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Ratiometric fluorescence immunoassay is known as a spectrofluorimetric variant for sensitive and quantitative analyses of biomarkers. However, there are few comparative studies between it and the traditional, single-wavelength-based immunoassay. In this work, a ratiometric fluorescence immunoassay was designed via the fluorescence resonance energy transfer (FRET) in a system comprising MnO2 nanosheets, o-phenylenediamine (OPD), and fluorescent carbon nanodots (FCNs). The MnO2 nanosheet oxidizes OPD, producing a fluorescence peak at 580 nm, while FCNs exhibit a fluorescence peak at 490 nm. Under the effect of FRET, the fluorescence at 580 nm increases significantly while the fluorescence at 490 nm is reduced. The FRET effect was confirmed by our fluorescence lifetime measurements. We optimized the experimental conditions that include solution pH, OPD concentration, and reaction time. By electrostatically coating the MnO2 nanosheets with antibody molecules and using the resultant materials as a detection probe, the ratiometric fluorescence sensing was extended to the detection of alpha fetoprotein (AFP) in human serum samples on a 96-well titer plate. Our ratiometric fluorescence immunoassay possesses a good dynamic range (from 0.14 to 130.43 pM). The detection limit was estimated to be 0.13 pM (0.009 ng/mL), which is 7.6-fold lower than that of the single-wavelength-based immunoassay. The measurement results of serum samples from healthy and patient donors are highly comparable to those measured by chemiluminescence for the same donors, demonstrating the viability for serological assays of important cancer markers.

A variety of compounds, including Ni-, Fe-, and Co-containing layered double hydroxides (LDHs), have been explored as catalysts for the oxygen evolution reaction (OER). However, few can meet the in...

Transition metal borides are one of the most promising electrocatalysts for oxygen evolution reaction (OER), but the precise role of boron is still not understood. Herein, we demonstrate experimentally and computationally that boron facilitates OER by modulating the interaction energies of the reaction intermediates. Through a simple chemical reduction in an externally applied magnetic field, one-dimensional (1D) Fe2B nanowires (NWs) can be directly deposited onto a three-dimensional (3D) nickel foam (NF). OER in alkaline solution converts interconnected Fe2B NWs to a thicker and larger network of metaborate- and FeOOH-covered Fe2B NWs. The Fe2B NWs/NF-based catalyst lowers the overpotential to 276 mV at 10 mA cm−2 (normalized to the electrochemical surface area). This intrinsic catalytic activity ranks top among the transition-metal-based compounds. This catalyst is highly stable, showing only 6 mV change in overpotential over continuous testing for 11 h. Such high efficiency is mainly attributed to the catalyst's unique electronic structure, accelerated charge transport, and hydrophilic surface. The remarkable stability stems from the increase in corrosion resistance by the metaborate species in the catalyst.

Despite its simplicity and specificity, enzyme-linked immunosorbent assay (ELISA) requires conjugation of the enzyme to an antibody and preservation of enzymatic activity during storage and assay. ...

The aberrant autoxidation of norepinephrine (NE) in the presence of oxygen, which is accelerated by Fe(III), has been linked to the pathogenesis of the Parkinson's disease (PD). Adenosine triphosphate (ATP), as a neurotransmitter whose release can be stimulated by tissue damage and oxidative stress, is co-stored and often co-released with NE in presynaptic terminals. We have shown previously that ATP inhibits the iron-catalyzed dopamine oxidation, thereby decreasing the production of certain neurotoxins such as 6-hydroxydopamine. Whether ATP plays a similar role in Fe(III)-catalyzed NE oxidation and how it maintains the NE stability have not been investigated. Here, we studied the coordination in a ternary complex among NE, Fe(III), and ATP, and found that Fe(III) is coordinated as a octahedral center by NE and ATP. Voltammetry and mass spectrometry were employed to examine this ternary complex's modulation of the NE autoxidation. NE-Fe(III)-ATP plays a protective role to modulate the autoxidation and Fe(III)-catalyzed oxidation of NE. The ternary complex can be detected in the substantia nigra (SN), locus coeruleus (LC), and striatum regions of C57BL/6 wild-type mice. In contrast, the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse brains displayed a significant decrease of the ternary complex in the SN region and an increase in the LC and striatum areas. We posit that the ternary complex is produced by noradrenergic neurons as a protective regulator against neuronal damage and oxidative stress, contributing to the lower vulnerability of LC neurons with respect to that of SN neurons.

A polythiophene-based solar cell (PTSC) is constructed by photoelectrochemically polymerizing thiophene onto an ultrathin compact TiO2 layer (150 nm thick) covered with a sub-monolayer of tethered 3-{5-[ N, N-bis(4-diphenylamino)phenyl]thieno[3,2- b]thiophen-2-yl}-2-cyano-acrylic acid dye (ca. 10% coverage). The influence of morphology and thickness of the PT film on the photocurrent generated by the PTSC was investigated. With a 270 nm thick PT film and 2,2',7,7'-tetrakis( N, N-di(4-methoxyphenyl)amino)-9,9'-spirobifluorene serving as the hole-transport material, the PTSC exhibited a short-circuit current density JSC of 12.90 ± 0.63 mA/cm2, an open-circuit voltage VOC of 0.81 ± 0.01 V, and a fill factor of 0.72 ± 0.01. The high conversion efficiency (7.52 ± 0.58%) of the PTSC is attributed to the controlled PT growth along the ordered and spatially accessible dye molecules at the compact TiO2 layer, which facilitates charge transfer, prevents the hole/electron recombination, and simplifies the polymer solar cell construction with a stable and easily processable material.

The Ni2+-histidine (His) chelation yields a more uniform and predicable orientation of immobilized protein molecules than an amine-coupling reaction in surface plasmon resonance (SPR) analyses. However, the gradual dissociation of His-tagged proteins leads to a long and sloped baseline, which adversely affects kinetic studies. Furthermore, as shown in this work for the first time, the strong binding affinity between the histidine-rich Fc domain of immunoglobulin-type antibodies and Ni-nitrilotriacetic acid (NTA) interferes with the kinetic studies of these antibodies and their His-tagged antigens. By performing an amine-coupling reaction immediately after the Ni2+-His chelation, essentially all of the Ni2+-tethered protein molecules can be covalently linked to the carboxyl groups on the underlying carboxymethylated dextran surface. The sequential injections of pH 8.6 phosphate-buffered saline provided additional time to ensure a higher amine coupling efficiency and reverted NHS esters on the protein molecules to carboxyl groups. The application of our method to antibody/antigen interactions is demonstrated with the kinetic analysis of His-tagged t-DARPP protein/anti-t-DARPP interactions. In a separate experiment, the highly efficient immobilization method resulted in a higher immobilization density of His-tagged human carbonic anhydrase (HCA) II, affording accurate kinetic measurements for the binding of 4-carboxybenzenesulfonamide. In addition, the higher HCA II density enhanced the SPR sensitivity, allowing 4-carboxybenzenesulfonamide to be determined with a remarkable detection limit (14 nM).

