Determination of ribonuclease H surface enzyme kinetics by surface plasmon resonance imaging and surface plasmon fluorescence spectroscopy

The kinetics of the ribonuclease H (RNase H) surface hydrolysis of RNA-DNA heteroduplexes formed on DNA microarrays was studied using a combination of real-time surface plasmon resonance imaging (SPRI) and surface plasmon fluorescence spectroscopy (SPFS). Time-dependent SPRI and SPFS data at various enzyme concentrations were quantitatively analyzed using a simple model that couples diffusion, enzyme adsorption, and surface enzyme kinetics. This model is characterized by a set of three rate constants, enzyme adsorption (k(a)), enzyme desorption (k(d)), enzyme catalysis (k(cat)), and one dimensionless diffusion parameter (beta). Values of k(a) = 3.15 (+/-0.20) x 10(6) M(-1).s(-1), k(d) = 0.10 (+/-0.05) s(-1), and k(cat) = 0.95 (+/-0.10) s(-1) were determined from fitting all of the SPRI and SPFS data sets. One of the most interesting kinetic parameters is the surface RNase H hydrolysis reaction rate constant (k(cat)), which was found to be approximately 10 times slower than that observed in solution, but approximately 100 times faster than that recently observed for the exonuclease III surface hydrolysis of double-stranded DNA microarrays (k(cat) = 0.009 s(-1)). Moreover, the surface coverage of the intermediate enzyme-substrate complex (ES) was found to be extremely small during the course of the reaction because k(cat) is much larger than the product of k(a) and the bulk enzyme concentration.     

Characterization and optimization of peptide arrays for the study of epitope-antibody interactions using surface plasmon resonance imaging

The characterization of peptide arrays on gold surfaces designed for the study of peptide-antibody interactions using surface plasmon resonance (SPR) imaging is described. A two-step process was used to prepare the peptide arrays: (i) a set of parallel microchannels was used to deliver chemical reagents to covalently attach peptide probes to the surface by a thiol-disulfide exchange reaction; (ii) a second microchannel with a wraparound design was used as a small-volume flow cell (5 microL) to introduce antibody solutions to the peptide surface. As a demonstration, the interactions of the FLAG epitope tag and monoclonal anti-FLAG M2 were monitored by SPR imaging using a peptide array. This peptide-antibody pair was studied because of its importance as a means to purify fusion proteins. The surface coverage of the FLAG peptide was precisely controlled by creating the peptide arrays on mixed monolayers of alkanethiols containing an amine-terminated surface and an inert alkanethiol. The mole fraction of peptide epitopes was also controlled by reacting solutions containing FLAG peptide and the non-interacting peptide HA or cysteine. By studying variants based on the FLAG binding motif, it was possible to distinguish peptides differing by a single amino acid substitution using SPR imaging. In addition, quantitative analysis of the signal was accomplished using the peptide array to simultaneously determine the binding constants of the antibody-peptide interactions for four peptides. The binding constant, K(ads), for the FLAG peptide was measured and found to be 1.5 x 10(8) M(-1) while variants made by the substitution of alanine for residues based on the binding motif had binding constants of 2.8 x 10(7), 5.0 x 10(6), and 2.0 x 10(6) M(-1).     

Microdialysis sampling method for evaluation of binding kinetics of small molecules to macromolecules

Here we present an application of microdialysis sampling for evaluation of the binding kinetics of small molecules to macromolecules. It is label-free, and no immobilization of any interaction partner is required. The method was established by the coupling of a binding reaction with a membrane transport in a miniature and dynamic microdialysis sampling system. A theoretical model was established to describe the quantitative relationship between the binding kinetics of small ligands to macromolecules and the enhanced mass transport of small ligands and was applied to estimate the binding kinetics. To demonstrate the proof-of-principle, we examined the binding kinetics of an abundant plasma protein human serum albumin (HSA) and a representative drug ketoprofen as an example. The primary binding constant of ketoprofen to HSA was estimated as 1.63 (+/-0.12) x 10(6) M(-1). The estimated association and dissociation rate constants (k1 and k(-1)) were about 3.71 x 10(5) M(-1) s(-1) and 0.227 s(-1), respectively. The results suggest a fast binding of ketoprofen to HSA and a fast dissociation of the formed complex, which are consistent with the reversible binding property of drug and HSA (k(-1) in the order of s(-1)). This is the first report on binding-kinetics measurement using microdialysis sampling.     

