An evaluation of a rapid portable gold-nanotechnology measuring SARS-CoV-2 IgM, IgA and IgG antibody concentrations against spike 1 (S1), spike 2 (S) and nucleocapsid (N) was conducted using serum samples from 74 patients tested for SARS-CoV-2 RNA on admission to hospital, and 47 historical control patients from March 2019. 59 patients were RNA(+) and 15 were RNA(-). A serum (±) classification was derived for all three antigens and a quantitative serological profile was obtained. Serum(+) was identified in 30% (95% CI 11-48) of initially RNA(-) patients, in 36% (95% CI 17-54) of RNA(+) patients before 10 days, 77% (95% CI 67-87) between 10 and 20 days and 95% (95% CI 86-100) after 21 days. The patient-level diagnostic accuracy relative to RNA(±) after 10 days displayed 88% sensitivity (95% CI 75-95) and 75% specificity (95% CI 22-99), although specificity compared with historical controls was 100% (95%CI 91-100). This study provides robust support for further evaluation and validation of this novel technology in a clinical setting and highlights challenges inherent in assessment of serological tests for an emerging disease such as COVID-19.

Recombinant protein A/G (PAG) has a sequence coding for eight IgG binding sites and has enhanced interspecies affinity. High-frequency sampling of a PAG titration with IgG produces concentration profiles that are sensitive to the kinetic availability of the binding sites. The full kinetic model developed here for IgG binding sequentially to PAG shows only two distinct kinetic processes, describing an initial rapid association of two antibodies to PAG with a rate constant k-fast = (1.86 ± 0.08) × 106 M-1 s-1 and a slower antibody binding process to all remaining sites, k-slow = (1.24 ± 0.05) × 104 M-1 s-1. At equilibrium (after 1 h), the maximum IgG occupancy of PAG is 2.8 ± 0.5, conflicting with the genetic evidence of eight binding sites and suggesting significant steric hindrance of the neighboring IgG binding sites. The phosphate-buffered saline (PBS) solution defines a standard system setting, and this may be compared with other settings. The mean association rate of PAG-IgGn in the standard setting is 282 ± 20% higher than when PAG is tethered to a surface. A systems biology approach requires that a model parameter set that defines a system in a standard setting should be transferable to another system. The transfer of parameters between settings may be performed using activity coefficients characterizing an effective concentration of species in a system, ai = γici. The activity correction, γ, for the eight-site occupancy is γ = 0.35 ± 0.06, and mapping from the standard setting to the solution setting suggests γPAG-IgG = 0.4 ± 0.03. The role of activity coefficients and transferability of kinetic parameters between system settings is discussed. (Graph Presented).

Introduction the aim of this study was to identify patient factors including serum biomarkers that may predict response to neoadjuvant chemoradiotherapy (CRT) in patients with locally advanced rectal cancer staged on magnetic resonance imaging. Prediction of response may be helpful when selecting patients for a non-operative programme. Methods a retrospective review was carried out of patients undergoing neoadjuvant CRT for rectal cancer, conducted at the Royal Devon and Exeter Hospital. All patients were managed through the multidisciplinary team. Receiver operating characteristic (ROC) curve analysis was undertaken to assess the ability of biomarkers to predict response to neoadjuvant CRT. The biomarkers assessed included neutrophils, lymphocytes, monocytes, haemoglobin, platelets, C-reactive protein and carcinoembryonic antigen. Results Seventy-three patients underwent neoadjuvant CRT between January 2006 and December 2011. Nine (12.3%) of these experienced a clinical complete response and were managed with a 'watch and wait' approach. An additional ten patients (13.7%) had a pathological complete response following surgery. Using ROC curve analysis, the biomarkers with the largest area under the curve (AUC) were pre-CRT haemoglobin and post-CRT lymphocyte concentrations, producing AUC values of 0.673 and 0.618 respectively for clinical complete response. Pre-CRT haemoglobin and neutrophil concentrations produced the highest AUC values for pathological complete response at 0.591 and 0.614 respectively. Conclusions None of the assessed biomarkers offer the ability to predict response to neoadjuvant CRT in patients with rectal cancer. They cannot therefore assist in identifying complete clinical or pathological responders who could be considered for a non-operative, observational approach.

UNLABELLED: Burkholderia mallei are Gram-negative bacteria, responsible for the disease glanders. B. mallei has recently been classified as a Tier 1 agent owing to the fact that this bacterial species can be weaponised for aerosol release, has a high mortality rate and demonstrates multi-drug resistance. Furthermore, there is no licensed vaccine available against this pathogen. Lipopolysaccharide (LPS) has previously been identified as playing an important role in generating host protection against Burkholderia infection. In this study, we present gold nanoparticles (AuNPs) functionalised with a glycoconjugate vaccine against glanders. AuNPs were covalently coupled with one of three different protein carriers (TetHc, Hcp1 and FliC) followed by conjugation to LPS purified from a non-virulent clonal relative, B. thailandensis. Glycoconjugated LPS generated significantly higher antibody titres compared with LPS alone. Further, they improved protection against a lethal inhalation challenge of B. mallei in the murine model of infection. FROM THE CLINICAL EDITOR: Burkholderia mallei is associated with multi-drug resistance, high mortality and potentials for weaponization through aerosol inhalation. The authors of this study present gold nanoparticles (AuNPs) functionalized with a glycoconjugate vaccine against this Gram negative bacterium demonstrating promising results in a murine model even with the aerosolized form of B. Mallei.