An enzyme-free titer plate-based colorimetric assay utilizing functionalized mesoporous silica nanoparticles (MSNs) entrapping pH-indicator molecules has been developed. Pores in the silica nanoparticles were functionalized with phenyltrimethyloxysilane so that pH indicator molecules (thymolphthalein or TP in the present case) can be tightly entrapped through π-π conjugation. To detect prostate specific antigen (PSA), the TP-containing MSNs were coated with polyethylenimine (PEI), which favors the attachment of the negatively charged secondary anti-PSA antibody. The entrapped thymolphthalein molecules can be readily released from the pores with a simple addition of alkaline solution. The resultant bifunctional MSNs were used for signal-amplified detection of PSA captured by the primary antibody preimmobilized in the wells of a plate. Our method possesses a wide dynamic range (0.5 to 8000 pg/mL) wherein the adsorption of the bifunctional MSNs obeys a modified Langmuir isotherm. A detection limit (LOD) down to as low as 0.36 pg/mL can be attained. Owing to the size uniformity of the MSNs and the obviation of enzyme molecules employed in the enzyme-linked immunosorbent assay (ELISA), excellent reproducibility (RSD = 1.12%) was achieved. The selective detection of PSA in human serum samples demonstrates the amenability of our method to detect important biomarkers in complex biological samples, whereas the performance of the assay in a titer plate ensures high throughput and obviates the use of expensive instruments. Both of these features are prerequisites for clinical settings wherein a great number of samples need to be analyzed in a timely fashion.

Two six-port injector valves and one selector valve commonly used in flow injection analysis are combined with a surface plasmon resonance (SPR) instrument wherein solutions introduced from the two inlets counter-flow inside the flow cell. The system is versatile as the same or different solutions can be rapidly and repeatedly introduced to the two fluidic channels in series or in parallel. Unlike most commercial SPR instruments employing a single injector valve, solutions separately injected from the two injector valves can be readily exchanged (

Pyrene derivatives are among the most widely used organic fluorescent photoprobes. Many of them are photosensitizers for hole injection. Pyrenes, however, are mostly UV absorbers, limiting their utility for photonic applications. Nitration of pyrene shifts its absorption to the visible region. Conversely, nitration of pyrene that is already derivatized for covalent labeling, produces mixtures of isomers that are challenging to separate. We present a robust procedure for attaining isomerically pure nitropyrenes. NMR analysis provides unequivocal assignments of the regioisomers and of the structures of the disubstituted nitropyrenes. The added substituents negligibly affect the electronic properties of the nitropyrenes. Photoexcited nitropyrenes undergo efficient triplet formation, making them an attractive choice for triplet sensitizers and photooxidants. Hence, facile and reliable preparation of disubstituted nitropyrenes provides venues for exploring their electronic and photonic utility.

Necroptosis has been found to be involved in the pathogenesis of some lung diseases, but its role in hyperoxic acute lung injury (HALI) is still unclear. This study aimed to investigate contribution of necroptosis to the pathogenesis of HALI induced by hyperbaric hyperoxia exposure in a rat model. Rats were divided into control group, HALI group, Nec-1 (necroptosis inhibitor) group and edaravone group. Rats were exposed to pure oxygen at 250 kPa for 6 h to induce HALI. At 30 min before hyperoxia exposure, rats were intraperitoneally injected with Nec-1 or edaravone, and sacrificed at 24 h after hyperoxia exposure. Lung injury was evaluated by histology, lung water to dry ratio (W/D) and bronchoalveolar lavage fluid (BALF) biochemistry; the serum and plasma oxidative stress, expression of RIP1, RIP3 and MLKL, and interaction between RIP1 and RIP3 were determined. Results showed hyperoxia exposure significantly caused damage to lung and increased necroptotic cells and the expression of RIP1, RIP3 and MLKL. Edaravone pre-treatment not only inhibited the oxidative stress in HALI, but also reduced necroptotic cells, decreased the expression of RIP1, RIP3 and MLKL and improved lung pathology. Nec-1 pretreatment inhibited necroptosis and improved lung pathology, but had little influence on oxidative stress. This study suggests hyperoxia exposure induces oxidative stress may activate necroptosis, involving in the pathology of HALI, and strategies targeting necroptosis may become promising treatments for HALI.

Solar cells constructed with well-ordered polythiophene (PT) or other π-conjugated polymer films are known to yield better photovoltaic characteristics. We recently constructed a PT-based solar cell (PTSC) wherein the photoactive PT film was grown photoelectrochemically along a dye anchor, 3-{5-[N,N-bis(4-diphenylamino)phenyl]thieno[3,2-b]thiophen-2-yl}-2-cyano-acrylic acid (C207) pre-adsorbed onto a compact TiO2 layer. In an effort to understand the effects of other important factors contributing to the PTSC performance, we compared ex-situ and in-situ doping of ordered PT films with three different alkylammonium cations, tetrabutylammonium (TBA+), tetraethylammonium (TEA+), and tetramethylammonium (TMA+). The PT films that have undergone such ex- and in-situ doping both show higher open-circuit voltage (Voc) and short-circuit current density (Jsc) than their pristine counterparts. The optimal conversion efficiency (η) of 7.53 ± 0.58% was achieved via in-situ PT doping with TMA+. The incident photon-to-electron conversion efficiency (IPCE), electrochemical impedance, and electron lifetime measurements all indicate that in-situ doping is less affected by the cation size and can lead to a higher doping level than the ex-situ doping method. The IPCE also benefits from the use of a thin TiO2 layer, as more light reaches the PT film to generate photohole/electron pairs. With the combined use of a thin layer of TiO2 and an ordered PT film containing a large number of dopant ions, the photohole/electron recombination is significantly suppressed.

Although gold nanorods (GNRs) have been prepared with a wide range of methods for their uses as novel diagnostic and therapeutic agents, the synthesis of monodispersed GNRs with high yields and size tunability still requires further improvements. We report on a simple one-pot method for preparing highly monodispersed GNRs using phenols (e.g., hydroquinone, 1,2,3-trihydroxybenzene, and 1,2,4-trihydroxybenzene) as the reducing agent and NaBH4 as the initiating reactant. Fine-tuning of the LSPR peak position of phenols-reduced GNRs from 550 to 1150 nm is accomplished by regulating the silver ion concentrations. The size of GNRs produced via phenols reduction can also be controlled by changing the NaBH4 concentration. By systematically optimizing the concentrations of the reagents involved in the one-pot synthesis of GNRs, the yield (in many cases exceeding 90%) is significantly higher than that prepared with the commonly used reductant (e.g., ascorbic acid). The improved efficiency and controllability cut do...

Cleavage of amyloid precursor protein (APP) by the β-site APP cleaving enzyme 1 (BACE1) is a key step in the formation of amyloid beta (Aβ) peptide, the main component of amyloid plaques in Alzheimer's disease (AD). Suppression of BACE1 activity has thus become an efficient way for the treatment of AD. In this study, BACE1 in the absence or presence of BACE1 inhibitors was exposed to the biotinylated peptide substrate-modified electrode. This step was followed by the attachment of ferrocene (Fc)-capped gold nanoparticle/streptavidin conjugates. Due to the blockage of the BACE1 activity by select inhibitors, well-defined voltammetric peaks of high signal intensity were obtained. However, featureless voltammogram was obtained upon initiating the cleavage reaction. The proposed method is simple, sensitive, and suitable for monitoring of BACE1 activity and screening of BACE1 inhibitors.