Using the intrinsic chirality of a molecule as a label-free probe to detect molecular adsorption to a surface by second harmonic generation

Chiral second harmonic generation (C-SHG) has been used for the label-free detection of (R)-(+)-1,1'-bi-2-naphthol (RBN) and (S)-(+)-1,1'-bi-2-naphthol (SBN) binding to planar-supported lipid bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphotidylcholine (POPC) based on the intrinsic chirality of the molecules. C-SHG adsorption isotherms of RBN and SBN reveal Langmuir adsorption behavior with binding constants of 2.7 +/- 0.2 x 10(5) M(-1) and 3.0 +/- 0.1 x 10(5) M(-1), respectively. The kinetics of RBN binding to a POPC bilayer was also measured. It was determined that the adsorption rate for RBN was 5.7 +/- 0.4 x 10(3) s(-1)M(-1) and the desorption rate was 2.1 +/- 0.8 x 10(-2) s(-1). From the kinetic data a binding constant of 2.7 +/- 1.0 x 10(5) M(-1) was calculated, which agrees well with the thermodynamic measurement. The C-SHG technique was correlated with surface tension measurements in order to determine the RBN surface excess within the POPC membrane. The maximum surface excess of RBN in a monolayer of POPC was 4.3 +/- 0.5 x 10(-11) mol cm2. Using the maximum surface excess in conjunction with the C-SHG binding data a lower limit of detection of 1.5 +/- 0.1 x 10(-13) mols cm(-2) was calculated. The results of these studies show that C-SHG is a powerful tool for the study of chiral molecular interactions at surfaces.     

Surface plasmon resonance imaging measurements of DNA and RNA hybridization adsorption onto DNA microarrays

Surface plasmon resonance (SPR) imaging is a surface-sensitive spectroscopic technique for measuring interactions between unlabeled biological molecules with arrays of surface-bound species. In this paper, SPR imaging is used to quantitatively detect the hybridization adsorption of short (18-base) unlabeled DNA oligonucleotides at low concentration, as well as, for the first time, the hybridization adsorption of unlabeled RNA oligonucleotides and larger 16S ribosomal RNA (rRNA) isolated from the microbe Escherichia coli onto a DNA array. For the hybridization adsorption of both DNA and RNA oligonucleotides, a detection limit of 10 nM is reported; for large (1,500-base) 16S rRNA molecules, concentrations as low as 2 nM are detected. The covalent attachment of thiol-DNA probes to the gold surface leads to high surface probe density (10(12) molecules/cm2) and excellent probe stability that enables more than 25 cycles of hybridization and denaturing without loss in signal or specificity. Fresnel calculations are used to show that changes in percent reflectivity as measured by SPR imaging are linear with respect to surface coverage of adsorbed DNA oligonucleotides. Data from SPR imaging is used to construct a quantitative adsorption isotherm of the hybridization adsorption on a surface. DNA and RNA 18-mer oligonucleotide hybridization adsorption is found to follow a Langmuir isotherm with an adsorption coefficient of 1.8 x 10(7) M(-1).     

Pulsed laser photofragment emission for detection of mercuric chloride

The viability of pulsed laser photofragment emission (PFE) is evaluated for the in situ measurement of vapor-phase mercuric chloride (HgCl(2)) concentration in combustion flue gas. Dispersed emissions from both the Hg (6(3)P(1)) and HgCl (B(2)Sigma(+)) photoproducts are presented, and the dependence of the HgCl(2) PFE signal originating from Hg (6(3)P(1)) on the collisional environment is examined for buffer-gas mixtures of N(2), O(2), and CO(2). Integrated PFE intensity measurements as a function of buffer gas pressure support the assumption that the primary effect of the relevant flue gas constituents is to quench emission from Hg (6(3)P(1)). The quenching rate constants for PFE from HgCl(2) were measured to be 1.37 (+/-0.16) x 10(5) Torr(-1) s(-1) for N(2), 9.35 (+/-0.25) x 10(6) Torr(-1) s(-1) for O(2), and 1.49 (+/-0.29) x 10(6) Torr(-1) s(-1) for CO(2). These values are in good accord with literature values for the quenching of Hg (6(3)P(1)). The emission cross section for Hg (6(3)P(1)) generated by photodissociation of HgCl(2) in 760 Torr N(2) is found to be 1.0 (+/-0.2) x 10(-25) m(2) by comparing the PFE signal to N(2) Raman scattering.     

