The reconstitution of an apo-flavoenzyme, apo-glucose oxidase, on a 1.4-nanometer gold nanocrystal functionalized with the cofactor flavin adenine dinucleotide and integrated into a conductive film yields a bioelectrocatalytic system with exceptional electrical contact with the electrode support. The electron transfer turnover rate of the reconstituted bioelectrocatalyst is approximately 5000 per second, compared with the rate at which molecular oxygen, the natural cosubstrate of the enzyme, accepts electrons (approximately 700 per second). The gold nanoparticle acts as an electron relay or "electrical nanoplug" for the alignment of the enzyme on the conductive support and for the electrical wiring of its redox-active center.

Blue, gold, and DNA: A methylene blue (MB) tagged, thrombin‐binding DNA aptamer immobilized on a gold surface undergoes a large conformational change upon target binding (see schematic representation; eT: electron transfer). This folding produces a large, readily measurable change in redox current and allows the electrochemical detection of thrombin in blood serum.

The catalytic enlargement of aptamer-functionalized Au nanoparticles amplifies the optical detection of aptamer-thrombin complexes in solution and on surfaces.

Thrombin binding stabilizes the alternative G-quadruplex conformation of the aptamer, liberating the methylene blue (MB)-tagged oligonucleotide to produce a flexible, single-stranded DNA element. This allows the MB tag to collide with the gold electrode surface, producing a readily detectable Faradaic current at thrombin concentrations as low as approximately 3 nM.

Recent years have seen the development of a number of reagentless, electrochemical sensors based on the target-induced folding or unfolding of electrode-bound oligonucleotides, with examples reported to date, including sensors for the detection of specific nucleic acids, proteins, small molecules and inorganic ions. These devices, which are often termed electrochemical DNA (E-DNA) and E-AB (electrochemical, aptamer-based) sensors, are comprised of an oligonucleotide probe modified with a redox reporter (in this protocol methylene blue) at one terminus and attached to a gold electrode via a thiol-gold bond at the other. Binding of an analyte to the oligonucleotide probe changes its structure and dynamics, which, in turn, influences the efficiency of electron transfer to the interrogating electrode. This class of sensors perform well even when challenged directly with blood serum, soil and other complex, multicomponent sample matrices. This protocol describes the fabrication of E-DNA and E-AB sensors. The protocol can be completed in 12 h.

Electrochemical, aptamer-based (E-AB) sensors, which are comprised of an electrode modified with surface immobilized, redox-tagged DNA aptamers, have emerged as a promising new biosensor platform. In order to further improve this technology we have systematically studied the effects of probe (aptamer) packing density, the AC frequency used to interrogate the sensor, and the nature of the self-assembled monolayer (SAM) used to passivate the electrode on the performance of representative E-AB sensors directed against the small molecule cocaine and the protein thrombin. We find that, by controlling the concentration of aptamer employed during sensor fabrication, we can control the density of probe DNA molecules on the electrode surface over an order of magnitude range. Over this range, the gain of the cocaine sensor varies from 60% to 200%, with maximum gain observed near the lowest probe densities. In contrast, over a similar range, the signal change of the thrombin sensor varies from 16% to 42% and optimal signaling is observed at intermediate densities. Above cut-offs at low hertz frequencies, neither sensor displays any significant dependence on the frequency of the alternating potential employed in their interrogation. Finally, we find that E-AB signal gain is sensitive to the nature of the alkanethiol SAM employed to passivate the interrogating electrode; while thinner SAMs lead to higher absolute sensor currents, reducing the length of the SAM from 6-carbons to 2-carbons reduces the observed signal gain of our cocaine sensor 10-fold. We demonstrate that fabrication and operational parameters can be varied to achieve optimal sensor performance and that these can serve as a basic outline for future sensor fabrication.

The development of specific ion sensors is linked to pressing needs for the rapid detection of toxic metals. [1][2][3][4] Of particular interest has been the detection of lead (Pb 2+ ), an important pollutant with major routes of human exposure arising from lead-based paints and contaminated soils and foodstuffs. 5 Because of the often severe effects of lead toxicity, which include renal malfunction and the inhibition of brain development, 6 allowable juvenile serum lead levels are just 100 parts-per-billion (ppb). 7 Current protocols for the detection of lead require inductively coupled plasma mass spectroscopy (ICP/MS) (see Supporting Information (SI)), a rather complex laboratory technique. Motivated by the desire for rapid, portable means of quantifying low-level lead contamination, recent years have seen the development of fluorescent 8 and colorimetric 9 sensors achieving parts-per-billion detection limits. These optical methods, however, suffer from possible drawbacks including potential false signals arising from contaminating colorants, fluorophores and quenchers, and, frequently, a reliance on cumbersome optical equipment. Electrochemical methods, in contrast, benefit from the impressive miniaturization of modern microelectronics, the relative paucity of electroactive contaminants, and the relative stability and environmental insensitivity of electroactive labels and thus are less likely to suffer from these potential drawbacks. 10 Unfortunately, however, the electrochemical lead detection methods reported to date require complex, multistep protocols involving the reductive deposition of metallic lead followed by anodic stripping voltammetry. 10 Here we propose a simpler electrochemical approach based on the highly specific, metal-induced activation of a lead-requiring DNAzyme.DNAzymes are catalytic DNA sequences isolated via in vitro selection. 11,12 Cofactor-dependent DNAzymes can often be generated from this approach by adding varying cofactors and cofactor concentrations during the selection process. Using a lead-dependent DNAzyme produced by this method, several groups have created optical lead sensors that couple the presence of this cofactor with catalytic activities producing fluorescent or colorometric outputs. 8,9 Here we adapt this same lead-dependent DNAzyme in an electrochemical biosensor that achieves parts-per-billion (nanomolar) sensitivity and excellent selectivity in a single, convenient measurement step.The Pb 2+ -requiring DNAzyme we have employed, the "8-17" DNAzyme, is a sequence-specific nuclease acting on a singlestranded DNA substrate containing a single, sessile ribo-adenine (indicated by arrows in Scheme 1). 8a,13 The sensor consists of a methylene-blue (MB) modified version of this catalytic DNA strand (1) hybridized to its complementary, 20-base substrate oligonucleotide (2). This complex, which is chemi-absorbed to a gold electrode via a 5′ terminal thiol on the catalytic strand, 14 is relatively rigid, presumably preventing the MB from approaching the electrode to...

DNA and telomerase activity are detected by a DNAzyme generated upon hybridization and opening of a functional catalytic beacon.