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...

Mercury ions can easily pass through biological membranes and cause serious damage to the central nervous and endocrine systems. [1] Therefore, imaging of Hg 2+ ions in living cells is crucial for the elucidation of their biological effects. Fluorescence spectroscopy has become a powerful tool for sensing and imaging trace amounts of samples because of its simplicity and sensitivity. [2] Thus, the development of fluorescent Hg 2+ probes, [3] particularly those that have practical application in living cells, [4] has attracted much attention. Most reported examples of fluorescent sensing of Hg 2+ ions in living cells function by the enhancement of fluorescence signals. However, as the change in fluorescence intensity is the only detection signal, factors such as instrumental efficiency, environmental conditions, and the probe concentration can interfere with the signal output. [5] Ratiometric sensors can eliminate most or all ambiguities by selfcalibration of two emission bands. [6] Ratiometric probes can be designed to function following two mechanisms: intramolecular charge transfer (ICT) and fluorescence resonance energy transfer (FRET). ICT probes have been frequently reported and some work well under physiological conditions. Two aspects which potentially influence the accuracy of ICT probes are: 1) Binding of the target ions promotes or inhibits ICT interactions, which results in remarkable shifts of the sensors absorption maxima; but if multiple excitation wavelengths are used to match the different excitation maxima, their difference in efficiency may be a potential origin of inaccuracy. 2) Relatively broad fluorescence spectra are often observed for ICT fluorophores; in a significant number of cases the broad fluorescence spectra before and after binding target ions have a high degree of overlap (or in an extreme case, a broad spectrum with high intensity completely covers one with lower intensity), which makes it difficult to accurately determine the ratio of the two fluorescence peaks. Theoretically, the above problems can be avoided by using a FRET-based sensor for which the single excitation wavelength of a donor fluorophore results in emission of the acceptor at a longer wavelength. [7] Herein we present a BODIPY-rhodamine (BODIPY = boron-dipyrromethene) FRET "off-on" system 3 as a ratiometric and intracellular Hg 2+ sensor. A leuco-rhodamine derivative was chosen as a sensitive and selective chemosensor for Hg 2+ ions. This was inspired by Tae and co-workers as well as other research groups, [8] , who used these leuco derivatives with unconjugated structures as fluorogenic and chromogenic sensors. A highly efficient ring-opening reaction induced by Hg 2+ generates the long-wavelength rhodamine fluorophore which can act as the energy acceptor. BODIPY [9] was chosen as the energy donor because its intense fluorescence is insensitive to environmental factors and its fluorescence spectrum matches well with the absorption spectrum of rhodamine. The choice of the connection between the donor and acceptor ...

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.