Nonregeneration protocol for surface plasmon resonance: study of high-affinity interaction with high-density biosensors

Surface plasmon resonance (SPR) has been used in determining kinetics and thermodynamics of biological interaction in the past decades. One difficulty encountered in this technology is the need for a proper regeneration, which means the removal of analytes from the bound complexes to regenerate the activity of the ligands. Regeneration is not always practical since the harsh regeneration reagents may destroy the bioactivity of the ligands. It is even more difficult for complexes with high affinity constants. In this paper, we report a nonregeneration protocol for SPR techniques in which subsequent ligand/analyte interactions can be measured without regeneration; thus ligand biological activity could be retained. Kinetics, binding models, and mathematics of this protocol are discussed in detail using rabbit IgG as the analyte and engineered recombinant antibody A10B single-chain fragment variables (scFv) as the ligand. The affinity constant of rabbit IgG binding with A10B scFv measured by using a nonregeneration protocol was (2.5 +/- 0.2) x 10(7) M(-1), which was comparable with the value determined with a conventional regeneration SPR method ((2.2 +/- 1.5) x 10(7) M(-1)) and quartz crystal microbalance (1.9 x 10(7) M(-1)). A paradigm of streptavidin-biotin binding was analyzed to validate this protocol. The affinity constant for each binding subunit of streptavidin to the immobilized biotin was determined to be (7.3 +/- 0.2) x 10(6) M(-1), which was comparable with the solution-based value of 2 x 10(7) M(-1). The nonregeneration protocol requires a relatively high ligand density on the biosensor surface so that more data points can be obtained before surface saturation. The small size of scFv enables them to be constructed in the biosensors for such purpose.     

Quantitative methods for spatially resolved adsorption/desorption measurements in real time by surface plasmon resonance microscopy

A simple method for converting local reflectivity changes measured in surface plasmon resonance (SPR) microscopy to effective adlayer thicknesses and absolute surface coverages of adsorbed species is presented. For a range of high-contrast angles near the SPR resonance where the local metal surface's reflectivity changes linearly with angle, the change in reflectivity at fixed angle is proportional to the change in effective refractive index (eta(eff)) near the surface. This change in eta(eff) can be converted to absolute adsorbate coverage using methods developed for quantitative SPR spectroscopy. A measurement of the change in reflectivity due to changes in refractive index of bulk solutions, i.e., percent reflectivity change per refractive index unit (RIU), is the only calibration required. Application of this method is demonstrated for protein adsorption onto protein/DNA arrays on gold from aqueous solution using an SPR microscope operating at 633 nm. A detection limit of 0.072% change in absolute reflectivity is found for simultaneous measurements of all 200 microm x 200 microm areas within the 24-mm(2) light beam with 1-s time averaging. This corresponds to a change in effective refractive index of 1.8 x 10(-5) and a detection limit for protein adsorption of 1.2 ng/cm(2) (approximately 0.5 pg in a 200-microm spot). The linear dynamic range is Deltaeta(eff) = approximately 0.011 RIU or approximately 720 ng/cm(2) of adsorbed protein. Using a nearby spot as a reference channel, one can correct for instrumental drift and changes in refractive index of the solutions in the flow cell.     

