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

2011, Analytical Chemistry

b S Supporting Information E lectrochemical DNA biosensors (E-DNA) appear to be a promising alternative to optical sensors for the specific detection of oligonucleotide sequences. 1À3 These devices are composed of a redox-reporter-modified DNA probe immobilized on a gold electrode. Hybridization-linked changes in the flexibility of this probe (due to specific conformational changes 1,4,5 or an increase in the amount of double-helix 6 ) alter the rate with which electrons are transferred from the redox reporter, leading to a readily detectable change in Faradaic current upon voltammetric interrogation. 7,8 Because E-DNA sensors are driven by electrochemistry, rather than optical detection methods, they can be integrated into microfluidic devices, powered by inexpensive, hand-held electronics, and easily multiplexed. 9À11 Moreover, because their signaling is predicated on a binding-induced change in the physical properties of the probe DNA, rather than to adsorption of analytes to the sensor surface, E-DNA sensors are relatively insensitive to the nonspecific adsorption of interferants and are selective enough to deploy directly in complex clinical and environmental samples, such as blood or soil extracts. 12,13 E-DNA sensors thus appear well suited for point of care medical diagnostics, as well as portable analysis systems for forensics and food quality control. Although E-DNA sensors have a wide variety of positive attributes, many of the diverse E-DNA probe architectures described to date operate in a signal-off fashion, meaning that the measured current decreases as the concentration of analyte DNA increases. 14 For example, first generation E-DNA sensors employ a stem-loop architecture 1,14 such that, when a complementary target oligonucleotide is introduced, hybridization causes the stem to open. This moves the redox reporter further from the electrode surface, reducing electron transfer. This signal-off mechanism significantly limits the gain of the sensor: the maximum possible ABSTRACT: Electrochemical DNA (E-DNA) sensors, which are rapid, reagentless, and readily integrated into microelectronics and microfluidics, appear to be 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.