Towards maintenance-free biosensors for hundreds of bind/release cycles.

A single aptamer bioreceptor layer was formed using a common streptavidin-biotin immobilization strategy and employed for 100-365 bind/release cycles. Chemically induced aptamer unfolding and release of its bound target was accomplished using alkaline solutions with high salt concentrations or deionized (DI) water. The use of DI water scavenged from the ambient atmosphere represents a first step towards maintenance-free biosensors that do not require the storage of liquid reagents. The aptamer binding affinity was determined by surface plasmon resonance and found to be almost constant over 100-365 bind/release cycles with a variation of less than 5% relative standard deviation. This reversible operation of biosensors based on immobilized aptamers without storage of liquid reagents introduces a conceptually new perspective in biosensing. Such new biosensing capability will be important for distributed sensor networks, sensors in resource-limited settings, and wearable sensor applications.

Selective sensing of vapors of similar dielectric constants using peptide-capped gold nanoparticles on individual multivariable transducers.

Peptide-capped AYSSGAPPMPPF gold nanoparticles were demonstrated for highly selective chemical vapor sensing using individual multivariable inductor-capacitor-resistor (LCR) resonators. Their multivariable response was achieved by measuring their resonance impedance spectra followed by multivariate spectral analysis. Detection of model toxic vapors and chemical agent simulants, such as acetonitrile, dichloromethane and methyl salicylate, was performed. Dichloromethane (dielectric constant εr = 9.1) and methyl salicylate (εr = 9.0) were discriminated using a single sensor. These sensing materials coupled to multivariable transducers can provide numerous opportunities for tailoring the vapor response selectivity based on the diversity of the amino acid composition of the peptides, and by the modulation of the nature of peptide-nanoparticle interactions through designed combinations of hydrophobic and hydrophilic amino acids.

Wireless sensors and sensor networks for homeland security applications.

New sensor technologies for homeland security applications must meet the key requirements of sensitivity to detect agents below risk levels, selectivity to provide minimal false-alarm rates, and response speed to operate in high throughput environments, such as airports, sea ports, and other public places. Chemical detection using existing sensor systems is facing a major challenge of selectivity. In this review, we provide a brief summary of chemical threats of homeland security importance; focus in detail on modern concepts in chemical sensing; examine the origins of the most significant unmet needs in existing chemical sensors; and, analyze opportunities, specific requirements, and challenges for wireless chemical sensors and wireless sensor networks (WSNs). We further review a new approach for selective chemical sensing that involves the combination of a sensing material that has different response mechanisms to different species of interest, with a transducer that has a multi-variable signal-transduction ability. This new selective chemical-sensing approach was realized using an attractive ubiquitous platform of battery-free passive radio-frequency identification (RFID) tags adapted for chemical sensing. We illustrate the performance of RFID sensors developed in measurements of toxic industrial materials, humidity-independent detection of toxic vapors, and detection of chemical-agent simulants, explosives, and strong oxidizers.

Battery-free radio frequency identification (RFID) sensors for food quality and safety.

Market demands for new sensors for food quality and safety stimulate the development of new sensing technologies that can provide an unobtrusive sensor form, battery-free operation, and minimal sensor cost. Intelligent labeling of food products to indicate and report their freshness and other conditions is one important possible application of such new sensors. This study applied passive (battery-free) radio frequency identification (RFID) sensors for the highly sensitive and selective detection of food freshness and bacterial growth. In these sensors, the electric field generated in the RFID sensor antenna extends from the plane of the RFID sensor and is affected by the ambient environment, providing the opportunity for sensing. This environment may be in the form of a food sample within the electric field of the sensing region or a sensing film deposited onto the sensor antenna. Examples of applications include monitoring of milk freshness, fish freshness, and bacterial growth in a solution. Unlike other food freshness monitoring approaches that require a thin film battery for operation of an RFID sensor and fabrication of custom-made sensors, the passive RFID sensing approach developed here combines the advantages of both battery-free and cost-effective sensor design and offers response selectivity that is impossible to achieve with other individual sensors.