Five monoclonal antibodies, mAb7.3, mAb29.3, mAb46.3, mAb12.3 and mAb36.3, raised to the LcrV virulence factor from Yersinia pestis were characterised for their Fab affinity against the purified protein and their Fc affinity to Protein A/G as a proxy for the FcγR receptor. Kinetic measurements were performed label-free in a localised particle plasmon array reader. The Fc-ProteinA/G complex first-order half-life was determined for each antibody and fell in the range of 0.8-3.8. h. The Fab first-order half-lives had ranged from 3.4 to 9.2. h although two antibodies, mAb12.3 and mAb36.3, showed low affinity interactions. Competitive binding studies of mixtures of the Fab-active antibodies were performed to measure the relative binding efficiency of one antibody in the presence of the other. A geometric relative positioning of the epitopes of mAb7.3, mAb29.3 and mAb46.3 was determined based on the footprint locus of the antibody and the percentage of competitive binding. The two known protective antibodies mAb7.3 and mAb29.3 showed greater interference, indicating epitopes close to one another compared to the non-protective mAb46.3 antibody. The Fab-Fc complex half-life screen and epitope mapping are potentially useful tools in the screening of therapeutic antibodies or vaccine candidates.

© 2015 Springer-Verlag Berlin Heidelberg a unified approach to affinity screening for Fab and Fc interactions of an antibody for its antigen and FcγR receptor has been developed. An antigen array is used for the Fab affinity and cross-reactivity screening and protein A/G proxy is the FcγR receptor. The affinities are derived using a simple 1:1 binding model with a consistent error analysis. The association and dissociation kinetics are measured over optimised times for accurate determination. The Fab/Fc affinities are derived for ten antibodies: mAb-actin (mouse), pAb-BSA (sheep), pAb-collagen V (rabbit), pAb-CRP (goat), mAb-F1 (mouse), mAbs (mouse) 7.3, 12.3, 29.3, 36.3 and 46.3 raised against LcrV in Yersinia pestis. The rate of the dissociation of antigen–antibody complexes relates directly to their immunological function as does the Fc-FcγR complex and a new half-life plot has been defined with a Fab/Fc half-life range of 17–470 min. The upper half-life value points to surface avidity. Two antibodies that are protective as an immunotherapy define a Fab half-life. > 250 min and an Fc half-life. > 50 min as characteristics of ideal interactions which can form the basis of an antibody screen for immunotherapy.

We report the whole-transcriptome response of Escherichia coli bacteria to acute treatment with silver nanoparticles (AgNPs) or silver ions [Ag(I)] as silver nitrate using gene expression microarrays. In total, 188 genes were regulated by both silver treatments, 161 were up-regulated and 27 were down-regulated. Significant regulation was observed for heat shock response genes in line with protein denaturation associated with protein structure vulnerability indicating Ag(I)-labile -SH bonds. Disruption to iron-sulphur clusters led to the positive regulation of iron-sulphur assembly systems and the expression of genes for iron and sulphate homeostasis. Further, Ag ions induced a redox stress response associated with large (>600-fold) up-regulation of the E. coli soxS transcriptional regulator gene. Ag(I) is isoelectronic with Cu(I), and genes associated with copper homeostasis were positively regulated indicating Ag(I)-activation of copper signalling. Differential gene expression was observed for the silver nitrate and AgNP silver delivery. Nanoparticle delivery of Ag(I) induced the differential regulation of 379 genes; 309 genes were uniquely regulated by silver nanoparticles and 70 genes were uniquely regulated by silver nitrate. The differential silver nanoparticle-silver nitrate response indicates that the toxic effect of labile Ag(I) in the system depends upon the mechanism of delivery to the target cell.

Electroluminescent assays for epitopes on the complement components C3dg, terminal complement complex (TCC) and factor B/Bb (fB/Bb) have been developed with capture and detection antibodies to produce detection limits C3dg=91±9ng/mL, TCC=3±0.1ng/mL and fB=55.7±0.1ng/mL. The assay performance was assessed against a series of zymosan and heat aggregated IgG (HAIgG) in vitro activations of complement using a calibrated activated complement serum (ACS) as calibration standard. The ACS standard was stable within 20% accuracy over a 6-month period with freeze-thaw cycles as required. Differential activation of the complement cascade was observed for TCC showing a pseudo-first order formation half-life of 3.5h after activation with zymosan. The C3dg activation fragment indicates a 10% total activation for both activation agents. The kinetic-epitope analysis for fB indicates that the capture epitope is on the fB/Bb protein fragment which can then become covered by the formation of C3bBb or C3bBbP complexes during the time course of the cascade.

Electroluminescent assays for epitopes on the complement components C3dg, terminal complement complex (TCC) and factor B/Bb (fB/Bb) have been developed with capture and detection antibodies to produce detection limits C3dg=91±9ng/mL, TCC=3±0.1ng/mL and fB=55.7±0.1ng/mL. The assay performance was assessed against a series of zymosan and heat aggregated IgG (HAIgG) in vitro activations of complement using a calibrated activated complement serum (ACS) as calibration standard. The ACS standard was stable within 20% accuracy over a 6-month period with freeze-thaw cycles as required. Differential activation of the complement cascade was observed for TCC showing a pseudo-first order formation half-life of 3.5h after activation with zymosan. The C3dg activation fragment indicates a 10% total activation for both activation agents. The kinetic-epitope analysis for fB indicates that the capture epitope is on the fB/Bb protein fragment which can then become covered by the formation of C3bBb or C3bBbP complexes during the time course of the cascade. Copyright © 2013 Elsevier B.V. All rights reserved.

