This thesis describes a new approach to biomolecular analysis, called the volume-amplified magnetic nanobead detection assay (VAM-DNA). It is a sensitive, specific magnetic bioassay that offers a potential platform for the development of low-cost, easy-to-use diagnostic devices. The VAM-NDA consists of three basic steps: biomolecular target recognition, enzymatic amplification of the probe-target complex using the rolling circle amplification (RCA) technique, and addition of target complementary probe-tagged magnetic nanobeads which exhibit Brownian relaxation behavior. Target detection is demonstrated by measuring the frequency-dependent complex magnetization of the magnetic beads. The binding of the RCA products (target DNA-sequence coils) to the bead surface causes a dramatic increase in the bead size, corresponding essentially to the size of the DNA coil (typically around one micrometer). This causes a decrease in the Brownian relaxation frequency, since it is inversely proportional to the hydrodynamic size of the beads. The concentration of the DNA coils is monitored by measuring the decrease in amplitude of the Brownian relaxation peaks of free beads. The parameters oligonucleotide surface coverage, bead concentration, bead size and RCA times were investigated in this thesis to characterize features of the assay. It was found that all of these parameters affect the outcome and efficiency of the assay. The possibility of implementing the assay on a portable, highly sensitive AC susceptometer platform was also investigated. The performance of the assay under these circumstances was compared with that using a superconducting quantum interference device (SQUID); the sensitivity of the assay was similar for both platforms. It is concluded that, the VAM-NDA opens up the possibility to perform biomolecular detection in point-of-care and outpatient settings on portable platforms similar to the one tested in this thesis. Finally, the VAM-NDA was used to detect Escherichia coli bacteria and the spores of Bacillus globigii, the non-pathogenic simulant of Bacillus anthracis. A limit of detection of at least 50 bacteria or spores was achieved. This shows that the assay has great potential for sensitive detection of biomolecules in both environmental and biomedical applications.

In this study, the magnetically-induced vibrations of functionalized magnetic particle suspensions were probed for the development of a novel optical spectroscopy technique. Through this work (1) the frequency-dependence of the faraday rotation in ferrofluids and (2) the extension of this system to elucidating particle size and conformation as an alternative immunossay to costly and labor/time intensive Western Blotting and ELISA has been shown. With its sensitivity and specificity, this method has proven to be a promising multi-functional tool in biosensing, diagnostic, and therapeutic nanomedicine efforts. Due to its ubiquitous nature in all optically-transparent materials, the farady rotation, or circular birefringence, was developed as a robust and sensitive nanoscale biomolecule characterization technique through Brownian relaxation studies of particle suspensions. Current efforts have shown the applicability of this phenomenon in solid, pure liquid, and colloidal samples as well as simultaneous advancements of magnetic nanoparticle research in the magnetometric and magneto-optical regimes. By merging these two fields, a clinically relevant spectroscopy (fd-FRS, Frequency Domain Faraday Rotation Spectroscopy) was developed based on a newly revised model stemming from Debye relazation theory. Through this work, an optical bench with a variable permeability core electromagnet and a frequency-domain lock-in amplifier setup (DC to 20 kHz) have been used to distinguish between Fe3O4-core nanoparticles with functionalization layers of PEG4/PEG8 polymer with future applications involving the Anti-BSA/BSA antibody/antigen couple. Particle concentrations down to 500 nM (magnetic nanoparticles) and 0.01 Volume % (magnetic beads) were studied with diameters ranging from 200 nm to 1μm. currently, the characteristic peak corresponding to the out-of-phase relazation of the suspended particles has been elusive, despite a wide particle size distribution and the use of a balanced photodetector. Future work will involved highly monodisperse samples, faster scan times, and thermal characterization applications of fs-FRS.

Micro- or nanometer sized magnetic particles (beads) currently have a vast range of life science applications in, for example, bioseparation techniques, cancer therapy, development of contrast agents and biosensing techniques. In the latter field, magnetic beads offer several unique advantages, including minimal background signals, physical and chemical stability and low manufacturing costs. Because of these properties, magnetic biosensing techniques are potential candidates for low-cost, easy-to-use molecular diagnostic devices. This doctoral thesis focuses mainly on the proof of principle and further development of a new magnetic biosensor platform for detection of DNA targets, a potential candidate for a new generation of low-cost, easy-to-use diagnostic devices: the Volume-Amplified Magnetic Nanobead Detection Assay (VAM-NDA). The VAM-NDA principle combines target recognition by padlock probe ligation followed by rolling circle amplification (RCA) of the reacted probes with changes in Brownian relaxation behaviour of magnetic nanobeads (typically ~100 nm in diameter) induced by a change in hydrodynamic bead volume. More specifically, the RCA products (coils, typically ~1 μm in diameter) are detected magnetically by adding magnetic beads tagged with detection probes complementary to part of the repeating RCA-coil sequence. Thus, depending on the target concentration, a certain quantity of beads binds to the coils by base-pair hybridisation (bead immobilisation), resulting in a dramatic bead volume increase, which is then detected by measuring the complex magnetisation spectrum. Use of a commercial SQUID magnetometer for measuring complex magnetisation resulted in a detection limit in the low pM range for DNA targets with excellent quantification accuracy. Simultaneous multiplexing was also evaluated. The stability and aging of typical commercial ferrofluids (suspensions of magnetic beads) were investigated by measuring the complex magnetisation of and interbead interactions in oligonucleotide-functionalised ferrofluids. In summary, the bead surface characteristics were found to have a strong impact on the measured dynamic magnetic properties.