DNAzyme catalytic beacon sensors that resist temperature-dependent variations.

The temperature-dependent variability of a Pb2+-specific 8-17E DNAzyme catalytic beacon sensor has been addressed through the introduction of mismatches in the DNAzyme, and the resulting sensors resist temperature-dependent variations from 4 to 30 degrees C.

Activity, folding and Z-DNA formation of the 8-17 DNAzyme in the presence of monovalent ions.

The effect of monovalent ions on both the reactivity and global folding of the 8-17 DNAzyme is investigated, and the results are compared with those of the hammerhead ribozyme, which has similar size and secondary structure. In contrast to the hammerhead ribozyme, the 8-17 DNAzyme activity is not detectable in the presence of 4 M K(+), Rb(+), or Cs(+) or in the presence of 80 mM, [Co(NH(3))(6)](3+). Only 4 M Li(+), NH(4)(+) and, to a lesser extent, Na(+) conferred detectable activity. The observed rate constants (k(obs) approximately 10(-3) min(-1) for Li(+) and NH(4)(+)) are approximately 1000-fold lower than that in the presence of 10 mM Mg(2+), and approximately 200,000-fold slower than that in the presence of 100 microM Pb(2+). Since the hammerhead ribozyme displays monovalent ion-dependent activity that is often within approximately 10-fold of divalent metal ion-dependent activity, these results suggest that the 8-17 DNAzyme, obtained by in vitro selections, has evolved to have a more stringent divalent metal ion requirement for high activity as compared to the naturally occurring ribozymes, making the 8-17 DNAzyme an excellent choice as a Pb(2+) sensor with high selectivity. In contrast to the activity data, folding was observed in the presence of all the monovalent ions investigated, although those monovalent ions that do not support DNAzyme activity have weaker binding affinity (K(d) approximately 0.35 M for Rb(+) and Cs(+)), while those that confer DNAzyme activity possess stronger affinity (K(d) approximately 0.22 M for Li(+), Na(+) and NH(4)(+)). In addition, a correlation between metal ion charge density, binding affinity and enzyme activity was found among mono- and divalent metal ions except Pb(2+); higher charge density resulted in stronger affinity and higher activity, suggesting that the observed folding and activity is at least partially due to electrostatic interactions between ions and the DNAzyme. Finally, circular dichroism (CD) study has revealed Z-DNA formation with the monovalent metal ions, Zn(2+) and Mg(2+); the K(d) values obtained using CD were in the same range as those obtained from folding studies using FRET. However, Z-DNA formation was not observed with Pb(2+). These results indicate that Pb(2+)-dependent function follows a different mechanism from the monovalent metal ions and other divalent metal ions; in the presence of latter metal ions, metal-ion dependent folding and structural changes, including formation of Z-DNA, play an important role in the catalytic function of the 8-17 DNAzyme.

Probing metal binding in the 8-17 DNAzyme by TbIII luminescence spectroscopy.

Metal-dependent cleavage activities of the 8-17 DNAzyme were found to be inhibited by Tb(III) ions, and the apparent inhibition constant in the presence of 100 microM of Zn(II) was measured to be 3.3+/-0.3 microM. The apparent inhibition constants increased linearly with increasing Zn(II) concentration, and the inhibition effect could be fully rescued with addition of active metal ions, indicating that Tb(III) is a competitive inhibitor and that the effect is completely reversible. The sensitized Tb(III) luminescence at 543 nm was dramatically enhanced when Tb(III) was added to the DNAzyme-substrate complex. With an inactive DNAzyme in which the GT wobble pair was replaced with a GC Watson-Crick base pair, the luminescence enhancement was slightly decreased. In addition, when the DNAzyme strand was replaced with a complete complementary strand to the substrate, no significant luminescence enhancement was observed. These observations suggest that Tb(III) may bind to an unpaired region of the DNAzyme, with the GT wobble pair playing a role. Luminescence lifetime measurements in D(2)O and H(2)O suggested that Tb(III) bound to DNAzyme is coordinated by 6.7+/-0.2 water molecules and two or three functional groups from the DNAzyme. Divalent metal ions competed for the Tb(III) binding site(s) in the order Co(II)>Zn(II)>Mn(II)>Pb(II)>Ca(II) approximately Mg(II). This order closely follows the order of DNAzyme activity, with the exception of Pb(II). These results indicate that Pb(II), the most active metal ion, competes for Tb(III) binding differently from other metal ions such as Zn(II), suggesting that Pb(II) may bind to a different site from that for the other metal ions including Zn(II) and Tb(III).

