Morpholino oligonucleotides in responsive hydrogels for microRNA sensing

In recent years, microRNA (miRNA) has garnered a high level of interest in the field of biosensor development. MiRNA are a class of small, circulating RNA sequences that are essential for healthy control of protein expression. The variation of levels of specific miRNAs has been linked with over 150 diseases since the turn of the millennium, including cancers, cardiovascular diseases, parasitic infections and neurological disorders. The improved prognosis from early detection is stark, but established methods of miRNA detection suffer from poor sensitivity, low throughput, and require specialised laboratory equipment and trained staff to perform the time-consuming techniques. A simple, cheap and sensitive miRNA point of care sensor would be an invaluable tool in healthcare. This thesis presents the continued optimisation of a miRNA sensing hydrogel with oligonucleotide crosslinks that are selectively cleaved in the presence of the target miRNA sequence. This selective reduction in crosslink density was transduced by a change in the swelling profile of the hydrogel and intelligent crosslink design used to control the swelling response for detecting a miRNA sequence, a short RNA (sRNA) sequence, or a small molecule using an aptamer. Morpholino oligonucleotides (MOs), an uncharged DNA analogue, were functionalised with an acrylamide moiety and used as responsive crosslinks for miRNA sequence detection in a world’s first MO crosslinked hydrogel. The MO crosslinks offered significant improvements over DNA crosslinked hydrogels through improved thermal stability, no salt requirement and 1000-fold improved sensitivity, facilitating a wider range of sensing conditions. Analysis was also achieved using a mobile phone camera and laptop, demonstrating portability. Carbon nanoparticles (CNP) were suspended in the hydrogels to act as a conductive component. As the hydrogel swells the distance between the particles is increased until there is no conductive pathway, resulting in an increase in the hydrogel’s resistance. Numerous reproducibility challenges were identified with regards to gel delamination and CNP leaching partly due to inefficient UV photoinitiation of the pigment composite pre-gel solution. SEM imaging identified inconsistent composite homogeneity with areas of higher CNP and gel density in DNA crosslinked composites, while MO crosslinked composites were homogenous and less conductive. Inkjet printing of the composite material using an electrostatic dispersion as the conductive component was made possible using MO crosslinks with no salt and ammonium persulfate with TEMED in place of UV initiation. Optimised synthesis resulted in homogenous conductive composites far more robust and reproducible than the UV initiated CNP composite. However, MO solution viscosity resulted in improper aspiration and inaccurate deposition. Potential solutions and improvements are suggested, facilitated by the improvements offered by MO crosslinks.