Quantitative studies of allosteric effects by biointeraction chromatography: analysis of protein binding for low-solubility drugs

A new chromatographic method was developed for characterizing allosteric interactions between an immobilized binding agent and low-solubility compounds. This approach was illustrated by using it to characterize the interactions between tamoxifen and warfarin during their binding to the protein human serum albumin (HSA), with beta-cyclodextrin being employed as a solubilizing agent for these drugs. It was confirmed in this work through several experiments that warfarin had a single binding site on HSA with an association equilibrium constant of (2-5) x 10(5) M(-1) (average, 3.9 x 10(5) M(-1)) at 37 degrees C, in agreement with previous reports. It was also found that tamoxifen had a single major binding site on HSA, with an association equilibrium constant of (3-4) x 10(7) M(-1) (average, 3.5 x 10(7) M(-1)) at 37 degrees C. When warfarin was used as a mobile-phase additive in competition studies with tamoxifen, this had a positive allosteric effect on tamoxifen/HSA binding, giving a coupling constant of 2.3 (+/-0.3). Competitive studies using tamoxifen as a mobile-phase additive indicated that tamoxifen had a negative allosteric effect on warfarin/HSA binding, providing a coupling constant of 0.79 (+/-0.03). A unique feature of the technique described in this report was its ability to independently examine both directions of the warfarin/tamoxifen allosteric interaction. This approach is not limited to warfarin, tamoxifen, and HSA but can also be used to study other solutes and binding agents.     

Plant toxic and non-toxic nature of organic dyes through adsorption mechanism on cellulose surface

Effluents releasing from dyeing industries directly affect the soil, water, plant and human life. Among these dyes, plant poisoning, soil polluting and water polluting nature of organic dyes are not yet identified. The plant poisoning and non-poisoning organic dyes are identified through adsorption mechanism of cationic malachite green (MG) and anionic methyl orange (MO) on brinjal plant root powder (cellulose). The positive ΔH(o) (44 kJ mol(-1)) of MG higher than 40 kJ mol(-1) confirmed the adsorption of MG on cellulose is chemisorption and the negative ΔH(o) (-11 kJ mol(-1)) less than 40 kJ mol(-1) showed that the adsorption of MO on cellulose is physisorption. The ΔG(o) values for the adsorption of MG and MO on BPR are not much increased with increase of temperature which indicated that the adsorption is independent of the temperature. The entropy change for the adsorption of MG and MO has proved that the MG (+ΔS(o)) has less disorder at the adsorption interface and MO (-ΔS(o)) has the high disorder at the adsorption interface. The recovery of both dyes has been studied in water at 80°C on BPR surface and observed that the MO recovery is 95% and MG is 10%. The poor desorption of MG is due to the strong chemisorption on BPR (cellulose) surface proves its plant poisoning nature. The high recovery of MO due to physisorption mechanism proves that MO is not poisoning the plant.     

Improving the instrumental resolution of sensors based on localized surface plasmon resonance

The colorimetric variations induced upon changes in interfacial refractive index of nanoscale noble metal structures exhibiting localized surface plasmon resonance (LSPR) provides a convenient means of label-free, affinity-based detection of biomolecular recognition reactions. However, despite being similar in nature to conventional SPR, LSPR has so far suffered from significantly lower data quality in terms of its signal-to-noise ratio (S/N) in typical biomolecular recognition analysis. In this work, generic data analysis algorithms and a simple experimental setup that provide a S/N upon protein binding that is comparable to that of state-of-the art SPR systems are presented. Specifically, it is demonstrated how temporal variations (rate approximately 0.5 Hz) in parameters proportional to the resonance peak position can be recorded simultaneously, yielding a peak position precision of <5 x 10(-4) nm and an extinction noise level of <5 x 10(-6) absorbance units (Abs). This, in turn, is shown to provide a S/N of approximately 2000 (equivalent to a detection limit of <0.1 ng/cm(2)) for typical protein binding reactions. Furthermore, the importance of utilizing changes in both peak position and magnitude is highlighted by comparing different LSPR active noble metal architectures that respond differently to bulk and interfacial refractive index changes.     

