinted mould. Au was then sputter-coated over the replicated pattern. To this end, FE-SEM was then used to verify the nano-gemeotries of these substrates. All arrays are generally uniform over large area, with less than 15% inter- as well as intra-variations in cavity diameters. FDTD simulations were carried out using the dimensional information obtained via FE-SEM. Simulated electromagnetic fields verified the plasmonic hot-spots at the bottom of the cavities as intended. Experimentally, substrates with in-cavity substructures were shown to exhibit the largest SERS enhancement factors in consistent with simulation prediction.
For the current proof-of-principle study, Au cavities without in-cavity sub-structures were used for drug-membrane interaction studies. Prior to membrane-deposition, the substrate was rendered hydrophilic via surface functionalization with 6-mercapto-1-hexanol. Lipid bilayers were then deposited using a Langmuir Blodgett/vesicle fusion method developed in the Keyes group. Both the EIS and SERS techniques were used to assess the integrity of the suspended membranes, which was shown to last up to 21hrs. Such an excellent stability was attributed to the ‘cushioning effect’ brought about by the rounded-edge around the mouth of the cavity supports. Permeability test using SERS with a membrane-impermeable probe confirmed proper sealing of the cavities, suggesting the absence of any leaky defects in the membrane.
Three analytical approaches (Electrochemical impedance spectroscopy, Surface Enhanced Raman Spectroscopy, and Metal-enhanced Fluorescence measurements) were used to study drug transport across the membrane. Various drugs were investigated in the studies, including Ibuprofen, Diclofenac, Doxorubicin, Daunorubicin, and cell-penetrating peptides. Through the combined use of the spectroscopic techniques mentioned above, insights with regard to the transport mechanism was obtained. This includes the effect of membrane charges, drug-induced fluidity changes in the membrane and pore-formation. Lastly, the release mechanism of large molecules, such as oligonucleotides, from their delivery vector (Lipoplex in the current case) was examined in the context of gene-therapy. Various crucial intermediate stages in the release process were observed, suggesting the potential of the current technique in the rational design of gene-delivery vector.
An IDF is currently being filed for CLIMB. All experimental data will be published soon in scientific journals. Several funding programmes, such as the Wellcome career-development fellowship, are currently being pursued as a follow-on with the backing of the experimental outcomes, especially in the area of drug-delivery across blood-brain-barrier and gene-delivery. A business model has also been devised, and both Keyes and Dr. Kho are keen to seek early-stage investment.
