Label free detection of 5′hydroxymethylcytosine within CpG islands using optical sensors

Abstract

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.

Applications of Optical Microcavity Resonators in Analytical Chemistry

“…Detection of methylation patterns in DNA sequences also promises to provide insight into the epigenetic regulation of gene transcription, ultimately controlling cell and organismal phenotype (105). Hawk and Armani (106) reported a microtoroid cavity modified with methylation-specific antibodies and used it to detect methylated DNA in buffer without the need for PCR amplification or fluorescent tagging.…”

“…Output can be monitored with a photodetector, measuring current over time. Though the alignment can be arduous, this method allows for highly efficient coupling [e.g., Refs (106, 120)]. ( B ) Grating couplers allow for on-chip coupling without the demands of precise fiber alignment, though at the cost of lower coupling efficiencies.…”

Electrochemical biosensor for detection of DNA hydroxymethylation based on glycosylation and alkaline phosphatase catalytic signal amplification

“…The linear regression equation can be expressed as I pa (mA) = 0.51logc (nM) + 1.46 with the correlation coefficient of 0.9975. The limit of detection (LOD) was calculated to be 1.43 pM (S/N = 3), which is lower than that obtained at HPLC-MS/MS analysis (20 pM) [33] and high than that at optical sensor (0.42 pM) [34]. In addition, it has been reported that the content of 5hmC in human urine samples were about 22.6 AE 13.7 nM [33], which is higher than our detection limit.…”

Low temperature co-fired ceramic packaging of CMOS capacitive sensor chip towards cell viability monitoring

“…Hence there is a growing interest in noninvasive, label-free, real-time, data-rich biosensing systems that measure electrical, optical, magnetic, or mass related properties of the biological sample. Such sensing techniques include surface plasmon resonance spectroscopy [1], electrochemical quartz crystal microbalance measurements [2], optical sensing [3], impedimetric sensing [4–6], and capacitive sensing [7–11]. …”