Single-step formation of a biorecognition layer for assaying histidine-tagged proteins

The purpose of this work was to develop a simple procedure for the creation of a specific biorecognition layer for histidine-tagged (His-tagged) molecules. Such a layer was prepared by the spontaneous fusion of vesicles containing readily available plain (DOPC) and iminodiacetic acid (DOGS-NTA) phospholipids on a silica surface resulting in the formation of an NTA-containing supported lipid bilayer. The frequency surface acoustic waveguide device which supports Love waves was used to follow the real-time formation of the biorecognition layer. The mole percent of the DOGS-NTA phospholipids in the supported bilayer was optimized by following the kinetics of the fusion for the different NTA-containing lipids. Fluorescently labeled lipids were used with observations of the fluorescence recovery after photobleaching to confirm the presence of lipid bilayers. After saturating all NTA-molecules with Ni(2+), the binding of a His-tagged protein fragment within the concentration range of 0.04 and 0.4 mM to a 5 mol % DOGS-NTA/DOPC was detected; binding curves were used to calculate the apparent association constant k(on) = 2.56 x 10(4) M(-)(1) s(-)(1), dissociation constant k(off) = 1.3 x 10(-)(3) s(-)(1), and equilibrium constant k(eq) = 1.97 x 10(7) M(-)(1). The described method could find significant applications as a generic technique for preparing biorecognition layers for His-tagged proteins. In addition, the acoustic waveguide device, which provides high sensitivity together with flexibility in terms of the substrate material used, is shown to be an attractive alternative to direct optical biosensors.     

Quartz crystal microbalance detection of glutathione-protected nanoclusters using antibody recognition

A quartz crystal microbalance (QCM) immunosensor was developed for the quantitative detection of glutathione-protected nanoclusters. Advantages intrinsic to QCM were employed to make it an attractive alternative to other immunosensing techniques. We have addressed challenges in the area of QCM mass sensing through experimental correlation between damping resistance and frequency change for a reliable mass measurement. Electrode functionalization was optimized with the use of protein A to immobilize and present polyclonal IgG for antigen binding. This method was developed for the detection of glutathione (antigen)-protected clusters of nanometer size with high surface area and thiolate valency. Quantitation of glutathione-nanocluster binding to immobilized polyclonal antibody provides equilibrium constants (K(a) = (3.6 +/- 0.2) x 10(5) M(-1)) and kinetic rate constants (k(f) = (5.4 +/- 0.7) x 10(1) M(-1) s(-1) and k(r) = (1.5 +/- 0.4) x10(-4) s(-1)) comparable to literature reports. These observations further imply that immunoreactive nanoparticles have potential in medical diagnostics and materials assembly.     

Microspotting streptavidin and double-stranded DNA arrays on gold for high-throughput studies of protein-DNA interactions by surface plasmon resonance microscopy

We present two strategies for microspotting 10 x 12 arrays of double-stranded DNAs (dsDNAs) onto a gold-coated glass slide for high-throughput studies of protein-DNA interactions by surface plasmon resonance (SPR) microscopy. Both methods use streptavidin (SA) as a linker layer between a biotin-containing mixed self-assembled monolayer (SAM) and biotinylated dsDNAs to produce arrays with high packing density. The primary mixed SAM is produced from biotin- and oligo(ethylene glycol)-terminated thiols bonded as thiolates onto the gold surface. In the first method, a robotic microspotter is used to deliver nanoliter droplets of dsDNA solution onto a uniform layer of this SA ( approximately 2 x 10(12) SA/cm(2)). SPR microscopy shows a density of (5-6) x 10(11) dsDNA/cm(2) (0.2-0.3 dsDNA/SA) in the array elements. The second method uses instead a microspotted array of this SA linker layer, onto which the microspots of dsDNA are added with spatial registry. SPR microscopy before addition of the dsDNA shows a SA coverage of 2 x 10(12) SA/cm(2) within the spots and a dsDNA density of 8.5 +/- 3.5 x 10(11) dsDNA/cm(2) (0.3-0.7 dsDNA/SA, depending on the length of dsDNA) after dsDNA spotting. We demonstrate the ability to simultaneously monitor protein binding with the SPR microscope in many 200-microm spots with 1-s time resolution and sensitivity to <1 pg of protein.     

