We report a reagentless, electrochemical sensor for the detection of double-stranded DNA targets that employs triplex-forming oligonucleotides (TFOs) as its recognition element. These sensors are based on redox-tagged TFO probes strongly chemisorbed onto an interrogating gold electrode. Upon the addition of the relevant double-stranded DNA target, the probe forms a rigid triplex structure via reverse Hoogsteen base pairing in the major groove. The formation of the triplex impedes contact between the probe’s redox moiety and the interrogating electrode, thus signaling the presence of the target. We first demonstrated the proof of principle of this approach by using a well-characterized 22-base polypurine TFO sequence that readily detects a synthetic, double-stranded DNA target. We then confirmed the generalizability of our platform with a second probe, a 19-base polypyrimidine TFO sequence that targets a polypurine tract (PPT) sequence conserved in all HIV-1 strains. Both sensors rapidly and specifically detect their double-stranded DNA targets at concentrations as low as ~10 nM and are selective enough to be employed directly in complex sample matrices such as blood serum. Moreover, to demonstrate real-world applicability of this new sensor platform, we have successfully detected unpurified, double-stranded PCR amplicons containing the relevant conserved HIV-1 sequence.

E-DNA sensors are a reagentless, electrochemical oligonucleotide sensing platform based on a redox-tag modified, electrode-bound probe DNA. Because E-DNA signaling is linked to hybridization-linked changes in the dynamics of this probe, sensor performance is likely dependent on the nature of the self-assembled monolayer coating the electrode. We have investigated this question by characterizing the gain, specificity, response time and shelf-life of E-DNA sensors fabricated using a range of co-adsorbates, including both charged and neutral alkane thiols. We find that, among the thiols tested, the positively charged cysteamine gives rise to the largest and most rapid response to target and leads to significantly improved storage stability. The best mismatch specificity, however, is achieved with mercaptoethanesulfonic and mercaptoundecanol, presumably due to the destabilizing effects of, respectively, the negative charge and steric bulk of these co-adsorbates. These results demonstrate that a careful choice of co-adsorbate chemistry can lead to significant improvements in the performance of this broad class of electrochemical DNA sensors.

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These results provide clear insights into the advantages offered by the use of a microdialysis-based CGM system that additionally relies on the detection of hydrogen peroxide at low operating potential. GlucoMen Day may become the CGM system of choice for those patients who require either regular administration of drugs or their glycemia to be tightly controlled in the intensive care unit or similar environments.

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