Aptamers are artificial oligonucleotide receptors originated from in vitro selection (SELEX). 1 In principle, aptamers with high specificity and affinity can be selected for any given target, ranging from small molecules to large proteins and even cells. 2 Therefore, aptamers are widely recognized as highly promising tools for a variety of important applications. 3,4 Aptamers are particularly useful as the biosensing element as they are chemically stable, readily available, and offer high flexibility in biosensor design. [5][6][7][8][9][10][11] Recently, Heeger, Plaxco, and others developed a series of novel electrochemical aptamer-based (E-AB) sensors for thrombin, cocaine, and potassium, [12][13][14] an analogous version to the electrochemical DNA (E-DNA) sensor. 8,15 These E-AB sensors are based on binding-induced conformational changes of redox-tagged and surface-confined aptamers, which have proven highly sensitive and selective. 12-14 Also, because E-AB sensors are electrochemistrybased, they are inherently fast, portable, and cost-effective. However, since E-AB relies on unique structures of aptamers, these sensors have to be designed case-by-case for different aptamertarget pairs. As a step further, Xiao et al. recently developed a potentially generalizable E-AB sensor for thrombin by using targetinduced strand displacement. 16 Here we report a target-responsive electrochemical aptamer switch (TREAS), which is a signal-on sensor featuring both generalizability and simplicity in design, toward reagentless detection of adenosine triphosphate (ATP) with high sensitivity and selectivity.We employed an in vitro selected 27-base anti-ATP aptamer, which possesses high affinity for ATP while not for its analogues, cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP). 17 The anti-ATP aptamer dually labeled with 3′-SH and 5′-ferrocene is self-assembled on gold electrodes in its duplex form (Scheme 1). We reason that ferrocene is distal to the electrode surface, thus cannot efficiently exchange electrons with the underlying electrodes due to large distance separation (∼10 nm) in this eT OFF state. In the presence of the target ATP, the tertiary aptamer structure is stabilized, which responsively denatures the duplex and liberates the complementary DNA, similar to the aptamer structural switch in solution. 18 As a consequence of this structural switch from the duplex to the tertiary aptamer structure (duplex-to-aptamer), the ferrocene moiety approaches the electrode surface and generates measurable electrochemical signals (eT ON). Of note, compared to the E-AB thrombin sensor reported by Xiao et al., 16 our TREAS is similarly generalizable while it has several advantages. First, the sensor architecture is simpler. The sensing DNA strand of E-AB contains three parts, aptamer region (for recognition), duplex region (structural support), and spacer region (linkage), thus is inherently longer than the corresponding TREAS sensing strand (only aptamer sequence). Second, TREAS has two we...

We have demonstrated a novel sensing strategy employing single-stranded probe DNA, unmodified gold nanoparticles, and a positively charged, water-soluble conjugated polyelectrolyte to detect a broad range of targets including nucleic acid (DNA) sequences, proteins, small molecules, and inorganic ions. This nearly "universal" biosensor approach is based on the observation that, while the conjugated polyelectrolyte specifically inhibits the ability of single-stranded DNA to prevent the aggregation of gold-nanoparticles, no such inhibition is observed with double-stranded or otherwise "folded" DNA structures. Colorimetric assays employing this mechanism for the detection of hybridization are sensitive and convenient-picomolar concentrations of target DNA are readily detected with the naked eye, and the sensor works even when challenged with complex sample matrices such as blood serum. Likewise, by employing the binding-induced folding or association of aptamers we have generalized the approach to the specific and convenient detection of proteins, small molecules, and inorganic ions. Finally, this new biosensor approach is quite straightforward and can be completed in minutes without significant equipment or training overhead.biosensor | aptamer | visual detection | thrombin detection | cocaine detection G old nanoparticle colorimetric biosensors have seen significant applications in diagnostics, environmental monitoring, and antibioterrorism supporting unaided, visual readout (1-12). Commonly, the relevant nanoparticles are covalently modified with either a probe DNA or an aptamer such that hybridization (13-16) or aptamer-target interactions (17-27), for example the scanometric method developed by Mirkin (25), which is a very sensitive and specific tool, crosslink them, inducing aggregation. The second broad approach utilizes unmodified nanoparticles. (28-30) These two approaches, however, suffer from timeconsuming (20-40 h of assembly) and relatively poor (low nanomolar) detection limits, respectively. Here, a unique, colorimetric sensing strategy employing a simple but selective combination of a single-stranded DNA probe, a positively charged, water-soluble conjugated polyelectrolyte, and unmodified gold nanoparticles is demonstrated. The universality of this method allows detection of a broad range of targets, including nucleic acid (DNA) sequences, proteins, small molecules, and ino rganic ions. Our approach is rapid (turnaround time is 5-10 min) and sensitive (picomolar concentrations of target DNA are readily detected with the naked eye, even in complex sample matrices like blood serum). Hence, an operator with minimum scientific overhead can easily employ this technique.Generally, the gold nanoparticle applications typically rely on a quantitative coupling between target recognition and the aggregation of the nanoparticles, which, in turn, leads to a dramatic change in the photonic properties-and thus the color-of the nanoparticle solution. This colorimetric "readout" avoids the relative complexity inherent...

A limitation of many traditional approaches to the detection of specific oligonucleotide sequences, such as molecular beacons, is that each target strand hybridizes with (and thus activates) only a single copy of the relevant probe sequence. This 1:1 hybridization ratio limits the gain of most approaches and thus their sensitivity. Here we demonstrate a nuclease-amplified DNA detection scheme in which exonuclease III is used to "recycle" target molecules, thus leading to greatly improved sensitivity relative to, for example, traditional molecular beacons without any significant restriction in the choice of target sequences. The exonuclease-amplified assay can detect target DNA at concentrations as low as 10 pM when performed at 37 degrees C, which represents a significant improvement over the equivalent molecular beacon alone. Moreover, at 4 degrees C we can obtain a detection limit as low as 20 aM, albeit at the cost of a 24 h incubation period. Finally, our assay can be easily interrogated with the naked eye and is thus amenable to deployment in the developing world, where fluorometric detection is more problematic.

We herein demonstrate a sandwich assay based on single aptamer sequences is suitable for the direct detection of small molecule targets in blood serum and other complex matrices. By splitting an aptamer into two pieces, we convert a single affinity reagent into a two-component system in which the presence of the target drives formation of a complex comprised of the target and the two halves of the aptamer. To demonstrate the utility of this approach we have used single anticocaine and anti-ATP aptamers to fabricate electrochemical sensors directed against the representative small molecules cocaine and ATP. Both targets are detected at low micromolar concentrations, in seconds, and in a convenient, general, readily reusable, electrochemical format. Moreover, both sensors are selective enough to deploy directly in blood, crude cellular lysates and other complex sample matrices.