Parkinson's disease (PD) is characterized by the progressive degeneration of dopaminergic cells, which implicates a role of dopamine (DA) in the etiology of PD. A possible DA degradation pathway is the Fe(III)-catalyzed oxidation of DA by oxygen, which produces neuronal toxins as side products. We investigated how ATP, an abundant and ubiquitous molecule in cellular milieu, affects the catalytic oxidation reaction of dopamine. For the first time, a unique, highly stable DA-Fe(III)-ATP ternary complex was formed and characterized in vitro. ATP as a ligand shifts the catecholate-Fe(III) ligand metal charge transfer (LMCT) band to a longer wavelength and the redox potentials of both DA and the Fe(III) center in the ternary complex. Remarkably, the additional ligation by ATP was found to significantly reverse the catalytic effect of the Fe(III) center on the DA oxidation. The reversal is attributed to the full occupation of the Fe(III) coordination sites by ATP and DA, which blocks O2 from accessing the Fe(III) center and its further reaction with DA. The biological relevance of this complex is strongly implicated by the identification of the ternary complex in the substantia nigra of rat brain and its attenuation of cytotoxicity of the Fe(III)-DA complex. Since ATP deficiency accompanies PD and neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) induced PD, deficiency of ATP and the resultant impairment toward the inhibition of the Fe(III)-catalyzed DA oxidation may contribute to the pathogenesis of PD. Our finding provides new insight into the pathways of DA oxidation and its relationship with synaptic activity.

Aggregates of amyloid beta (Aβ) peptides are believed to be responsible for the neuropathology of Alzheimer’s disease. In this work, ferrocene (Fc) is attached to the aggregating core of the Aβ peptides, KLVFFAE. Inhibition of Fc-KLVFFAE aggregation by curcumin, a natural compound, was monitored by HPLC-electrochemical detection (HPLC-EC). The Fc oxidation current is dependent on the incubation condition and curcumin can retain Fc-KLVFFAE in its monomeric form. We demonstrate that tagging Fc to KLVFFAE affords a cost-effective and electroactive mimicry of Aβ(142) and HPLC-EC is suitable for sensitive, reproducible, and facile screening of drugs for inhibiting the aggregation of Aβ peptides.

One of the hurdles in the discovery of antibiotics is the difficulty of linking antibacterial compounds to their cellular targets. Our laboratory has employed a genome-wide approach of over-expressing essential genes in order to identify cellular targets of antibacterial inhibitors. Our objective in this project was to develop and validate a more sensitive disk diffusion based platform of target identification (Target Identification Platform for Antibacterials version 2; TIPA II) using a collection of cell clones in an Escherichia coli mutant (AS19) host with increased outer membrane permeability. Five known antibiotics/inhibitors and 28 boron heterocycles were tested by TIPA II assay, in conjunction with the original assay TIPA. The TIPA II was more sensitive than TIPA because eight boron heterocycles previously found to be inactive to AG1 cells in TIPA assays exhibited activity to AS19 cells. For 15 boron heterocycles, resistant colonies were observed within the zones of inhibition only on the inducing plates in TIPA II assays. DNA sequencing confirmed that resistant clones harbor plasmids with fabI gene as insert, indicating that these boron heterocycles all target enoyl ACP reductase. Additionally, cell-based assays and dose response curved obtained indicated that for two boron heterocycle inhibitors, the fabI cell clone in AG1 (wild-type) host cells exhibited at least 11 fold more resistance under induced conditions than under non-induced conditions. Moreover, TIPA II also identified cellular targets of known antibacterial inhibitors triclosan, phosphomycin, trimethoprim, diazaborine and thiolactomycin, further validating the utility of the new system.

MicroRNAs (miRNAs), acting as oncogenes or tumor suppressors in humans, play a key role in regulating gene expression and are believed to be important for developing novel therapeutic treatments and clinical prognoses. Due to their short lengths (17-25 nucleotides) and extremely low concentrations (typicallypicomolar) in biological samples, quantification of miRNAs has been challenging to conventional biochemical methods, such as Northern blotting, microarray, and quantitative polymerase chain reaction (qPCR). In this work, a biotinylated miRNA (biotin-miRNA) whose sequence is the same as that of a miRNA target is introduced into samples of interest and allowed to compete with the miRNA target for the oligonucleotide (ODN) probe preimmobilized onto an electrode. Voltammetric quantification of the miRNA target was accomplished after complexation of the biotin-miRNA with ferrocene (Fc)-capped gold nanoparticle/streptavidin conjugates. The Fc oxidation current was found to be inversely proportional to the concentration of target miRNA between 10 fM and 2.0 pM. The method is highly reproducible (relative standard deviation (RSD)5%), regenerable (at least 8 regeneration/assay cycles without discernible signal decrease), and selective (with sequence specificity down to a single nucleotide mismatch). The low detection levels (10 fM or 0.1 attomoles of miRNA in a 10 μL solution) allow the direct quantification of miRNA-182, a marker correlated to the progression of glioma in patients, to be performed in serum samples without sample pretreatment and RNA extraction and enrichment. The concentration of miRNA-182 in glioma patients was found to be 3.1 times as high as that in healthy persons, a conclusion in excellent agreement with a separate qPCR measurement of the expression level. The obviations of the requirement of an internal reference in qPCR, simplicity, and cost-effectiveness are other additional advantages of this method for detection of nucleic acids in clinical samples.

Carboxymethylated dextran (CMD)-coated magnetic iron oxide nanoparticles (MNPs) were synthesized using a co-precipitation method. Compared to neutral dextran coated MNPs, the CMD coating provides good dispersity and colloidal stability to the CMD-MNPs. In particular, the carboxyl groups on the CMD can be readily activated for covalent attachment of antibody molecules. The superparamagnetic property of the antibody-covered CMD-MNPs enables the captured antigen to be separated from the sample solution and CMD coating significantly reduces the nonspecific binding of the nanoparticles. Regeneration of the anti-BSA antibody-covered CMD-MNPs with NaOH does not significantly decrease the antibody activity, and the repeated magnetic separation and washing steps cause only small loss of the starting materials. The method was found to be highly reproducible (RSDs for BSA adsorption and desorption are between 0.78% and 5.1%). The anti-BSA antibody-covered CMD-MNPs possess good selectivity and are able to capture protein antigens from real samples.

