MicroRNAs (miRNAs) are single-stranded, short (~ 22–25 mers long), non-coding nucleotides that hold the key to studying the biology of diseases. Indeed, deficiencies or excesses in miRNA expression have been related to a number of diseases ranging from myocardial infarction to autoimmune disease. Despite the potential of miRNAs to serve as diagnostic biomarkers, their low cellular abundance and susceptibility to environmental degradability, make it challenging to realize their sensitive detection. Photoelectrochemical (PEC) biosensors with their remarkable sensitivity and specificity hold great promise for the sensitive bioanalysis of such trace biomarkers. However, these PEC biosensors are highly susceptible to variable backgrounds caused by biological matrices. In response, multi-species ratiometric or differential assays, combining two or more PEC signals under multiple excitation wavelength and voltages, have been implemented to reduce the effect of interference and experimental variations, thus enhancing accuracy and analysis reliability at trace analyte concentrations. Although effective, this multi-species approach increases the complexity of the instrumentation and the calibration algorithms needed to deal with the varying baseline signals exhibited by such systems. In response to this, a differential PEC strategy encompassing the use of a single wavelength excited titanium-dioxide/gold (TiO<sub>2</sub>/Au) system and two subsequent and correlated hybridization events – first with an unlabeled target and then with a signal amplification barcode (SAB) featuring plasmonic gold (Au) – was harnessed to detect unlabeled target DNA in both buffer and urine. This strategy exhibited a limit-of-detection (LOD) of 3 fM in buffer and 5 fM in diluted urine, demonstrating significant improvement over a conventional signal-off strategy using a single binding event (11 fM in buffer and 72 fM in urine). Additionally, this assay enhanced the analytical sensitivity by a factor of three as compared to an analogous assay that did not use differential signaling and demonstrated the ability to distinguish between sequence mismatches. This work offers a new strategy for enhancing the performance metrics of PEC biosensors that are key to facilitating their use in clinical decision making. Furthermore, given the target length (25-mer) and concentration range (1 fM–100 pM) probed, the assay presented here is ideally-suited for the analysis of short nucleotides such as miRNA.

Enhancing the sensitivity of photoelectrochemical in nucleotide biosensing using plasmonic barcodes and differential signal readout

ACS Fall 2021

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