Change in the dynamics of single-stranded DNA or RNA probes tethered to an Au electrode on immunospecific binding to the analyte is a versatile approach to quantify a variety of molecules, such as heavy metal ions, pesticides, proteins, and nucleic acids (NAs). A widely studied approach is the electrochemical beacon method where the redox of a dye attached to the probe decreases as its proximity to the underlying electrode changes on binding. The limit of quantification (LOQ) defined by the semilog dependence of the signal on target concentration is in the picomolar range. Here, a method was studied where, by differential reflectivity, multiple reactions were measured on a monolith electrode. An alternative contrast mechanism was discovered, which led to an approach to enhance the LOQ to 10 aM and increase the dynamic range to 7 orders of magnitude using similar probes and binding conditions. Quantitative analysis on sequences with the G−C fraction ranging from 37 to 72% was performed. The approach will allow for the development of a label-free, enzyme-free microarray to detect biomolecules including NAs and proteins on a single electrode at quantification from 10 aM to 0.1 nM with high specificity.

Quantitative dysregulation in small nucleic acids (NA), such as microRNA (miRNA), extracted from minimally invasive biopsies, such as, blood, stool, urine, nose, throat, are promising biomarker for diseases diagnosis and management. We quantify the effect of the extra step of poly(A) ligation for cDNA synthesis and small size of the NA on the limit of quantification (LOQ) of quantitative PCR (qPCR), the gold standard to measure copy number. It was discovered that for small NA, the cycle threshold, Ct that is proportional to ‐log[c], where [c] is the concentration of the target NA exhibits a sharp transition. The results indicate that although the limit of detection (LOD) of qPCR can be in femtomolar range, the LOQ is significantly reduced by well over three orders of magnitude, in picomolar range. Specifically, the study reveals that the PCR product length is the primary reason the limitation on LOQ and is explicitly shown to be an important consideration for primer design for qPCR in general.