In a traditional sandwich assay, a DNA target hybridizes to a single copy of the signal probe. Here we employ a modified signal probe containing a methylene blue (a redox moiety) label and a “sticky end.” When a DNA target hybridizes this signal probe, the sticky end remains free to hybridize another target leading to the creation of a supersandwich structure containing multiple labels. This leads to large signal amplification upon monitoring by voltammetry.

Single‐step DNA detection: A microfluidic electrochemical loop mediated isothermal amplification platform is reported for rapid, sensitive, and quantitative detection of pathogen genomic DNA at the point of care (see picture). DNA amplification was electrochemically monitored in real time within a monolithic microfluidic device, thus enabling the detection of as few as 16 copies of Salmonella genomic DNA through a single‐step process in less than an hour.

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.

The diagnosis, prevention, and treatment of many illnesses, including infectious and autoimmune diseases, would benefit from the ability to measure specific antibodies directly at the point of care. Thus motivated, we designed a wash-free, electrochemical method for the rapid, quantitative detection of specific antibodies directly in undiluted, unprocessed blood serum. Our approach employs short, contiguous polypeptide epitopes coupled to the distal end of an electrode-bound nucleic acid “scaffold” modified with a reporting methylene blue. The binding of the relevant antibody to the epitope reduces the efficiency with which the redox reporter approaches, and thus exchanges electrons with, the underlying sensor electrode, producing readily measurable change in current. To demonstrate the versatility of the approach we fabricated a set of six such sensors, each aimed at the detection of a different monoclonal antibody. All six sensors are sensitive (sub-nanomolar detection limits), rapid (equilibration time constants ~8 min), and specific (no appreciable cross reactivity with the targets of the other five). When deployed in a millimeter-scale, an 18-pixel array with each of the six sensors in triplicate, support the simultaneous measurement of the concentrations of multiple antibodies in a single, sub-milliliter sample volume. The described sensor platform thus appears be a relatively general approach to the rapid and specific quantification of antibodies in clinical materials.

DNA‐Detektion: Ein mikrofluidisches elektrochemisches Amplifizierungsverfahren zur schnellen, empfindlichen und quantitativen Detektion von pathogener DNA im patientennahen Bereich wird vorgestellt (siehe Bild). Die DNA‐Amplifizierung wurde elektrochemisch in Echtzeit in einem einzigen Mikrofluidikgerät beobachtet, und weniger als 16 Kopien der DNA von Salmonellen konnten in weniger als einer Stunde in einem einstufigen Verfahren detektiert werden.