This
paper reports the preparation of versatile electrochemical
biosensing platforms for the simple, rapid, and PCR-independent detection
of the most frequent DNA methylation marks (5-methylcytosine, 5-mC,
and/or 5-hydroxymethylcytosine, 5-hmC) both at global and gene-specific
levels. The implemented strategies, relying on the smart coupling
of immuno-magnetic beads (MBs), specific DNA probes and amperometric
detection at screen-printed carbon electrodes (SPCEs), provided sensitive
and selective determination of the target methylated DNAs in less
than 90 min with a great reproducibility and demonstrated feasibility
for the simultaneous detection of the same or different cytosine epimarks
both at global level and in different loci of the same gene or in
different genes. The bioplatforms were applied to determine global
methylation events in paraffin-embedded colorectal tissues and specific
methylation at promoters of tumor suppressor genes in genomic DNA
extracted from cancer cells and paraffin-embedded colorectal tissues,
and in serum without previous DNA extraction from cancer patients.
The characterization of the humoral response in Alzheimer's disease (AD) patients might aid in detecting the disease at early stages. We have combined phage display and protein microarrays to identify AD autoantibodies and their target biomarkers. After enrichment of the T7 phage display libraries from AD and healthy brain tissue mRNA in AD-specific phages, 1536 monoclonal phages were printed on microarrays to probe them with 8 AD and 8 healthy control sera. A total of 57 phages showed higher seroreactivity in AD. In total, 13 out of the 44 unique sequences displayed on the phages were selected for validation using 68 AD and 52 healthy control sera. Peptides from Anthrax toxin receptor 1, Nuclear protein 1, Glycogen phosphorylase, and Olfactory receptor 8J1 expressed in bacteria as HaloTag fusion proteins showed a statistically significant ability to discriminate between AD patients and controls. The identified panel of AD autoantibodies might provide new insights into the blood-based diagnosis of the disease.
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