Development of functional nanoparticle-biomolecule conjugates based biosensors

This thesis is composed of two parts: Part I is focused on studying how quantum dot (QD) surface small-molecule capping ligands affect its Förster resonance energy transfer (FRET) with a fluorescent protein (FP); Part II is
focused on developing an ultrasensitve DNA sensing technology by combining magnetic nanoparticle (MNP) and enzymatic signal amplification.
Part I
The FRET between a CdSe/ZnS core/shell QD capped with three different small-molecule ligands and a hexa-histidine (His6)-tagged FP (mCherry) has been studied. Results show that small-molecule ligands strongly affect the
FRET behaviours between QD and FP. The QD-FP self-assembly process is fast (complete in minutes at low nM concentration), strong (with Kd ~ 1 nM), suggesting that the QD-His6-tagged biomolecule self-assembly is an
effective approach for making compact QD-bioconjugates which may have a wide range of sensing and biomedical applications.
PART II
I have developed a facile, rapid, and sensitive DNA sensor by combining the efficient MNP-based target capture with the highly efficient signal amplification power of enzymes to achieve ultra-sensitivity. This sensor works efficiently in both pure buffer and complex media (such as 10%
human serum in buffer). Moreover, this developed approach is able to quantitate two distinct DNA strands in a homogenous phase at the same time with a detection limit of ~5 pM. Second, I have developed a novel, highly sensitive and selective approach for label-free DNA detection and single-nucleotide polymorphism (SNP) discrimination by combining target-recycled ligation (TRL), MNP assisted
target capture/ separation, and efficient enzymatic amplification. This approach possesses a detection limit of 600 fM unlabelled DNA targets and offers exquisitely high discrimination ratio (up to > 380 fold) between a
perfect-match mutant and its single-base mismatch DNA target. Furthermore, it can quantitate the rare cancer mutant in large excesses of coexisting wildtype
DNA down to 0.75%.