Lectin binding has been studied using the particle plasmon light-scattering properties of gold nanoparticles printed into an array format. Performance of the kinetic assay is evaluated from a detailed analysis of the binding of concanavalin a (ConA) and wheat germ agglutinin (WGA) to their target monosaccharides indicating affinity constants in the order of KD ∼10 nM for the lectin-monosaccharide interaction. The detection limits for the lectins following a 200 s injection time were determined as 10 ng/mL or 0.23 nM and 100 ng/mL or 0.93 nM, respectively. Subsequently, a nine-lectin screen was performed on the porcine and human fibrinogen glycoproteins. The observed spectra of lectin-protein specific binding rates result in characteristic patterns that evidently correlate with the structure of the glycans and allow one to distinguish between glycosylation of the porcine and human fibrinogens. The array technology has the potential to perform a multilectin screen of large numbers of proteins providing information on protein glycosylation and their microheterogeneity.

Kinetics of seed-mediated chemical growth of gold nanoparticles on silica substrates coated with a thin layer of graphene-derivative materials were studied with the ultimate goal of employing the developed graphene-nanoparticle composites in light-scattering imaging biosensor applications. The type of the surface coating and chemical conditions influence the surface number density and size of the grown nanoparticles. Importantly, when used as biosensor bases, the substrates coated with protein reduced graphene oxide have shown beneficial biological compatibility towards immobilized antibodies resulting in about 20-fold improvement in antigen capture by the antibodies tethered to the sensor surface in immunoassay performed on the multiplex photonic biosensor platform. © 2014 the Partner Organisations.

The Lensless microscope has a large field of view and allows the capture of the diffraction pattern from a large number of cells simultaneously. A simple algorithm to measure intensity changes in the Airy Disc First Fringe (ADFF) has been derived to follow the growth characteristics of the unicellular yeast Schizosaccharomyces pombe. The performance of the algorithm is calibrated using comparison between optical image and ADFF analysis of polystyrene microspheres with known dimensions and has an accuracy of 5% over all lengths above the diffraction-limited measurements. We have observed the growth characteristics of S. pombe for N=100 cells to determine the growth phenotype distributions of Length (L(t=0)) and width (W(t=0)) on arrival at the surface, lag phase adjustment to the new growth conditions (B), the length at birth, LB, and cell cycle length, tcell. The observed cell width distribution has a median width of 3.9 (±0.1) µm, as expected, but a non-normal distribution. Similarly, all growth parameters studied, L(t=0), LB and cell cycle time are phenotypes with non-normal distributions but with medians consistent with the literature values.

A whole-cell biosensor assay for dual ZnO nanoparticle toxicity mechanisms has been developed based on the transcriptional response of Escherichia coli to: (1) Zn(2+) from ZnO nanoparticle dissolution with genes zntA (Zn(2+) efflux) and znuABC (Zn(2+) uptake); and (2) redox stress from ZnO nanoparticle photo-electron production under ultraviolet light with genes soxS and katG. Both processes occur in a dispersion of ZnO nanoparticles leading to toxicity. ZnO nanoparticle dissolution was measured independently by ICP-MS and photo-radical generation was confirmed by the stochiometric reduction of the redox dye, 2, 6-dichloroindolphenol (DCPIP). The whole-cell biosensor can detect both toxicity mechanisms and is a species-specific assay capable of discriminating between ZnO nanoparticles and the Zn(2+) dissolution product.

We have used the formation of a bio-probe stack with up to 24 steps on gold nanoparticle and continuous gold surfaces to characterize the penetration depth of the plasmon field in a non-invasive manner by only involving biomolecules from standard bio-assays. An alternating anti-goat rabbit IgG and anti-rabbit IgG bio-probe stack is polymerized on protein A/G functionalized gold surfaces. The change in plasmon excitation angle or light scattering decreases exponentially with each stacking step although the bio-integrity of the antibody epitope is maintained. The exponential decay in the derived kinetic parameters is attributed to the change in the penetration depth and the step size is calibrated using a commercial continuous gold surface plasmon resonance surface to be 17.5 ± 0.8 nm, consistent with the expected dimension of the antibody. The penetration depth of the gold spherical nanoparticles of diameter 90 ± 13 nm is determined to be 93 ± 10 nm.

The efficacy of 15 nm gold nanoparticles (AuNP) coated with Yersinia pestis F1-antigen, as an immunogen in mice, has been assessed. The nanoparticles were decorated with F1-antigen using N-hydroxysuccinimide and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride coupling chemistry. Mice given AuNP-F1 in alhydrogel generated the greatest IgG antibody response to F1-antigen when compared with mice given AuNP-F1 in PBS or given unconjugated F1-antigen in PBS or alhydrogel. Compared with unconjugated F1-antigen, the IgG2a response was enhanced in mice dosed with AuNP-F1 in PBS (p < 0.05) but not in mice immunised with AuNP-F1 in alhydrogel. All treatment groups developed a memory response to F1-antigen, the polarity of which was inflenced by formulation in alhydrogel. The sera raised against F1-antigen coupled to AuNPs was able to competitively bind to rF1-antigen, displacing protective macaque sera. © 2012 Elsevier Ltd.