The oligopeptide transporter PepT1 expressed in inflamed colonic epithelial cells transports small bacterial peptides, such as muramyl dipeptide (MDP) and l-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP) into cells. The innate immune system uses various proteins to sense pathogen-associated molecular patterns. Nucleotide-binding oligomerization domain (NOD)-like receptors of which there are more than 20 related family members are present in the cytosol and recognize intracellular ligands. NOD proteins mediate NF-κB activation via receptor-interacting serine/threonine-protein kinase 2 (RICK or RIPK). The specific ligands for some NOD-like receptors have been identified. NOD type 1 (NOD1) is activated by peptides that contain a diaminophilic acid, such as the PepT1 substrate Tri-DAP. In other words, PepT1 transport activity plays an important role in controlling intracellular loading of ligands for NOD1 in turn determining the activation level of downstream inflammatory pathways. However, no direct interaction between Tri-DAP and NOD1 has been identified. In the present work, surface plasmon resonance and atomic force microscopy experiments showed direct binding between NOD1 and Tri-DAP with a K(d) value of 34.5 μM. In contrast, no significant binding was evident between muramyl dipeptide and NOD1. Furthermore, leucine-rich region (LRR)-truncated NOD1 did not interact with Tri-DAP, indicating that Tri-DAP interacts with the LRR domain of NOD1. Next, we examined binding between RICK and NOD1 proteins and found that such binding was significant with a K(d) value of 4.13 μM. However, NOD1/RICK binding was of higher affinity (K(d) of 3.26 μM) when NOD1 was prebound to Tri-DAP. Furthermore, RICK phosphorylation activity was increased when NOD was prebound to Tri-DAP. In conclusion, we have shown that Tri-DAP interacts directly with the LRR domain of NOD1 and consequently increases RICK/NOD1 association and RICK phosphorylation activity.

A simple, rapid, and sensitive determination of total free thiol groups in biological samples using cerium (IV) as a fluorescence probe is reported. The protocol is based on the oxidation of thiols by Ce(IV) and the formation the Ce(III) disulfide complex, which gives a fluorescence enhancement of Ce(III) at 352 nm. Using glutathione (GSH) and cysteine as model compounds, incubation with Ce(IV) at 25 °C for 6 min results in fluorescence, whose intensity is proportional to the thiol concentration in the range of 1.00–160 nM. The detection limits for GSH and cysteine are 0.05 and 0.08 nM, respectively. Other common metal ions and amino acids have little interference to the thiol detection. Cu(II) was used as a fluorescence quencher to eliminate potential interference from tryptophan. The method has been successfully applied to assays of free thiol contents in pig liver tissue samples, with a RSD lower than 2.5% and recovery between 100.6% and 102.3%.

This paper describes the construction of a mixed monolayer of ferrocenylalkanethiol and encapsulated horseradish peroxidase (HRP) at a gold electrode for amperometric detection of H(2)O(2) at trace levels. By tuning the alkanethiol chain lengths that tether the HRP enzyme and the ferrocenylalkanethiol (FcC(11)SH) mediator, facile electron transfer between FcC(11)SH and HRP can be achieved. Unlike most HRP-based electrochemical sensors, which rely on HRP-facilitated H(2)O(2) reduction (to H(2)O), the electrocatalytic current is resulted from an HRP-catalyzed oxidation reaction of H(2)O(2) (to O(2)). Upon optimizing other experimental conditions (surface coverage ratio, pH, and flow rate), the electrocatalytic reaction proceeding at the electrode was used to attain a low amperometric detection level (0.64 nM) and a dynamic range spanning over 3 orders of magnitude. Not only does the thin hydrophilic porous HRP capsule allow facile electron transfer, it also enables H(2)O(2) to permeate. More significantly, the enzymatic activity of the encapsulated HRP is retained for a considerably longer period (3 weeks) than naked HRP molecules attached to an electrode or those wired to a redox polymer thin film. By comparing to electrodes modified with denatured HRP that are subsequently encapsulated or embedded in a poly-L-lysine matrix, it is concluded that the encapsulation has significantly preserved the native structure of HRP and therefore its enzymatic activity. The electrode covered with FcC(11)SH and encapsulated HRP is shown to be capable of rapidly and reproducibly detecting H(2)O(2) present in complex sample media.

Glutathione (GSH) tagged with a ferrocene (Fc) label at its C-terminal was synthesized via coupling ferrocenyl amine to glutathione using o-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium (HBTU)/1-hydroxybenzotrizole (HOBt). The presence of Fc yielded well defined voltammetric signals, rendering the Fc-tagged GSH (GSH-Fc) suitable for electrochemical studies of GSH binding to other biological species. The interaction of GSH-Fc with bovine serum albumin (BSA) was investigated, and a binding ratio of 1.41±0.06 (GSH-Fc/BSA) and an affinity constant Ka of 6.53±2.01×106 M−1 were determined. These results compare well with those measured by fluorescence using untagged GSH, suggesting that the attachment of Fc to GSH does not significantly perturb the GSH structure and binding behavior. By contrasting the binding behavior to several compounds that are known to conjugate to different domains of BSA, the voltammetric study confirmed that GSH-Fc binds at subdomain IIA of BSA with high affinity. The versatility of GSH-Fc for studying GSH binding to surface-confined proteins was also demonstrated with the GSH binding to electroinactive Zn-metallothionein (Zn7-MT) through hydrogen binding at the region between the Zn7-MT α and β domains.

The interaction of amyloid-beta (Abeta) and redox-active metals, two important biomarkers present in the senile plaques of Alzheimer's disease (AD) brain, has been suggested to enhance the Abeta aggregation or facilitate the generation of reactive oxygen species (ROS). This study investigates the nature of the interaction between the metal-binding domain of Abeta, viz., Abeta(1-16), and the Fe(III) or Fe(II) complex with nitrilotriacetic acid (NTA). Using electrospray ionization mass spectrometry (ESI-MS), the formation of a ternary complex of Abeta(1-16), Fe(III), and NTA with a stoichiometry of 1:1:1 was identified. MS also revealed that the NTA moiety can be detached via collision-induced dissociation. The cumulative dissociation constants of both Abeta-Fe(III)-NTA and Abeta-Fe(II)-NTA complexes were deduced to be 6.3 x 10(-21) and 5.0 x 10(-12) M(2), respectively, via measurement of the fluorescence quenching of the sole tyrosine residue on Abeta upon formation of the complex. The redox properties of these two complexes were investigated by cyclic voltammetry. The redox potential of the Abeta-Fe(III)-NTA complex was found to be 0.03 V versus Ag/AgCl, which is negatively shifted by 0.54 V when compared to the redox potential of free Fe(III)/Fe(II). Despite such a large potential modulation, the redox potential of the Abeta-Fe(III)-NTA complex is still sufficiently high for a range of redox reactions with cellular species to occur. The Abeta-Fe(II)-NTA complex electrogenerated from the Abeta-Fe(III)-NTA complex was also found to catalyze the reduction of oxygen to produce H(2)O(2). These findings provide significant insight into the role of iron and Abeta in the development of AD. The binding of iron by Abeta modulates the redox potential to a level at which its redox cycling occurs. In the presence of a biological reductant (antioxidant), redox cycling of iron could disrupt the redox balance within the cellular milieu. As a consequence, not only is ROS continuously produced, but oxygen and biological reductants can also be depleted. A cascade of biological processes can therefore be affected. In addition, the strong binding affinity of Abeta toward Fe(III) and Fe(II) indicates Abeta could compete for iron against other iron-containing proteins. In particular, its strong affinity for Fe(II), which is 8 orders of magnitude stronger than that of transferrin, would greatly interfere with iron homeostasis.