Carbohydrate-protein interactions by "clicked" carbohydrate self-assembled monolayers

A Huisgen 1,3-dipolar cycloaddition "click chemistry" was employed to immobilize azido sugars (mannose, lactose, alpha-Gal) to fabricate carbohydrate self-assembled monolayers (SAMs) on gold. This fabrication was based on preformed SAM templates incorporated with alkyne terminal groups, which could further anchor the azido sugars to form well-packed, stable, and rigid sugar SAMs. The clicked mannose, lactose, and alpha-Gal trisaccharide SAMs were used in the analysis of specific carbohydrate-protein interactions (i.e., mannose-Con A; ECL-lactose, alpha-Gal-anti-Gal). The apparent affinity constant of Con A binding to mannose was (8.7 +/- 2.8) x 10(5) and (3.9 +/- 0.2) x 10(6) M(-1) measured by QCM and SPR, respectively. The apparent affinity constants of lactose binding with ECL and alpha-Gal binding with polyclonal anti-Gal antibody were determined to be (4.6 +/- 2.4) x 10(6) and (6.7 +/- 3.3) x 10(6) M(-1), respectively by QCM. SPR, QCM, AFM, and electrochemistry studies confirmed that the carbohydrate SAM sensors maintained the specificity to their corresponding lectins and nonspecific adsorption on the clicked carbohydrate surface was negligible. This study showed that the clicked carbohydrate SAMs in concert with nonlabel QCM or SPR offered a potent platform for high-throughput characterization of carbohydrate-protein interactions. Such a combination should complement other methods such as ITC and ELISA in a favorable manner and provide insightful knowledge for the corresponding complex glycobiological processes.     

Azadirachta indica (Neem) leaf powder as a biosorbent for removal of Cd(II) from aqueous medium

A biosorbent, Neem leaf powder (NLP), was prepared from the mature leaves of the Azadirachta indica (Neem) tree by initial cleaning, drying, grinding, washing to remove pigments and redrying. The powder was characterized with respect to specific surface area (21.45 m2g(-1)), surface topography and surface functional groups and the material was used as an adsorbent in a batch process to remove Cd(II) from aqueous medium under conditions of different concentrations, NLP loadings, pH, agitation time and temperature. Adsorption increased from 8.8% at pH 4.0 to 70.0% at pH 7.0 and 93.6% at pH 9.5, the higher values in alkaline medium being due to removal by precipitation. The adsorption was very fast initially and maximum adsorption was observed within 300 min of agitation. The kinetics of the interactions was tested with pseudo first order Lagergren equation (mean k(1)=1.2x10(-2)min(-1)), simple second order kinetics (mean k2=1.34x10(-3) gmg(-1)min(-1)), Elovich equation, liquid film diffusion model (mean k=1.39x10(-2)min(-1)) and intra-particle diffusion mechanism. The adsorption data gave good fits with Langmuir and Freundlich isotherms and yielded Langmuir monolayer capacity of 158mgg(-1) for the NLP and Freundlich adsorption capacity of 18.7 Lg(-1). A 2.0 g of NLP could remove 86% of Cd(II) at 293 K from a solution containing 158.8 mg Cd(II) per litre. The mean values of the thermodynamic parameters, DeltaH, DeltaS and DeltaG, at 293 K were -73.7 kJmol(-1), -0.24 Jmol(-1)K(-1) and -3.63 kJmol(-1), respectively, showing the adsorption process to be thermodynamically favourable. The results have established good potentiality for the Neem leaf powder to be used as a biosorbent for Cd(II).     

Design and synthesis of single-nanoparticle optical biosensors for imaging and characterization of single receptor molecules on single living cells

At the cellular level, a small number of protein molecules (receptors) can induce significant cellular responses, emphasizing the importance of molecular detection of trace amounts of protein on single living cells. In this study, we designed and synthesized silver nanoparticle biosensors (AgMMUA-IgG) by functionalizing 11.6 +/- 3.5-nm Ag nanoparticles with a mixed monolayer of 11-mercaptoundecanoic acid (MUA) and 6-mercapto-1-hexanol (1:3 mole ratio) and covalently conjugating IgG with MUA on the nanoparticle surface. We found that the nanoparticle biosensors preserve their biological activity and photostability and can be utilized to quantitatively detect individual receptor molecules (T-ZZ), map the distribution of receptors (0.21-0.37 molecule/microm(2)), and measure their binding affinity and kinetics at concentrations below their dissociation constant on single living cells in real time over hours. The dynamic range of detection is 0-50 molecules per cell. We also found that the binding rate (2-27 molecules/min) is highly dependent upon the coverage of receptors on living cells and their ligand concentration. The binding association and dissociation rate constants and affinity constant are k1 = (9.0 +/- 2.6) x 10(3) M(-1) s(-1), k(-1) = (3.0 +/- 0.4) x 10(-4) s(-1), and KB = (4.3 +/- 1.1) x 10(7) M(-1), respectively.     