Metal-dependent global folding and activity of the 8-17 DNAzyme studied by fluorescence resonance energy transfer.

The 8-17 DNAzyme is a DNA metalloenzyme catalyzing RNA transesterification in the presence of divalent metal ions, with activity following the order Pb2+ >> Zn2+ >>Mg2+. Since the DNAzyme has been used as a metal ion sensor, its metal-induced global folding was studied by fluorescence resonance energy transfer (FRET) by labeling the three stems of the DNAzyme with the Cy3/Cy5 FRET pair two stems at a time in order to gain deeper insight into the role of different metal ions in its structure and function. FRET results indicated that, in the presence of Zn2+ and Mg2+, the DNAzyme folds into a compact structure, stem III approaching a configuration defined by stems I and II without changing the angle between stems I and II. Correlations between metal-induced folding and activity were also studied. For Zn2+ and Mg2+, the metal ion with higher affinity for the DNAzyme in global folding (Kd(Zn) = 52.6 microM and Kd(Mg) = 1.36 mM) also displays higher affinity in activity (Kd(Zn) = 1.15 mM and Kd(Mg) = 53 mM) under the same conditions. Global folding was saturated at much lower concentrations of Zn2+ and Mg2+ than the cleavage activities, indicating the global folding of the DNAzyme occurs before the cleavage activity for those metal ions. Surprisingly, no Pb2+-dependent global folding was observed. These results suggest that for Pb2+ global folding of the DNAzyme may not be a necessary step in its function, which may contribute to the DNAzyme having the highest activity in the presence of Pb2+.

Kinetics of two-electron oxidations by the compound I derivative of chloroperoxidase, a model for cytochrome P450 oxidants.

[structure: see text] Rate constants for two-electron oxidation reactions of Compound I from chloroperoxidase (CPO) with a variety of substrates were measured by stopped-flow kinetic techniques. The thiolate ligand of CPO Compound I activates the iron-oxo species with the result that oxidation reactions are 2 to 3 orders of magnitude faster than oxidations by model iron(IV)-oxo porphyrin radical cations containing weaker binding counterions.

Spectroscopic characterizations of bridging cysteine ligand variants of an engineered Cu2(Scys)2 CuA azurin.

Bridging cysteine ligands of the Cu(A) center in an engineered Cu(A) azurin were replaced with serine, and the variants (Cys116Ser and Cys112Ser Cu(A) azurin) were characterized by mass spectrometry, as well as UV-vis and electron paramagnetic resonance (EPR) spectroscopic techniques. The replacements resulted in dramatically perturbed spectroscopic properties, indicating that the cysteines play a critical role in maintaining the structural integrity of the Cu center. The replacements at different cysteine residues resulted in different perturbations, even though the two cysteines are geometrically symmetrical in the primary coordination sphere with respect to the two copper ions. The Cys112Ser variant contains two distinct type 2 copper centers, while the Cys116Ser variant has one type 1 copper center with slight tetragonal distortion. Both the UV-vis and EPR spectra of the Cys116Ser variant change with pH, and the pK(a) of the transition is 6.0. A type 1 copper EPR spectrum with A(||) = 26 G was obtained at pH 7.0, while a type 2 copper EPR spectrum with A(||) = 140 G was found at pH 5.0. Interestingly, lowering the temperature from 290 to 85 K resulted in conversion of the Cys116Ser variant from a type 1 copper center to a type 2 copper center, suggesting rearrangement of the ligand around the copper or binding of an exogenous ligand at low temperature. This difference in mutation effects at different cysteines may be due to different constraints exerted on the two cysteines by hydrogen-bonding patterns in the ligand loop.