The preparation and characterization of patterns on atomically flat substrates (Si and Au(1 1 1)) composed of compact, bi- or tetra-layered dipalmitoyl phosphatidylserine (1,2-dipalmitoyl-sn-glycero-3-[phospho- l -serine] sodium salt, DPPS) regions and exposed substrate surfaces are described. Hydration of a monolayer or sub-monolayer of DPPS transferred onto a solid substrate using a Langmuir–Blodgett (LB) trough results in detachment of DPPS molecules weakly adhered to the substrate and their subsequent stacking onto DPPS molecules that are more strongly withheld by the substrate. The pattern and its thickness can be controlled by varying the surface pressure in which DPPS is transferred from the air/water interface in the LB trough and changing the surface properties of the substrates. Time-lapse atomic force microscopic imaging of phospholipase (PLA2) hydrolysis of the DPPS pattern and spectrofluorimetric measurements in the PLA2 solution used to digest the dansyl-DPPS/DPPS pattern reveal that the topmost layer (terrace) on the pattern is compact and defects mainly exist at the edges of the pattern. Moreover, upon extensive PLA2 digestion, a small amount of DPPS molecules remains at the substrate and the thickness of the residue corresponds to that of a bilayer. Quantification of the amount of DPPS molecules remaining at the surface via spectrofluorimetry indicated that very few lipid molecules, if any, were lost during the DPPS monolayer hydration and pattern formation.

Antimicrobial polypeptides, including lysozymes, have membrane perturbing activity and are well-documented effector molecules of innate immunity. In cystic fibrosis, a hereditary disease with frequent lung infection with Pseudomonas aeruginosa, the non-esterified fatty acid DA (docosahexaenoic acid), but not OA (oleic acid), is decreased, and DA supplementation has been shown to improve the clinical condition in these patients. We hypothesized that DA may, either alone or in conjunction with lysozyme, exert antibacterial action against Ps. aeruginosa. We found that DA and lysozyme synergistically inhibit the metabolic activity of Ps. aeruginosa, in contrast with OA. Electron microscopy and equilibrium dialysis suggest that DA accumulates in the bacterial membrane in the presence of lysozyme. Surface plasmon resonance with live bacteria and differential scanning calorimetry studies with bacterial model membranes reveal that, initially, DA facilitates lysozyme incorporation into the membrane, which in turn allows influx of more DA, leading to bacterial cell death. The present study elucidates a molecular basis for the synergistic action of non-esterified fatty acids and antimicrobial polypeptides, which may be dysfunctional in cystic fibrosis.

A novel fluorescent CdS-encapsulated DNA nanocomposite was synthesized via alternate adsorption of Cd(2+) and S(2-) onto the DNA template affixed inside an agarose gel. Confining DNA molecules in the gel matrix reduces the flexibility of the DNA strand, which facilitates the formation of a uniform coating of CdS onto the DNA template. The resultant rod-shaped nanocomposite (40-90 nm in width and 200-300 nm in length) is well dispersed in solution and fluoresces at 330 nm upon excitation at either 228 or 280 nm. The fluorescence is attributed to tiny particles present in the CdS coating. It was found that the fluorescence can be significantly quenched by trace amount of Hg(2+). The high selectivity toward Hg(2+) and the apparent change in the CdS coating upon exposure to Hg(2+) indicate that Hg(2+) has reacted with the CdS coating through formation of the much more insoluble HgS and the bridging S-Hg-S bonds at the surface. The extent of quenching is dependent on the concentration of Hg(2+) in the range of 0.04-13 microM, and a remarkable detection limit (8.6 nM at 30 degrees C and 4.3 nM at 50 degrees C) can be achieved. The feasibility of the method for the analysis of Hg(2+) in a wastewater sample was demonstrated with an excellent relative standard deviation (RSD, 3.4%). The method described herein is simple, selective, and sensitive and obviates the need of extensive sample pretreatment or special instrumentation.

At the air/buffer solution interface the kinetics of adsorption of amyloid beta peptide, Abeta(1-42), whose bulk concentration (submicromolar) is more than 2 orders of magnitude lower than that typically used in other in vitro aggregation studies, has been studied using a Langmuir-Blodgett trough. The pressure-time curves exhibit a lag phase, wherein the surface pressure essentially remains at zero, and a rising phase, corresponding to the Abeta adsorption at the interface. The duration of the lag phase was found to be highly dependent on both the Abeta bulk concentration and the solution temperature. A large activation energy (62.2 +/- 4.1 KJ/mol) was determined and the apparent adsorption rate constant was found to be linearly dependent on the Abeta bulk concentration. Attenuated total reflection-IR spectra of the adsorbed Abeta transferred to a solid substrate and circular dichroism measurements of Abeta in the solution layer near the interface reveal that the natively unstructured Abeta in the bulk undergo a conformation change (folding) to mainly the alpha-helical structure. The results suggest that, prior to the adsorption step, an equilibrium between Abeta conformations is established within the subsurface. The kinetic equation derived from this model confirms that the overall Abeta adsorption is kinetically controlled and the apparent rate constant is proportional to the Abeta bulk concentration. This model also indicates that interfaces such as cell membranes and lipid bilayers may facilitate Abeta aggregation/ fibrillation by providing a thin hydrophobic layer adjacent to the interface for the initial A/beta conformation change (misfolding) and accumulation. Such a preconcentration effect offers a plausible explanation of the fact that Abeta fibrillation occurs in vivo at nanomolar concentrations. Another important biological implication from our work is that Abeta misfolding may occur before its adsorption onto a cell membrane. This general kinetic model should also find applications in adsorption studies of other types of biomolecules whose overall kinetics exhibits a lag phase that is dependent on the bulk concentration of the adsorbate.

Small beta-amyloid (Abeta) 1-42 aggregates are toxic to neurons and may be the primary toxic species in Alzheimer's disease (AD). Methods to reduce the level of Abeta, prevent Abeta aggregation, and eliminate existing Abeta aggregates have been proposed for treatment of AD. A tricyclic pyrone named CP2 is found to prevent cell death associated with Abeta oligomers. We studied the possible mechanisms of neuroprotection by CP2. Surface plasmon resonance spectroscopy shows a direct binding of CP2 with Abeta42 oligomer. Circular dichroism spectroscopy reveals monomeric Abeta42 peptide remains as a random coil/alpha-helix structure in the presence of CP2 over 48 h. Atomic force microscopy studies show CP2 exhibits similar ability to inhibit Abeta42 aggregation as that of Congo red and curcumin. Atomic force microscopy closed-fluid cell study demonstrates that CP2 disaggregates Abeta42 oligomers and protofibrils. CP2 also blocks Abeta fibrillations using a protein quantification method. Treatment of 5x familial Alzheimer's disease mice, a robust Abeta42-producing animal model of AD, with a 2-week course of CP2 resulted in 40% and 50% decreases in non-fibrillar and fibrillar Abeta species, respectively. Our results suggest that CP2 might be beneficial to AD patients by preventing Abeta aggregation and disaggregating existing Abeta oligomers and protofibrils.