Nanoparticle-enhanced surface plasmon resonance detection of proteins at attomolar concentrations: comparing different nanoparticle shapes and sizes

The application of biofunctionalized nanoparticles possessing various shapes and sizes for the enhanced surface plasmon resonance (SPR) detection of a protein biomarker at attomolar concentrations is described. Three different gold nanoparticle shapes (cubic cages, rods and quasi-spherical) with each possessing at least one dimension in the 40-50 nm range were systematically compared. Each nanoparticle (NP) was covalently functionalized with an antibody (anti-thrombin) and used as part of a sandwich assay in conjunction with a Au SPR chip modified with a DNA-aptamer probe specific to thrombin. The concentration of each NP-antibody conjugate solution was first optimized prior to establishing that the quasi-spherical nanoparticles resulted in the greatest enhancement in sensitivity with the detection of thrombin at concentrations as low as 1 aM. When nanorod and nanocage antibody conjugates were instead used, the minimum target concentrations detected were 10 aM (rods) and 1 fM (cages). This is a significant improvement (>10(3)) on previous NP-enhanced SPR studies utilizing smaller (~15 nm) gold NP conjugates and is attributed to the functionalization of both the NP and chip surfaces resulting in low nonspecific adsorption as well as a combination of density increases and plasmonic coupling inducing large shifts in the local refractive index at the chip surface upon nanoparticle adsorption.     

In vivo measurement of Pu dissolution parameters of MOX aerosols and related uncertainties in the values of the dose per unit intake

The aim of this study was to compare dissolution parameter values for Pu from industrial MOX with different Pu contents. For this purpose, preliminary results obtained after inhalation exposure of rats to MOX containing 2.5% Pu are reported and compared to those obtained previously with MOX containing 5% Pu. Dissolution parameter values appear to increase when the amount of Pu decreases. Rapid fractions, f(r), of 4 x 10(-3) (s.d. = 2 x 10(-3)) and 1 x 10(-3) (s.d. = 6 x 10(-4)) and slow dissolution rates, s(s) of 2 x 10(-4) d(-1) (standard deviation, sigma = 5 x 10(-5)) and 5 x 10(-5) d(-1) (sigma = 1 x 10(-5)) were derived for MOX containing 2.5 and 5% of Pu, respectively. Simulations were performed to assess uncertainties on dose due to experimental errors. The relative standard deviations of the dose per unit intake (DPUI) due to f(r) (4-8%), are far less than those due to s(s) (about 20%), which is the main parameter altering the dose. Although quite different dissolution parameter values were derived, similar DPUIs were obtained for MOX aerosols containing 2.5 and 5% Pu which appear close to that for default Type S values.     

Adsorption and desorption kinetics of carbofuran in acid soils

Carbofuran adsorption and desorption were investigated in batch and stirred flow chamber (SFC) tests. The carbofuran adsorption capacity of the soils was found to be low and strongly dependent on their clay and organic carbon contents. Carbofuran sorption was due mainly (>80%) to fast adsorption processes governed by intraparticle diffusion. The adsorption kinetic constant for the pesticide ranged from 0.047 to 0.195 min(-1) and was highly correlated with constant n in the Freundlich equation (r=0.965, P<0.05). Batch tests showed carbofuran desorption to be highly variable and negatively correlated with eCEC and the clay content. The SFC tests showed that soil organic carbon (C) plays a key role in the irreversibility of carbofuran adsorption. Carbofuran desorption increased rapidly at C contents below 4%. The desorption kinetic constant for the compound (0.086-0.195 min(-1)) was generally higher than its adsorption kinetic constant; therefore, carbofuran is more rapidly desorbed than it is adsorbed in soil.