Significant research has been invested in correlating genetic variations with different disease probabilities. Recently, it has become apparent that other DNA modifications, such as the addition of a methyl
or hydroxymethyl group to cytosine, can also play a role. While these modifications do not change the
sequence, they can negatively impact the function. Therefore, it is critical to be able to both read the
genetic code and identify these modifications. Currently, the detection of hydroxymethylated cytosine (5′
hmC) and the two closely related variants, cytosine (C) and 5′methylcytosine (5′mC), relies on a combination of nucleotide modification steps, followed by PCR and gene sequencing. However, this approach
is not ideal because transcription errors which are inherent to the PCR process can be misinterpreted as
fluctuations in the relative C:5′mC:5′hmC concentrations. As such, an alternative method which does not
rely on PCR or nucleotide modification is desirable. One approach is based on label-free optical resonant
cavity sensors. In the present work, toroidal resonant cavity sensors are functionalized with antibodies to
enable label-free detection and discrimination between C, 5′mC, and 5′hmC in real-time without PCR.
Specifically, epoxide chemistry is used to covalently attach the 5′hmC antibody to the surface of the
cavity. Subsequently, to thoroughly characterize the sensor platform, detection of C, 5′mC, and 5′hmC is
performed over a concentration range from pM to nM. At low (pM) concentrations, the hydroxymethylated cytosine produces a significantly larger signal than the structurally similar epigenetic
markers; thus demonstrating the applicability of this platform.