Glutathione (GSH), in addition to serving as a redox buffer in cellular environment, has been suggested as a modulator in metal regulation and homeostasis by metallothioneins (MTs). The interactions of MTs with both GSH and its oxidized form GSSG have been shown to govern the direction of metal transfer. Common methods for the determination of zinc release from MTs modulated by GSH/GSSG either involve radioactive species or enzymes or are labor-intensive. In this study, upon separation of Zn 2þ from the reaction mixture of MTs and GSH with a centrifugal filter membrane, differential pulse voltammetry (DPV) was used for the Zn 2þ quantification. The same approach is extended to the studies of metal transfer between Zn7MT with a GSH/GSSG mixture and that between Zn7MT with GSSG. The concomitant conversion between the free thiol and disulfide bonds was confirmed with UVvis spectrophotometry. The results demonstrate that GSSG, GSH, and the GSH/GSSG mixture all modulate zinc release from Zn7MT. The percentage of zinc release increases in the order of GSH, GSSG, and the GSH/GSSG mixture. The new approach is demonstrated to be well suited for investigation of redox regulation of MT and its reaction with zinc-containing enzymes.

Self-assembled monolayers (SAMs) of ferrocene-labeled α-helical peptides were prepared on gold surfaces and studied using electrochemical surface plasmon resonance (EC-SPR). The leucine-rich peptides were synthesized with a cysteine sulfhydryl group either at the C- or N-terminus, enabling their immobilization onto gold surfaces with control of the direction of the molecular dipole moment. Two electroactive SAMs were studied, one in which all of the peptide dipole moments are oriented in the same direction (SAM1), and the other in which the peptide dipole moment of one peptide is aligned in the opposite direction to that of its surrounding peptide molecules (SAM2). Cyclic voltammetry combined with SPR measurements revealed that SAM reorientations concomitant with the oxidation of the ferrocene label were more significant in SAM2 than in SAM1. The substantially greater change in the peptide film thickness in the case of SAM2 is attributed to the electrostatic repulsion between the electrogenerated ferrocinium moiety and the positively charged gold surface. The greater permeability of SAM1 to electrolyte anions, on the other hand, appears to effectively neutralize this electrostatic repulsion. The film thickness change in SAM2 was estimated to be 0.25 ± 0.05 nm using numerical simulation. The timescale of the redox-induced SPR changes was established by chronoamperometry and time-resolved SPR measurements, followed by fitting of the SPR response to a stretched exponential function. The time constants measured for the anodic process were 16 and 6 ms for SAM1 and SAM2 respectively, indicating that the SAM thickness changes are notably fast.

Voltammetric studies of rabbit liver metallothioneins (MTs, containing both Zn and Cd ions) and Zn7-MT were carried out at Nafion-coated mercury film electrodes (NCMFEs). The accumulation of MT molecules into the NCMFEs enhances the voltammetric signals and the electrostatic interaction between the Nafion membrane and MT facilitates facile electron transfer reactions. Two well-defined redox waves, with reduction potential (Epc) values at −0.740 and −1.173 V, respectively, were observed. The peak at Epc =−0.740 V is attributable to the reduction of the Cd-MT complex, whereas that at Epc=−1.173 V was assigned to the reduction of the Zn-MT complex. Zn7-MT exhibits only one redox wave with Epc=−1.198 V. The NCMFE was found to be more advantageous than thin mercury film electrode (MFE), because the pristine metal ions in MTs (e.g., Cd2+ and/or Zn2+) are not significantly replaced by Hg2+. The NCMFE is also complementary to Nafion-coated bismuth film electrode in that it has a greater hydrogen overpotential, which allows the reduction of the Zn-MT complex to be clearly observed. Moreover, intermetallic compound formation between Cd and Zn appears to be less serious at NCMFEs. Consequently, the amounts of Cd and Zn deposited into the electrode upon the reduction reactions can be quantified more accurately.

The developmental transition from vegetative growth to flowering in Arabidopsis is associated with a precipitous decline in the activity of leaf ascorbate peroxidase (APx), an enzymatic scavenger of hydrogen peroxide, and an increase in specific lipid peroxidation leading to the accumulation of 13-hydroperoxy-9,11,15 (Z,E,Z) octadecatrienoic acid (13 HOO-FA). The appearance of this specific isomer suggests that it is of enzymatic origin and may represent the activation of an oxylipin signaling pathway. We thus hypothesized that leaf 13-lipoxygenase (LOX) activity increases at the floral transition and leads to the observed elevation of 13-HOO-FA levels. Leaf protein extracts were prepared from seven distinct life stages of Arabidopsis plants and used to assay for LOX activity. We report that leaf 13-LOX enzymatic activity increases two- to three-fold from the vegetative stage to the immediate post-floral transition stage. We found two forms of LOX activity in cell extracts and show that the higher pH optimum form is the isoenzyme activated. This increase is correlated with a small increase in H(2)O(2), perhaps resulting from the previously reported decline in leaf APx activity. Very low levels of exogenous H(2)O(2) activate the induced form in vegetative leaf extracts in vitro, suggesting that the floral transition-dependent APx decline and subsequent H(2)O(2) elevation are involved in activating plastid 13-LOX and thus a second messenger oxylipin pathway.

Affinity capillary electrophoresis (ACE) was used to study the interaction between ferulic acid (FA) and BSA. The interaction between FA and BSA was facilitated by injecting FA into a BSA-containing running buffer. Both mobility ratio and mobility shift assays were performed to deduce the binding constant (K(b)). However, the K(b )value obtained with the mobility ratio assay was only approximately 20% of that extracted from the mobility shift assay. The former assay yielded a K(b) value (5.6 +/- 0.4 x 10(4) M(-1)), which compares well with the result obtained with surface plasmon resonance (SPR) (5.1 +/- 0.6 x 10(4) M(-1)). The discrepancy between the mobility ratio and mobility shift assays suggests that the data extrapolation from the mobility ratio should be more reliable for cases when both changes in the EOF and viscosity of the running buffer are important. The work demonstrates that ACE, a solution-based technique, and SPR, a technique addressing interfacial processes, are highly complementary to each other and the comparative studies are confirmatory and allow binding constants to be accurately determined.

While an array of technologies based on radioactive labels or luminescent tags are dominant in modern biomedical research on DNA, surface plasmon resonance (SPR) and SPR imaging measurements are sensitive, rapid, and label-free. This review summarizes recent advances in the development of SPR and coupled techniques and their applications in DNA research, including the gene analysis at trace levels and studies of DNA–protein and DNA–drug interactions.

The amyloid cascade hypothesis links the amyloid-β (Aβ) peptide aggregation to neuronal cell damage and ultimately the etiology of Alzheimer's disease (AD). Although Aβ aggregation has been known to accelerate at cell membranes, the exact mechanism of Aβ peptide deposition and the involvement of extracellular species are still largely unclear. Using surface plasmon resonance (SPR) and atomic force microscopy (AFM), we demonstrate that Ca(2+) ions, in conjunction with lipid bilayer, lower the threshold concentration for Aβ aggregation (>a few micromolar in vitro) to physiological levels (low nanomolar). Circular dichroism spectroscopy reveals that Ca(2+) ions and the lipid bilayer concertedly accelerate the conformational change or misfolding of Aβ peptides. Molecular dynamics calculation indicates that Ca(2+) is sandwiched between Glu-22 of Aβ and the lipid phosphate group. SPR experiments conducted using an E22G mutant confirmed the strong interaction among Ca(2+), Aβ(1-42), and the phospholipid bilayer. With the C- and N-termini of the Aβ dimer fully exposed for the attachment of additional Aβ molecules, fibrils formed with the Ca(2+)-anchored Aβ nuclei appear to interact with lipid bilayers differently from those preformed in solution. Thus, similar to the role of Ca(2+) in enriching islet amyloid polypeptides in the pancreas of diabetic patients ( Biophys. J. 2013 , 104 , 173 - 184 ) and the "Ca(2+) bridge" in mediating membrane interaction with α-synuclein in the Parkinson's disease ( Biochemistry , 2006 , 45 , 10947 - 10956 ), the influence of Ca(2+) on the Aβ adsorption at cell membranes, which leads to neuronal membrane damage in AD, cannot be overlooked.