The efficacy of 15 nm gold nanoparticles (AuNP) coated with Yersinia pestis F1-antigen, as an immunogen in mice, has been assessed. The nanoparticles were decorated with F1-antigen using N-hydroxysuccinimide and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride coupling chemistry. Mice given AuNP-F1 in alhydrogel generated the greatest IgG antibody response to F1-antigen when compared with mice given AuNP-F1 in PBS or given unconjugated F1-antigen in PBS or alhydrogel. Compared with unconjugated F1-antigen, the IgG2a response was enhanced in mice dosed with AuNP-F1 in PBS (p

CD73 is a dimeric ecto-5'-nucleotidase that is expressed on the exterior side of the plasma membrane. CD73 has important regulatory functions in the extracellular metabolism of certain nucleoside monophosphates, in particular adenosine monophosphate, and has been linked to a number of pathological conditions such as cancer and myocardial ischaemia. Here, we present the crystal structure of a soluble form of human soluble CD73 (sCD73) at 2.2 Å resolution, a truncated form of CD73 that retains ecto-5'-nucleotidase activity. With this structure we obtained insight into the dimerisation of CD73, active site architecture, and a sense of secondary modifications of the protein. The crystal structure reveals a conserved loop that is directly involved in the dimer-dimer interaction showing that the two subunits of the dimer are not linked by disulfide bridges. Using biophotonic microarray imaging we were able to confirm glycosylation of the enzyme and show that the enzyme is decorated with a variety of oligosaccharide structures. The crystal structure of sCD73 will aid the design of inhibitors or activator molecules for the treatment of several diseases and prove useful in explaining the possible roles of single nucleotide polymorphisms in physiology and disease.

A label-free nanoparticle array platform has been used to detect total peanut allergen-specific binding from whole serum of patients suffering from peanut allergy. The serum from 10 patients was screened against a four-allergen panel of cat and dog dander, dust mite and peanut allergen protein Ara h1. The IgE and IgG contributions to the total specific-binding protein load to Ara h1 were identified using two secondary IgG- and IgE-specific antibodies and were found to contribute less than 50 % of the total specific protein load. The total mass of IgE, IgE and the unresolved specific-binding protein ΔsBP for Ara h1 provides a new serum profile for high-RAST-grade patients 5 and 6 with the IgG/IgE ratio of 4±2 and ΔsBP/IgE ratio of 17±11, neither of which is protective for the small patient cohort. © Springer-Verlag 2012.

A label-free nanoparticle array platform has been used to detect total peanut allergen-specific binding from whole serum of patients suffering from peanut allergy. The serum from 10 patients was screened against a four-allergen panel of cat and dog dander, dust mite and peanut allergen protein Ara h1. The IgE and IgG contributions to the total specific-binding protein load to Ara h1 were identified using two secondary IgG- and IgE-specific antibodies and were found to contribute less than 50 % of the total specific protein load. The total mass of IgE, IgE and the unresolved specific-binding protein ΔsBP for Ara h1 provides a new serum profile for high-RAST-grade patients 5 and 6 with the IgG/IgE ratio of 4 ± 2 and ΔsBP/IgE ratio of 17 ± 11, neither of which is protective for the small patient cohort.

This study investigated the dissolution-based toxicity mechanism for silver nanoparticles to Escherichia coli K12. The silver nanoparticles, synthesised in the vapour phase, are effective anti-bacterial agents against the Gram-negative bacterium, E. coli K12. The nanoparticles associate with the bacterial cell wall, appearing to interact with the outer and inner membranes, and then dissolve to release Ag(+) into the cell and affect a transcriptional response. The dissolution of these nanoparticles in a modified LB medium was measured by inductively coupled plasma mass spectrometry (ICP-MS) and has been shown to follow a simple first-order dissolution process proportional to the decreasing surface area of the nanoparticles. However, the resulting solution phase concentration of Ag(+), demonstrated by the ICP-MS data, is not sufficient to cause the observed effects, including inhibition of bacterial growth and the differential expression of Cu(+) stress response genes. These data indicate that dissolution at the cell membrane is the primary mechanism of action of silver nanoparticles, and the Ag(+) concentration released into the bulk solution phase has only limited anti-bacterial efficacy.

A gold nanoparticle, localised plasmon array biosensor using light scattering has been employed in the detection of allergen-specific antibodies in whole blood and sera. The array sensor was functionalized with four different allergens, cat dander (Fel d1), dust mite (Der p1), peanut allergen (Ara h1) and dog dander (Can f1) and immuno-kinetic assay was performed to detect their respective anti-allergen IgG antibodies. Specific positive responses to antibodies at a concentration of 25. nM were observed for Fel d1, Der p1, and Ara h1 allergens, while the can f1 channel served as a reference control. The sensitivity was further enhanced using a secondary anti-IgG detection antibodies to give a limit of detection of 2. nM. The results indicate the potential for nanoparticle scattering multiplexed arrays to screen unprepared blood samples at point-of-care for assays of complex samples such as the whole blood. © 2012.

