Ultra-sensitive measurements of magnetically labelled RCA products in a microfluidic channel using a high-Tc SQUID

Licentiate thesis, 2018

The development of a nucleic acid (deoxyribonucleic acid (DNA), ribonucleic acid (RNA)) bioassay based on magnetic nanoparticles (MNPs) with a high-Tc superconducting quantum interference device (SQUID) gradiometer as a magnetic readout is described. The specific binding of the MNPs to the target DNA molecules changes the MNPs size distribution and, therefore, the relaxation dynamics and is measured by magnetic ac susceptometry. The binding reactions are measured by SQUID gradiometer in a microfluidic channel with volume of 3 μL. The magnetic content sensitivity at the noise level of our SQUID is estimated to be 1.5e6 MNPs/sqrt(Hz) or 2.9e-10 emu/sqrt(Hz) in magnetic moment, corresponding to 2.5 ng of MNPs with
diameter of 100 nm.
Two different assay protocols are investigated for a magnetic nucleic acid biosensor. Padlock probes with suitable sequences are used as bioreceptors and circularize upon target recognition. The rolling circle amplification (RCA) provides the gain to the target molecule by copying the circularized padlock probe into a large concatemer. The specific binding of the MNPs to these large DNA coils changes their relaxation dynamics. These large DNA molecules can also digest into short monomers. The monomers can induce an agglutination if two MNP with matching sequence motifs to the two ends of the monomer are introduced. The agglutinated clusters would have large hydrodynamic size, thus, a different relaxation dynamics. The bioassay
has shown higher sensitivity using large DNA coils. Extrapolated sensitivity of the sensor to target analyte is estimated to be 66 fM of RCA coils. This is limit is equivalent to 1.0e5 target DNA molecules.

The method and instruments that are adopted and presented here are not limited to the Vibrio cholera bioanalyte and are generic and could in principle be used for other DNA or RNA viruses. The ultra-high magnetic sensitivity combined with the microfluidic sample handling is a critical step towards a magnetic bioassay for rapid detection of diseases at the point-of-care (POC). Future developments include implementation of all steps of the bioassay on a disposable lab-on-chip and eliminating the liquid nitrogen by operating the SQUID on a micro-cooler platform. These would make the magnetic bioassay promising for applications as a future nano-diagnostics unit.