Nafion-coated bismuth film electrodes (NCBiFEs) were used to investigate the redox behavior of and metal release from rabbit liver metallothioneins (MTs) in an acetate buffer. Owing to the permselective exchange between positively charged MT molecules and cations in Nafion and the absence of detectable MT adsorption at bismuth surface, a diffusion-controlled Nernstian redox wave of MTs (Epc=−0.869 V) was observed for the first time. The Nernstian behavior of the diffusing MTs is in contrast to the voltammetric responses of MTs at thin mercury films or Nafion-coated mercury film electrodes, which either result in the replacement of the metals originally present in MTs by mercury or lead to a noticeable MT adsorption. By avoiding these undesirable features, the NCBiFE provides an excellent milieu for voltammetric studies of different types of MTs and related isoforms, paving the way for studying the redox-modulated metal transfer of MTs and understanding the biological role of MTs in heavy metal detoxification and essential metal regulations.

A thiol-specific electroactive cross-linker, N-(2-ethyl-ferrocene)maleimide (Fc-Mi), has been used to tag surfaceconfined peptides containing cysteine residues or oligodeoxynucleotides (ODNs) whose 3’ ends have been modified with thiol groups. The peptides studied herein include both the oxidized and reduced forms of glutathione and a hexapeptide. Cyclic voltammograms (CVs) of the Fc-Mi groups attached to the surfaces were used to quantify the total number of cysteine residues that are tagged and/or can undergo facile electron transfer reactions with the underlying electrodes. A quartz crystal microbalance was used in conjunction with CV to estimate the total number of cysteine groups labeled by Fc-Mi per peptide molecule. By comparing to mass spectrometric studies, it is confirmed that not all of the Fc-Mi linked to the cysteine groups can participate in the electron transfer reactions. The methodology is further extended to the determination of ODN samples in a sandwich assay wherein the thiol linker on the 3’ end can be tagged with Fc-Mi. The analytical performance was evaluated through determinations of a complementary ODN target and targets with varying numbers of mismatching bases. ODN samples as low as 10 fmol can be detected. Such a low detection level is remarkable considering that no signal amplification scheme is involved in the current method. The approach is shown to be sequence- and/or structure-specific and does not require sophisticated instrumentation and complex experimental procedure.

Ferrocene derivatives containing primary amines and maleimide groups were attached covalently onto Nhydrosuccinimidyl (NHS)-terminated alkanethiol self-assembled monolayers (SAMs) and SAMs of alkanedithiol. The surface coverage and efficiencies of the two cross-linking reactions were evaluated with cyclic voltammetry. All the ferrocene derivatives attached onto the alkanethiol or alkanedithiol SAMs exhibit reversible redox waves. The surface coverage of the aminated ferrocene groups was compared to that of N-hydrosuccinimidyl (NHS)-terminated alkanethiol SAM. The covalent attachment of b-ferrocenylethylamine onto a 11,11’-dithio-bis(succinimidylundecanoate) SAM yielded an efficiency as high as 63.1%. The cross-linking efficiency of this reaction was found to increase with the nucleophilicity of the amino groups. SAMs of longer alkyl chains favor the attachment of a greater number of ferrocene derivatives. As for the Michael-type electrophilic addition between the sulfhydryl groups of the alkanedithiol SAMs and the ferrocenyl maleimide, the cross-linking efficiencies were found to range from 6.5% to 25.7%, depending on the alkanedithiol chain length. The difference in the efficiencies between the two types of crosslinking reactions might be partially attributable to the steric hindrance imposed by the SAMs and the relative sizes of the functional groups.

An approach to producing films of nanometer-sized copper oxide particulates, based on polyelectrolyte-mediated assembly of the precursor, copper(II)phthalocyanine tetrasulfonate (CPTS), is described. Multilayered CPTS and polydiallyldimethylammonium chloride (PDADMAC) were alternately assembled on different planar substrates via the layer-by-layer (LbL) procedure. The growth of CPTS multilayers was monitored by UV-visible spectrometry and quartz crystal microbalance (QCM) measurements. Both the UV-visible spectra and the QCM data showed that a fixed amount of CPTS could be attached to the substrate surface for a given adsorption cycle. Cyclic voltammograms at the CPTS/PDADMAC-covered gold electrode exhibited a decrease in peak currents with the layer number, indicating that the permeability of CPTS multilayers on the electrodes had diminished. When these CPTS multilayered films were calcined at elevated temperatures, uniform thin films composed of nanoparticulate copper oxide could be produced. Ellipsometry showed that the thickness of copper oxide nanoparticulate films could be precisely tailored by varying the thickness of CPTS multilayer films. The morphology and roughness of CPTS multilayer and copper oxide thin films were characterized by atomic force microscopy. X-ray diffraction (XRD) measurements indicated that these thin films contained both CuO and Cu2O nanoparticles. The preparation of such copper oxide thin films with the use of metal complex precursors represents a new route for the synthesis of inorganic oxide films with a controlled thickness.

The aggregation of alpha-synuclein has been implicated as a critical step in the development of Parkinson's disease. Parkinson's disease is a progressive neurodegenerative disorder caused by the loss of dopaminergic neurons from the substantia nigra; currently, no cure exists. Baicalein is a flavonoid with antioxidant properties; upon oxidation, it forms several products including quinones. We show here that low micromolar concentrations of baicalein, and especially its oxidized forms, inhibit the formation of alpha-synuclein fibrils. In addition, existing fibrils of alpha-synuclein are disaggregated by baicalein. The product of the inhibition reaction is predominantly a soluble oligomer of alpha-synuclein, in which the protein molecules have been covalently modified by baicalein quinone to form a Schiff base with a lysine side chain in alpha-synuclein. The binding of baicalein was abolished by conversion of the Tyr residues into Phe, demonstrating that Tyr is involved in the interaction of alpha-synuclein with baicalein. In disaggregation baicalein causes fragmentation throughout the length of the fibril. These observations suggest that baicalein and similar compounds may have potential as therapeutic leads in combating Parkinson's disease and that diets rich in flavonoids may be effective in preventing the disorder.

