Harmonic Analysis of Biomolecules
Non-linear and not time-based method for high sensitivity detection
- Non-linear harmonic method
- Independent of time
- Internal normalization eliminates errors
- Differentiation between ss and ds DNA
There is a growing interest in biosensors, both in research and commercial fields. An important step in the fabrication of successful surface-based biosensors is the immobilization of the biological capture probe that is selective towards a particular target of interest. However, especially surface-based approaches comprising a DNA self-assembled monolayer (SAM) of fluorophore decorated DNA strands face challenges of photobleaching and/or variations in surface density, which are the cause of a high level of variability leading to inaccurate measurements.
Researchers at The University of British Columbia have designed a spectroelectrochemical imaging method for biomolecules that overcomes the described issues of surface density variations and photobleaching, as it allows for normalization. In this ensemble spectroelectrochemical setup, a biomolecule is characterized based on its non-linear harmonic signature from the measured fluorescence-potential response. For example, a fluorophore labeled DNA SAM was prepared on a single crystal gold bead electrode, and its fluorescence intensity response towards a single frequency sinusoidal potential perturbation (163 Hz), which causes the DNA SAM to reorient and in turn changes the efficiency of fluorescent quenching by the gold electrode, was measured. Due to the chosen low frequency potential perturbation, the fluorescence is not limited by time constraints of the system, enabling the modelling of a non-linear response. The modulated fluorescence intensity data is composed of 1st, 2nd, and 3rd harmonic signals, which are different for single-strand (ss) and double-strand (dd) DNA. So this new approach makes the quantitative measurement of the shape of the fluorescence–potential response curve possible, thereby highlighting the differences between e.g. ssDNA and ds DNA self-assembled monolayers.