In this work, we develop a field-effect transistor with a two-dimensional channel made of a single graphene layer to achieve label-free detection of DNA hybridization down to attomolar concentration, while being able to discriminate a single nucleotide polymorphism (SNP). The SNP-level target specificity is achieved by immobilization of probe DNA on the graphene surface through a pyrene-derivative heterobifunctional linker. Biorecognition events result in a positive gate voltage shift of the graphene charge neutrality point. The graphene transistor biosensor displays a sensitivity of 24 mV/dec with a detection limit of 25 aM: the lowest target DNA concentration for which the sensor can discriminate between a perfect-match target sequence and SNP-containing one.

The inherent redox activity of dopamine enables its direct electrochemical in vivo analysis ( Venton , B. J.; Wightman, M. R. Anal. Chem. 2003, 75, 414A). However, dopamine analysis is complicated by the interference from other electrochemically active endogenous compounds present in the brain, including dopamine precursors and metabolites and other neurotransmitters (NT). Here we report an electrochemical RNA aptamer-based biosensor for analysis of dopamine in the presence of other NT. The biosensor exploits a specific binding of dopamine by the RNA aptamer, immobilized at a cysteamine-modified Au electrode, and further electrochemical oxidation of dopamine. Specific recognition of dopamine by the aptamer allowed a selective amperometric detection of dopamine within the physiologically relevant 100 nM to 5 μM range in the presence of competitive concentrations of catechol, epinephrine, norepinephrine, 3,4-dihydroxy-phenylalanine (L-DOPA), 3,4-dihydroxyphenylacetic acid (DOPAC), methyldopamine, and tyramine, which gave negligible signals under conditions of experiments (electroanalysis at 0.185 V vs Ag/AgCl). The interference from ascorbic and uric acids was eliminated by application of a Nafion-coated membrane. The aptasensor response time was <1 s, and the sensitivity of analysis was 62 nA μM(-1) cm(-2). The proposed design of the aptasensor, based on electrostatic interactions between the positively charged cysteamine-modified electrode and the negatively charged aptamer, may be used as a general strategy not to restrict the conformational freedom and binding properties of surface-bound aptamers and, thus, be applicable for the development of other aptasensors.

Electron transfer (ET) in redox-labeled double-stranded (ds) DNA tethered to electrodes through the alkanethiol linker at either the 3' or 5' DNA end and bearing methylene blue (MB) conjugated to the opposite end of DNA is shown to depend on the DNA end of tethering to electrodes. For 3' tethering, a nanoscale diffusion of the positively charged MB redox probe (and thus of the individual DNA molecules) to the negatively charged electrode surface provided the highest apparent diffusion and ET rates as a result of the tilting of 3'-tethered DNA (as compared to 5'-tethered DNA) versus the normal to the surface. Dynamic values of the tilting angle varied between 57 and 45° for 16-mer and 22-mer 3'-tethered DNA, and 5'-tethering was correlated with an upright orientation of DNA at the electrode surface. The values of the diffusion coefficient D(MB) corrected for tilting angles were similar for 5'- and 3'-tethered DNA and ranged between 5.4 × 10(-12) and 2.5 × 10(-12) cm(2) s(-1), whereas the ET rate constant k(ET)(dif) fit the 4.7 × 10(-6)-10.3 × 10(-6) cm s(-1) range for 22-mer and 16-mer dsDNA, respectively. Those values, when related to the nanometer (10(-7) cm) diffusion distances (the length of the studied DNA), allow relatively fast diffusion-limited ET at an apparent rate that may exceed the rate of the corresponding surface-confined ET process. This phenomenon is of particular importance for molecular electronics and electrochemical genosensor development.

The efficiency of DNA-based bioelectronic devices strongly depends on the way DNA molecules are linked to the electronic component. Commonly, DNA is tethered to metal electrodes via an alkanethiol linker representing an additional barrier for electron transport. Here we demonstrate that the replacement of the alkanethiol linker for a phosphorothioated adenosine tag increases the rate of DNA-mediated electron transfer (ET) up to 259 s(-1), representing the highest hitherto reported rate of electrochemically-modulated ET, and improves the stability of DNA-electrode surface binding. Both results offer pronounced technological and scientific benefits for DNA-based electronics.