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We report a strategy for the reagentless transduction of DNA hybridization into a readily detectable electrochemical signal by means of a conformational change analogous to the optical molecular beacon approach. The strategy involves an electroactive, ferrocene-tagged DNA stem-loop structure that self-assembles onto a gold electrode by means of facile gold-thiol chemistry. Hybridization induces a large conformational change in this surface-confined DNA structure, which in turn significantly alters the electron-transfer tunneling distance between the electrode and the redoxable label. The resulting change in electron transfer efficiency is readily measured by cyclic voltammetry at target DNA concentrations as low as 10 pM. In contrast to existing optical approaches, an electrochemical DNA (E-DNA) sensor built on this strategy can detect femtomoles of target DNA without employing cumbersome and expensive optics, light sources, or photodetectors. In contrast to previously reported electrochemical approaches, the E-DNA sensor achieves this impressive sensitivity without the use of exogenous reagents and without sacrificing selectivity or reusability. The E-DNA sensor thus offers the promise of convenient, reusable detection of picomolar DNA.

Spectroscopic studies have identified a number of proteins that appear to retain significant residual structure under even strongly denaturing conditions. Intrinsic viscosity, hydrodynamic radii, and small-angle x-ray scattering studies, in contrast, indicate that the dimensions of most chemically denatured proteins scale with polypeptide length by means of the power-law relationship expected for random-coil behavior. Here we further explore this discrepancy by expanding the length range of characterized denatured-state radii of gyration (RG) and by reexamining proteins that reportedly do not fit the expected dimensional scaling. We find that only 2 of 28 crosslink-free, prosthetic-group-free, chemically denatured polypeptides deviate significantly from a power-law relationship with polymer length. The RG of the remaining 26 polypeptides, which range from 16 to 549 residues, are well fitted (r2 = 0.988) by a power-law relationship with a best-fit exponent, 0.598 ± 0.028, coinciding closely with the 0.588 predicted for an excluded volume random coil. Therefore, it appears that the mean dimensions of the large majority of chemically denatured proteins are effectively indistinguishable from the mean dimensions of a random-coil ensemble

Whereas spectroscopic and chromatographic techniques for the detection of small organic molecules have achieved impressive results, these methods are generally slow and cumbersome, and thus the development of a general means for the real-time, electronic detection of such targets remains a compelling goal. Here we demonstrate a potentially general, label-free electronic method for the detection of small-molecule targets by building a rapid, reagentless biosensor for the detection of cocaine. The sensor, based on the electrochemical interrogation of a structure-switching aptamer, specifically detects micromolar cocaine in seconds. Because signal generation is based on binding-induced folding, the sensor is highly selective and works directly in blood serum and in the presence of commonly employed interferents and cutting agents, and because all of the sensor components are covalently attached to the electrode surface, the sensor is also reusable: we achieve >99% signal regeneration upon a brief, room temperature aqueous wash. Given recent advances in the generation of highly specific aptamers, this detection platform may be readily adapted for the detection of other small molecules of a wide range of clinically and environmentally relevant small molecules.

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.

Gold nanoparticles quench the fluorescence of cationic polyfluorene with Stern-Volmer constants (KSV) approaching 10 11 M ؊1 , several orders of magnitude larger than any previously reported conjugated polymer-quencher pair and 9 -10 orders of magnitude larger than small molecule dye-quencher pairs. The dependence of KSV on ionic strength, charge and conjugation length of the polymer, and the dimensions (and thus optical properties) of the nanoparticles suggests that three factors account for this extraordinary efficiency: (i) amplification of the quenching via rapid internal energy or electron transfer, (ii) electrostatic interactions between the cationic polymer and anionic nanoparticles, and (iii) the ability of gold nanoparticles to quench via efficient energy transfer. As a result of this extraordinarily high KSV, quenching can be observed even at subpicomolar concentrations of nanoparticles, suggesting that the combination of conjugated polymers with these nanomaterials can potentially lead to improved sensitivity in optical biosensors. (1). This is 5-6 orders of magnitude more efficient than the quenching of typical small molecule dye-quencher pairs (8), an effect that translates into greatly improved sensitivity in fluorescence-based assays (1, 2). Were further increases in K SV possible, they should thus translate directly into improved sensor performance.The observation that gold metal efficiently quenches the emission of many fluorophores (9-11) suggests that gold nanoparticles might serve as efficient quenchers of conjugated polymer fluorescence. Huang and Murray (12) have described the quenching of small molecule dyes by gold nanoparticles, and Dubertret et al. . This greatly increased K SV provides insights into the mechanisms that underlie the extraordinarily efficient quenching of these materials. More interestingly, versatile chemistry available for surface functionization of gold nanoparticles (14) makes the gold nanomaterials especially suitable for possible ligand tethering and therefore applicable for use in high-performance conjugated polymer-based sensors (1). Materials and MethodsThe water-soluble conjugated polymers and oligomers seen in Scheme 1 were synthesized at the University of California, Santa Barbara as described (15)(16)(17). Gold nanoparticles were obtained from either Sigma (5, 10, or 20 nm) or British Biocell International, Cardiff, U.K. (2 nm). Concentrations of gold nanoparticles were adapted from the data provided by the manufacturer. Absorption spectra were collected with a Shimadzu UV-2401PC UV-visible recording spectrophotometer, and photoluminescence (PL) spectra were collected with a PTI fluorometer (Photon Technology International, Lawrenceville, NJ). The quartz cuvettes were treated by hexamethyldisilazane to block nonspecific electrostatic absorption of cationic polymers to anionic quartz surfaces (18). The fluorescence spectra were obtained by exciting poly(9,9Ј-bis(6-N,N,N-trimethylammonium)-hexyl)-fluorene phenylene (PF) at 375 nm, oligofluorene at 320 nm, poly...

The fastest simple, single domain proteins fold a million times more rapidly than the slowest. Ultimately this broad kinetic spectrum is determined by the amino acid sequences that define these proteins, suggesting that the mechanisms that underlie folding may be almost as complex as the sequences that encode them. Here, however, we summarize recent experimental results which suggest that (1) despite a vast diversity of structures and functions, there are fundamental similarities in the folding mechanisms of single domain proteins and (2) rather than being highly sensitive to the finest details of sequence, their folding kinetics are determined primarily by the large-scale, redundant features of sequence that determine a protein's gross structural properties. That folding kinetics can be predicted using simple, empirical, structure-based rules suggests that the fundamental physics underlying folding may be quite straightforward and that a general and quantitative theory of protein folding rates and mechanisms (as opposed to unfolding rates and thus protein stability) may be near on the horizon.

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.