A gold nanoparticle, localised plasmon array biosensor using light scattering has been employed in the detection of allergen-specific antibodies in whole blood and sera. The array sensor was functionalized with four different allergens, cat dander (Fel d1), dust mite (Der p1), peanut allergen (Ara h1) and dog dander (Can f1) and immuno-kinetic assay was performed to detect their respective anti-allergen IgG antibodies. Specific positive responses to antibodies at a concentration of 25 nM were observed for Fel d1, Der p1, and Ara h1 allergens, while the can f1 channel served as a reference control. The sensitivity was further enhanced using a secondary anti-IgG detection antibodies to give a limit of detection of 2 nM. The results indicate the potential for nanoparticle scattering multiplexed arrays to screen unprepared blood samples at point-of-care for assays of complex samples such as the whole blood.

The effect of human serum albumin (HSA) on an immunokinetic assay for an antibody to bovine serum albumin has been determined in model serum solutions with HSA concentrations in the range 0 to 450 microM (0-30 mgml(-1)). The assay is performed on two plasmon-based detection platforms: a continuous gold surface and a nanoparticle-based array reader. The assay has a minimum detection concentration of 760+/-160 pM (120+/-25 ngml(-1)) in phosphate-buffered saline, falling to 2.5+/-0.7 nM (380+/-100 ngml(-1)) in physiological HSA concentration. The concentration of HSA correlates with the refractive index of the solution, and this may be used to calibrate assay response. The addition of the charged chaotrope SCN(-) in 150 mM concentration improves the reproducibility and consistency of the assay, with a minimum detection concentration of 2.9+/-0.5 nM (440+/-80 ngml(-1)). The effect of high concentrations of HSA on the immunokinetic assay can be corrected with a measurement of bulk refractive index in a reference channel.

A biophotonic array based on gold nanoparticles functionalized with antigen proteins has been used to determine the concentrations of the respective antibodies in solution. Four proteins-fibrinogen, bovine serum albumin, transferrin, and C-reactive protein-were used to construct a test array with the assay repeated a number of times. The antibody-antigen association and dissociation rate constants were determined for the antibody assays from a series of calibration experiments. The label-free determination of the unknown antibody concentrations was performed using two related kinetic analyses. From these results, the current array assay sensitivity is 250 ng ml(-1) with an accuracy of 15% using an 8-min kinetic measurement and a 16-spot averaged assay.

The aggregation kinetics have been observed for citrate-reduced 15 nm gold nanoparticles at the native silica and modified silica - water interfaces. At the native, negatively charged silica - water interface, two-phase adsorption is observed: a pseudo-Langmuirian adsorption phase and, after an acid wash to remove the citrate ligand from the adsorbed particles, another pseudo-Langmuirian adsorption phase. A kinetic analysis of these phases shows an average adsorption rate constant of (1.5 ± 0.4) × 10 6M-1s-1 with no measurable desorption. Another acid wash induces a nearly linear aggregation phase with a 4-fold faster nanoparticles deposition rate. SEM imaging of the aggregation-phase surface shows the formation of small nanoparticle clusters of < 10 particles. Adsorption on a positively charged, aminated silica surface shows a kinetic trace consistent with aggregations at low surface coverages and has a similar adsorption rate to the aggregation phases on the negatively charged surfaces. SEM imaging reveals clusters of many particles of >10. The refractive index sensitivity of the produced surfaces was measured by varying the analyte refractive index at each phase: the aggregated clusters on the positively charged surface are up to 4 times more sensitive. © 2009 American Chemical Society.

Bovine serum albumin antibodies (aBSA) have been screened from whole leporine anti serum on a biophotonic
array. The array was initially printed with seed gold nanoparticles into a 96-spot configuration,
and 130-nm gold nanoparticles were synthesised in situ on the surface of each spot. The gold nanoparticle
surface was then functionalized with the proteins bovine serum albumin (BSA), fibrinogen, and
immunoglobulin G (IgG) and with the amino acid glycine. The concentration of aBSA in the whole serum
was determined using a kinetic analysis of the time-dependent light scattering from the nanoparticles.
The aBSA–BSA kinetic parameters derived from the array are ka = (1.3 ± 0.3). 105 M 1 s 1,
kd = (4 ± 2). 10 4 s 1, and KD = 3 nM, which compare favorably with those from continuous gold surfaces.
The ultimate sensitivity of the array reader to the bulk refractive index (RI) is 1. 10 4 refractive
index units (RIU), corresponding to 1 lgml 1 for aBSA. The nanoparticles appear to be more sensitive
than the continuous gold surface to the aBSA binding event from whole serum, and this is interpreted
in terms of the difference in RI contrast in the plasmon fields.

Gold nanoparticles grown in situ from printed seed particles on a glass substrate have been fabricated into a biosensor array. The light-scattering properties of the resulting surfaces show sensitivity to changes in the local refractive index. Each array spot is functionalized with fibrinogen or bovine serum albumin and scattered radiation is used to monitor the refractive index change on label-free binding of the antibodies to their antigens from whole blood antiserum. Data were collected real-time and the association rate constants for the specific antibody-antigen binding were derived from a kinetic analysis. The minimum antibody concentration detection sensitivity is of 100 nM.