Ferrocene (Fc)-capped gold nanoparticle/streptavidin conjugates were attached onto electrodes covered with either biotinylated oligodeoxynucleotides (ODNs) duplexes or N-biotinoyl-N-(6-maleimidedohexanoyl)hydrazide that had been preadsorbed onto the reduced form of glutathione (GSH) immobilized onto a dithiobissuccinmidyl propionate self-assembled monolayer (SAM). Effects of the DNA probe and target strand lengths on the voltammetric behavior of the Fc groups residing at the nanoparticle conjugates were studied. The voltammetric behavior was also compared to that arising from the Fc groups present at the end of the ODN duplex in a sandwich complex (Wang et al. Anal. Chem. 2003, 75, 3941). It was found that shorter duplexes would lead to a more reversible voltammetric wave. However, the formation of duplexes with a greater number of base pairs will result in a lower detection level. For the analysis of a biotinylated 30mer target at a mixed SAM of a 30mer probe and hexanethiol, a concentration level as low as 0.25 pM could be detected (which is even lower than our previously reported detection level of 2 pM for an ODN sandwich complex). The detection level for GSH (1 nM) was found to be comparable to those of many other analytical techniques. The capability of our method for trace analysis of biotinylated biomolecules can be attributed to the enhanced voltammetric detection by the large number of Fc moieties present at each conjugate. The method described herein is simple, reproducible, versatile, and selective, and does not require sophisticated analytical instrumentation.

The use of nanosphere lithography to construct two-dimensional arrays of polystyrene (PS) particles coated with multilayered polyelectrolyte (PE) shells and truncated eggshell structures composed of PE thin layers is reported. The truncated eggshell PE structures were produced by extraction of the PS particle cores with toluene. The core-extraction process ruptures the apex of the PE coating and causes a slight expansion of the PE thin layers. Aniline hydrochloride was infiltrated into the PE shells and subsequently electropolymerized to yield an array of a composite containing polyaniline (PAni) and PE thin shells. Voltammetric, quartz crystal microbalance, and reflectance Fourier transform infrared spectroscopic measurements indicate that aniline monomers were confined within the thin PE shells and the electropolymerization occurred in the interior of the PE shell. The PE thickness governs the amount of infiltrated monomer and the ultimate loading of the PAni in the truncated eggshell structure. Surface-structure imaging by atomic force microscopy and scanning electron microscopy, carried out after each step of the fabrication process, shows the influence of the PE thickness on the organization and dimensions of the arrays. Thus, the PE thin shells composed of different layers can function as nanometer-sized vessels for the entrapment of charged species for further construction of composite materials and surface modifications. This approach affords a new avenue for the synthesis of new materials that combine the unique properties of conductive polymers and the controllability of template-directed surface reactions.

A detailed elucidation of the factors affecting the binding of anti-double-stranded (ds)-DNA antibody to ds-DNA immobilized on surfaces may provide insight into the pathogenicity of the systemic lupus erythematosus disease and lead to the development of surfaces for specific DNA and antibody recognition. In this work, the influence of the surface parameters on the attachment of antibody to DNA under physiologically relevant conditions was studied by magnetic alternating current mode atomic force microscopy, ellipsometry, and fluorescence spectroscopy. The DNA surface orientation (tethered vs flat lying), duplex surface density, antibody concentration, and reaction time were all found to affect the antibody/DNA conjugate formation. The mouse monoclonal antibody was specific toward ds-DNA over single-stranded DNA. The steric hindrance and reduced flexibility of surface-confined polynucleotide DNA molecules for the conjugate formation were examined by comparing the conjugates attached to calf thymus DNA preimmobilized onto a surface to those preformed in a homogeneous solution comprising DNA and antibody molecules and subsequently attached to the surface. Antibody aggregation was also found to be prevalent at surface sites where DNA/antibody conjugates existed.

Gold nanoparticle/streptavidin conjugates covered with 6-ferrocenylhexanethiol were attached onto a biotinylated DNA detection probe of a sandwich DNA complex. Due to the elasticity of the DNA strands, the ferrocene caps on gold nanoparticle/streptavidin conjugates are positioned in close proximity to the underlying electrode modified with a mixed DNA capture probe/hexanethiol self-assembled monolayer and can undergo reversible electron-transfer reactions. A detection level, down to 2.0 pM (10 amol for the 5 microL of sample needed) for oligodeoxynucleotide samples was obtained. The amplification of the voltammetric signals was attributed to the attachment of a large number of redox (ferrocene) markers per DNA duplex formed. The ferrocene oxidation current increased with the target concentration and began to level off at a target concentration of 10 nM. An Excellent linearity was found within the range between 6.9 and 150.0 pM and reasonable relative standard deviations (between 3.0 and 13.0%) were obtained. The amenability of this method to the analyses of polynucleotides (i.e., PCR products of the pre-S gene of hepatitis B virus in serum samples) was also demonstrated. The method is shown to be simple, selective, reproducible, and cost-effective and does not require labeling of the DNA targets.

Multilayers of poly(diallyldimethylammonium chloride) and poly(sodium 4-styrenesulfonate) precoated onto melamine formaldehyde (MF) particles via the layer-by-layer (LbL) self-assembly procedure were used as templates for the subsequent deposition of polyaniline (PANI). Transmission electron microscopic (TEM) and scanning electron microscopic (SEM) images reveal that the as-prepared PANI-containing polyelectrolyte (PE)−MF core−shell particles are narrowly dispersed and possess uniform surface morphology. Intact hollow PANI/PE capsules can also be produced by extracting the MF cores in an acidic solution. FTIR spectra of the hollow capsules indicate that PANI and PE are both present and suggest that the PANI exists in the emeraldine form. The incorporation of PANI in the shells was found to enhance the rigidity of these composite materials, and the PANI/PE-coated MF core−shell particles have comparable conductivities with other PANI-coated colloidal particles. The amount of PANI incorporation was also foun...

Scanning electrochemical microscopic (SECM) imaging of oligonucleotides and polynucleotides (poly[G] and calf thymus DNA) immobilized onto aldehyde-modified glass substrates was achieved through oxidation of guanine residues on the DNA molecules by SECM tip-generated Ru(bpy)33+. Several parameters affecting the tip current associated with the guanine oxidation reaction (e.g. tip/substrate separation, the amount of guanine residues, and the voltammetric parameters) were investigated. The relationship between the tip current and the DNA concentration used for immobilization was also studied. The potential of utilizing the tip-generated Ru(bpy)33+ to image DNA hybridization was demonstrated. This study demonstrates that SECM imaging based on guanine oxidation is straightforward and sensitive and obviates the necessity of labeling the DNA targets with an electroactive marker.

Well-ordered 2-D polyaniline (PAni) honeycomb films were produced by electropolymerization of aniline within the interstitial voids in assemblies of polystyrene (PS) nanospheres precoated with poly(diallyldimethylammonium chloride) and poly(sodium 4-styrenesulfonate) (PDADMAC/PSS) thin shells, followed by the extraction of the PS cores with toluene. Depositing polyelectrolyte (PE) layers onto the PS particles was found to greatly improve the uniformity of the PAni attachment onto the PS particle surface. Through a systematic variation of the PE layer number and the electropolymerization parameters, PAni honeycomb films with desirable pore sizes, pore wall widths, and film thicknesses can be fabricated. Atomic force microscopy (AFM) and scanning electron microscopy were used to study the surface features of the resulting PAni honeycomb films, whereas electrochemical quartz crystal microbalance (EQCM) and reflectance FTIR measurements were carried out to verify the redox states of the PAni films and the com...