No abstract

Steady-state and time-resolved fluorescence spectroscopy was used to follow the local and global changes in structure and dynamics during chemical and thermal denaturation of unlabeled human serum albumin (HSA) and HSA with an acrylodan moiety bound to Cys34. Acrylodan fluorescence was monitored to obtain information about unfolding processes in domain I, and the emission of the Trp residue at position 214 was used to examine domain II. In addition, Trp-to-acrylodan resonance energy transfer was examined to probe interdomain spatial relationships during unfolding. Increasing the temperature to less than 50 degrees C or adding less than 1.0 M GdHCl resulted in an initial, reversible separation of domains I and II. Denaturation by heating to 70 degrees C or by adding 2.0 M GdHCl resulted in irreversible unfolding of domain II. Further denaturation of HSA by either method resulted in irreversible unfolding of domain I. These results clearly demonstrate that HSA unfolds by a pathway involving at least three distinct steps. The low detection limits and high information content of dual probe fluorescence should allow this technique to be used to study the unfolding behavior of entrapped or immobilized HSA.

DNA enzymes are single-stranded DNA molecules with catalytic capabilities that are isolated from random-sequence DNA libraries by "in vitro selection". This new class of catalytic biomolecules has the potential of being used as unique molecular tools in a variety of innovative applications. Here we describe the creation and characterization of an RNA-cleaving autocatalytic DNA, DEC22-18, that uniquely links chemical catalysis with real-time fluorescence signaling capability in the same molecule. A trans-acting DNA molecule, DET22-18, was also developed from DEC22-18 that behaves as a true enzyme with a k(cat) of approximately 7 min(-1)-a rate constant that is the second largest ever reported for a DNA enzyme. It cleaves a chimeric RNA/DNA substrate at the lone RNA linkage surrounded by a closely spaced fluorophore-quencher pair-a unique structure that permits the synchronization of the chemical cleavage with fluorescence signaling. DET22-18 has a stem-loop structure and can be conjugated with DNA aptamers to form allosteric deoxyribozyme biosensors.

A reagentless bioactive paper-based solid-phase biosensor was developed for detection of acetylcholinesterase (AChE) inhibitors, including organophosphate pesticides. The assay strip is composed of a paper support (1 x 10 cm), onto which AChE and a chromogenic substrate, indophenyl acetate (IPA), were entrapped using biocompatible sol-gel derived silica inks in two different zones (e.g., sensing and substrate zones). The assay protocol involves first introducing the sample to the sensing zone via lateral flow of a pesticide-containing solution. Following an incubation period, the opposite end of the paper support is placed into distilled deionized water (ddH(2)O) to allow lateral flow in the opposite direction to move paper-bound IPA to the sensing area to initiate enzyme catalyzed hydrolysis of the substrate, causing a yellow-to-blue color change. The modified sensor is able to detect pesticides without the use of any external reagents with excellent detection limits (bendiocarb approximately 1 nM; carbaryl approximately 10 nM; paraoxon approximately 1 nM; malathion approximately 10 nM) and rapid response times (approximately 5 min). The sensor strip showed negligible matrix effects in detection of pesticides in spiked milk and apple juice samples. Bioactive paper-based assays on pesticide residues collected from food samples showed good agreement with a conventional mass spectrometric assay method. The bioactive paper assay should, therefore, be suitable for rapid screening of trace levels of organophosphate and carbamate pesticides in environmental and food samples.

We demonstrate a novel approach for rapid, selective, and sensitive detection of heavy metals using a solid-phase bioactive lab-on-paper sensor that is inkjet printed with sol-gel entrapped reagents to allow colorimetric visualization of the enzymatic activity of β-galactosidase (B-GAL). The bioactive paper assay is able to detect a range of heavy metals, either alone or as mixtures, in as little as 10 min, with detection limits as follows: Hg(II) = 0.001 ppm; Ag(I) = 0.002 ppm, Cu(II) = 0.020 ppm; Cd(II) = 0.020 ppm; Pb(II) = 0.140 ppm; Cr(VI) = 0.150 ppm; Ni(II) = 0.230 ppm. The paper-based assay was immune to interferences from nontoxic metal ions such as Na(+) or K(+), could be used to detect heavy metals that were spiked into tap water or lake water, and provided quantitative data that was in agreement with values obtained by atomic absorption. With the incorporation of standard chromogenic metal sensing reagents into a multiplexed bioactive paper sensor, it was possible to identify specific metals in mixtures, albeit with much lower detection limits than were obtained with the enzymatic assay. The paper-based sensor should be valuable for rapid, on-site screening of trace levels of heavy metals in resource limited areas and developing countries.