Detection of radiological materials in the solution phase is restricted by conventional radiation-counting techniques owing to extreme attenuation. Chemical sensing of the resultant radiological species such as uranyl UO(2)(2+) is possible on the surface of a plastic or glass fibre optic. A dihydroxy isoamethryin complex is tethered to the fibre surface which has a large extinction coefficient (119 000 M(-1) cm(-1) at lambda = 439 nm) and changes colour upon binding UO(2)(2+). The spectral changes are greater on the surface than in solution and binding is specific to UO(2)(2+) with small interferences from Gd(3+). Monitoring the spectral response in three detector bands in the red, green and blue enable the optical power change to be measured with sensitivities of 1 mdB, allowing UO(2)(2+) to be detected confidently at 50-100 ppb levels. Real-time kinetic analysis enables discrimination between the target species and possible interferents.

The adsorption kinetics of thioctic acid (ToA) self-assembled monolayer (SAM) formation has been observed by monitoring small changes in the extinction of a gold nanoparticle surface, interrogated by evanescent wave cavity ring-down spectroscopy. A direct measurement of the adsorption kinetics for charged and neutral SAM formation has been made and modeled and shows significant differences in the lateral interactions between surface moieties: the interaction parameter for the charged species is 20 times larger than for the neutral species. Titration of ToA SAMs deposited from basic conditions demonstrate a pH-switchable surface with charge densities varying from 0.1 to 1.0 e nm-2. Similar measurements were performed from SAMs deposited under acidic conditions producing charge densities of 1.8 e nm-2. The surface potential varies between -44 and -198 mV for all SAMs, which produces an interfacial pH 0.8 - 3.4 units more acidic than the bulk and interfacial capacitances varying from 5.5 to 88 μF cm-2. © 2007 American Chemical Society.

A self-assembled acid-functionalised monolayer on a gold surface has an interfacial pH 2.93 more acidic than the bulk and surface pK(a) very similar to that of the free acid.

A fibre optic platform has been fabricated for the field deployment of evanescent wave cavity ring-down spectroscopy with an absorbance sensitivity of 5 ppm. An optical cavity is fabricated by depositing high-reflectivity mirrors onto each end of the fibre and the evanescent field is exposed to the sample in a tapered region of the cavity. The decay time, τ, is dominated by the propagation loss of the radiation in the fibre optic and the loss of the tapered region. The multi-pass configuration can detect molecules adsorbing to the surface of the tapered region if they absorb radiation at the wavelength of the laser. An indicator molecule has been tethered to the glass surface to produce a colour change in response to the bulk pH producing an optical pH sensor with a sensitivity of 0.01 pH units. The fibre cavities have potential to form an optical sensor network to detect target molecules with presumptive detection on functionalised fibre surfaces.

Evanescent wave cavity ring-down spectroscopy (EW-CRDS) is used to observe the adsorption isotherm for hemoglobin (Hb) from controlled urine samples to assess the potential for rapid diagnosis in hemoglobinuria. The absorbance of Hb at 425 nm is monitored using an alexandrite laser-pumped, room temperature, LiF:F +2. color-center pulsed laser. A minimum absorbance detection level of 2.57×10 -4 is achieved, corresponding to a minimum detectable concentration of Hb in urea of 5.8 nM. A multilayered Hb biofilm is formed, and a minimum of eight layers are required to model the adsorption isotherm, allowing for cooperative binding within the layers and extending 56 nm into the interface. A binding constant for Hb to silica 18.23±7.58×10 6 M is derived, and a binding constant for Hb to Hb in subsequent layers is determined to be 5.631 ±0.432 ×10 5 M. Stoichiometric binding coefficients of 1.530±0.981 for layer one and 1.792±0.162 for subsequent layers suggest that cooperative binding both to the silica surface and between the layers of the biofilm is important. © 2005 Society of Photo-Optical Instrumentation Engineers.

Evanescent wave cavity ring-down spectroscopy (EW-CRDS) is used to observe the adsorption isotherm for hemoglobin (Hb) from controlled urine samples to assess the potential for rapid diagnosis in hemoglobinuria. The absorbance of Hb at 425 nm is monitored using an alexandrite laser-pumped, room temperature, LiF:F+2. color-center pulsed laser. A minimum absorbance detection level of 2.57×10-4is achieved, corresponding to a minimum detectable concentration of Hb in urea of 5.8 nM. A multilayered Hb biofilm is formed, and a minimum of eight layers are required to model the adsorption isotherm, allowing for cooperative binding within the layers and extending 56 nm into the interface. A binding constant for Hb to silica 18.23±7.58×106M is derived, and a binding constant for Hb to Hb in subsequent layers is determined to be 5.631 ±0.432 ×105M. Stoichiometric binding coefficients of 1.530±0.981 for layer one and 1.792±0.162 for subsequent layers suggest that cooperative binding both to the silica surface and between the layers of the biofilm is important. © 2005 Society of Photo-Optical Instrumentation Engineers.

