Electrochemical Biosensors Employing an Internal Electrode Attachment Site and Achieving Reversible, High Gain Detection of Specific Nucleic Acid Sequences

Abstract

Electrochemical DNA (E-DNA) sensors, which are rapid, reagentless and readily integrated into microelectronics and microfluidics, appear a promising alternative to optical methods for the detection of specific nucleic acid sequences. Keeping with this, a large number of distinct E-DNA architectures have been reported to date. Most, however, suffer from one or more drawbacks, including low signal gain (the relative signal change in the presence of complementary target), signal-off behavior (target binding reduces the signaling current, leading to poor gain and raising the possibility that sensor fouling or degradation can lead to false positives) or instability (degradation of the sensor during regeneration or storage). To remedy these problems, we report here the development of a signal-on E-DNA architecture that achieves both high signal gain and good stability. This new sensor employs a commercially synthesized, asymmetric hairpin DNA as its recognition and signaling probe, the shorter arm of which is labeled with a redox reporting methylene blue at its free end. Unlike all prior E-DNA architectures, in which the recognition probe is attached via a terminal functional group to its underlying electrode, the probe employed here is affixed using a thiol group located internally, in the turn region of the hairpin. Hybridization of a target DNA to the longer arm of the hairpin displaces the shorter arm, allowing the reporter to approach the electrode surface and transfer electrons. The resulting device achieves signal increases of ~800% at saturating target, a detection limit of just 50 pM and ready discrimination between perfectly matched sequences and those with single nucleotide polymorphisms. Moreover, because the hairpin probe is a single, fully covalent strand of DNA, it is robust to the high stringency washes necessary to remove the target, and thus these devices are fully reusable.

Methylene blue phosphoramidite for DNA labelling

“…[14][15][16][17][18] For instance, K. Plaxco et al reported on the design of DNA, protein, antibody and drug sensors based on bioreceptor modification using MB as an electrochemical indicator. [19][20][21][22][23] MB proved efficient for electron transfer and had good stability over time in such detection systems, even better than ferrocene. 24 To develop an efficient DNA sensor, the position of MB along the ODN used as a bioreceptor appears to be critical.…”

A reusable and label-free supersandwich biosensor for sensitive DNA detection by immobilizing target-triggered DNA concatamers on ternary self-assembled monolayer

“…However, the construction of most interface architectures is complex and time-consuming. To solve this problem, some novel strategies were proposed by introducing gentle regeneration of the interface architectures [23][24][25]. Although the reusability makes the biosensors attractive, the reused biosensors show limited sensitivity, generally around 10 pM level [26].…”

Toehold-mediated DNA displacement-based surface-enhanced Raman scattering DNA sensor utilizing an Au–Ag bimetallic nanodendrite substrate

“…A hairpin capture probe became an open-loop structure by hybridization with a target DNA and then a streptavidin coupled reporter strand was allowed to bind on the opened probe for mass amplification (LOD: 0.3 nM). Alternatively, two different electrochemical DNA (E-DNA) sensors using methylene blue as a redox reporter are presented [13,14]. In these sensors, the capture of a target DNA allows the reporter to approach the electrode surface and increases current through enhanced electron transfer (LODs: 50 pM and 400 fM).…”