Evanescent wave cavity ring-down spectroscopy (EW-CRDS) has been used to investigate the adsorption of crystal violet (CV+) to a charged silica-water interface as a function of bulk pH by the direct measurement of the absorbance of the CV+ chromophore. Absolute absorbances of order 10-4 have been routinely detected, showing significant variation in the structure of the silica-water interface. At low ionic strength, the interfacial absorbance of CV+ shows a monotonic increase with increasing pH. A simple competitive Langmuir adsorption model, which provides values for the silica surface parameters that are in broad agreement with the existing literature values, has been fit to the data. In addition, interfacial absorbance has been monitored as a function of pH for CV+ solutions maintained at high ionic strength with NaCl, KCl, and CaCl2. As pH increases, the CV+ interfacial absorbance exhibits a pronounced maximum, which occurs at pH 8.7 for Na+ and K+ and pH 7.9 for Ca2+, followed by a sharp decrease. This trend is attributed to competitive binding between the metal cations and CV+ to the silica surface binding site, and it has not been observed in previous measurements using second-harmonic generation. The simple Langmuir model, however, does not accurately describe the high ionic strength behavior.

A series of careful double resonance Zeeman experiments were performed on the 21.8 GHz doublet leading to an assignment of the quantum numbers involved in this transition. A similar spin Hamiltonian analysis of the hyperfine structure of the 15.2 GHz multiplet also suggested assignments for this transition. Significant results were derived from an extensive search of the microwave and millimeter-wave region of the spectrum with 6-170 GHz radiation.

The full interaction potential between Ne(1S) and Ne+ (2P), determined by least-squares fitting of potential parameters to spectroscopic data from the near-dissociation microwave spectra of the Ne2+ complex, was discussed. The interaction potential incorporated the potential curves for six electronic states correlating with Ne(1S) + Ne1 (2P) and the couplings between them. The analysis showed that the Ne2+ ions were vibrationally and rotationally hot.

We have used an ion beam method to observe 276 transitions in the microwave electronic spectrum of the long-range Ne. Ne+ ionic complex, involving levels which lie within 10.2 cm-1 of the lowest dissociation limit. An electric field dissociation technique has been used to access directly levels with binding energies of up to 6.8 cm-1 and we have been able to construct an experimental energy level pattern for at least 17 vibration-rotation progressions in this region of the potential. Microwave- microwave double resonance and Zeeman effect measurements have been crucial in establishing this energy level pattern. The levels are populated by the vertical electron impact ionisation of the neutral neon dimer, produced by means of a nozzle beam, with some population of higher rotational levels of the dimer ion probably resulting from larger cluster fragmentation. A case (c) effective Hamiltonian analysis has been successful in describing the levels in six of the progressions but a full theoretical analysis requires a coupled-states calculation. Analysis of Zeeman splittings for the microwave lines identifies the J values of the levels involved. The observed g-factors provide information about the transition from case (c) to case (e) coupling as both the rotational quantum number increases and the energy levels become more weakly bound.

We have observed a microwave spectrum of the HeKr+ ion in which all of the observed levels lie within a few cm-1 of either the first or second dissociation limit. We use an ion beam technique in which HeKr+ ions, formed by electron impact, are mass analyzed. Passage of the ion beam through an electric field lens results in selective fragmentation of energy levels lying close to dissociation. Kr+ ions formed in the lens are separated from all other ions by means of an electrostatic analyzer, and are detected with an electron multiplier. Microwave radiation induces transitions which result in population transfer and produce detected changes in the electric field-induced Kr+ fragment ion current. Additional transitions have been detected by a microwave-microwave double resonance method, and we have also made extensive use of the Zeeman effects produced by small applied coaxial magnetic fields to identify the J quantum numbers of the levels involved. Coupled channel calculations of the bound states of the He⋯Kr+ ion are carried out, fully including all the couplings between different electronic states correlating with He+Kr+ (2P3/2 and 2P1/2)- the calculations allow the spectra to be assigned to pure rotational transitions involving levels in the X, A1, and A2 states that lie within 2.5 cm-1 of the dissociation limits. Because of a systematic near degeneracy between vibrational levels in the X and A1 states, the long-range He⋯Kr+ ion provides a very good example of Hund's case (e) in the form introduced by Mulliken, in which there are no projection quantum numbers onto the interatomic axis. Mulliken's case (e) is rather different from the Rydberg case (e) described by Lefebvre-Brion, and this is the first time that Mulliken's case (e) has been observed. The spectra allow the interaction potential for He⋯Kr+ to be determined accurately, for the first time, by least-squares fitting of potential parameters to the experimental line frequencies and g factors. The resulting interaction potential (designated MALI) is compared with that previously determined for He⋯Ar+: the He⋯Kr+ potential is significantly shallower, because the long-range ion-induced dipole C4 coefficient is the same for the two systems but the larger Kr+ ion prevents the He atom approaching as close. © 1996 American Institute of Physics.

We report the first spectroscopic observation of the HeN+ molecular ion. We use a technique in which a mass-selected ion beam is exposed to tunable microwave radiation, and transitions involving the weakly bound long-range levels are detected by means of state-selective electric field dissociation, producing N+ fragment ions. Three of the observed microwave transitions exhibit 14N hyperfine structure, whilst a fourth transition consists of a single unsplit line. Preliminary observations of Zeeman effects are also described. The results are discussed in terms of molecular energy levels lying close to one or other of the three He(1S) + N+(3P) dissociation asymptotes, which are split by spin-orbit coupling.

We report the first spectroscopic observations of the HeH+2 molecular ion. We use an ion beam technique in which mass-selected ions are exposed to microwave radiation, and transitions involving weakly bound long-range levels are detected by means of state-selective electric field dissociation. One of the observed transitions exhibits complex proton nuclear hyperfine structure; another shows a simple field-free spectrum but extensive Zeeman structure in the presence of an applied magnetic field. The results are discussed in terms of coupled-channel calculations using a recent ab initio potential surface.

Ion-beam techniques provide opportunities for extremely sensitive spectroscopic studies of molecular ions. Transitions involving the energy levels lying close to the dissociation limit may be detected through a range of indirect methods; electric field dissociation has proved to be particularly valuable. This review describes IR and microwave studies of a range of molecular ions, mostly diatomic; we compare the different detection methods and the molecular information each provides. These investigations have revealed the first electronic spectra of the H2+ and D2+ ions, leading to detailed characterisations of the long-range H⋯H+ and D⋯D+ charge-induced-dipole states, and also providing accurate data with which to test ab initio calculations. In the case of the HD+ ion the high spectroscopic resolution achieved enabled the nuclear hyperfine interactions to be measured; these revealed marked asymmetry in the electron distribution as the dissociation asymptote is approached. H3+, the only polyatomic molecule to be investigated in our laboratory so far, exhibits an extraordinarily rich and complex IR predissociation spectrum, which arises from metastable periodic states embedded in a chaotic sea of levels lying above the lowest dissociation limit. Other examples of predissociation spectra involving rotationally quasibound levels are described. In recent work the first 37 bound levels of the long-range He⋯Ar+ complex, counting from the dissociation limit downwards, have been identified and described theoretically. In this very weakly bound region the Born-Oppenheimer approximation collapses because of extremely strong coupling between the electronic and nuclear rotational motions. Finally, we describe new experiments using a combined neutral-ion beam source which has enabled the first state-to-state resonant spectra of the Ar2+ and Ne2+ molecular ions to be observed.

We describe a new ion source in which a nozzle beam of neutral molecules is ionised by an electron beam and the resulting molecular ion beam is studied by microwave spectroscopic methods. This ionisation technique results in significant population of the near-dissociation levels of the molecular ion, so that a sensitive state-selective electric field dissociation method can be used to detect the spectra. We have observed the first state-to-state resonant electronic spectra of the Ar2+ and Ne2+ ions. © 1995.

We have measured and interpreted a microwave spectrum of the HeAr + ion in which all of the observed energy levels lie within 8 cm -1 of the lowest dissociation limit, He(1S)+Ar +(2P3/2). We use an ion beam technique in which the HeAr+ ions are formed by electron impact, accelerated to kilovolt potentials, and mass-analyzed. After passage through an appropriate section of waveguide, the ions enter an electric field lens in which state-selective fragmentation occurs; the Ar+ ions produced in the lens are separated from all other ions by means of an electrostatic analyser and detected with an electron multiplier. Microwave transitions induced in the waveguide section result in population transfer which produces detected changes in the electric field-induced Ar+ fragment current. Many transitions have also been observed by a microwave-microwave double resonance technique. We have observed 68 lines spanning the frequency range 6-170 GHz; no immediately recognizable pattern is apparent. We have measured the Zeeman splitting produced by a small axial magnetic field for almost every line, which enables us to determine the values of the total angular momentum J involved in each transition, and also effective g factors for the two levels involved. We are therefore able to construct a purely experimental pattern of 37 levels lying within 8 cm-1 of the dissociation limit. The data are treated first by means of a conventional effective Hamiltonian in a case (c) basis, which allows electronic and vibrational quantum numbers to be assigned to most of the levels; the assignments are approximate, however, because very strong rotational-electronic coupling undermines the Born-Oppenheimer approximation. A more complete theoretical treatment is then presented, using the coupled-channel method in a case (e) representation to calculate the energy levels without making the Born-Oppenheimer approximation. The microwave transition frequencies and g-factors are fitted, together with earlier ultraviolet spectra, to provide a new interaction potential (designated MAL1) for He interacting with Ar +(2P3/2 and 2P1/2). The MAL1 potential is substantially more accurate than previous potentials, especially in the long-range region and for the A12Π3/2 state, which had not been observed before. An important new feature of the MAL1 potential is that the long-range C6 coefficient is strongly anisotropic, so that the different electronic curves have substantially different C6 coefficients. © 1995 American Institute of Physics.

We have extended our earlier microwave and IR measurements of the 2pσu-1sσg electronic spectrum of D2+ into the millimetre-wave region of the spectrum, and report the measurement of a further eight vibration-rotation components. The measured transition frequencies and the nuclear hyperfine splittings are very close to those predicted by the best ab initio calculations.

Chemielectron and chemiion spectra have been recorded for the reaction of oxygen atoms with 2-butyne. Two chemielectron bands have been observed with band maxima at 0.06 ± 0.04 and 0.27 ± 0.06 eV whose relative intensity depends on the relative partial pressure of the reactants used. The chemiion spectra show that HCO+ and CH3CO+ are primary ions, although the relative intensity of the ion signals is affected by ion-molecule reactions in the reaction cell used. The experimental evidence suggests that the two chemielectron bands can be assigned to the associative ionization reactions O + CH(X+Π) → HCO+ + e- and O + CH3C → CH3CO+ + e-. It is not possible to determine conclusively the electronic state of CH3C involved in the O + CH3C chemiionization reaction, although some evidence is presented that indicates that the CH3C(á4A2) + O(3P) reaction gives rise to the 0.27-eV chemielectron band. © 1994 American Chemical Society.

We have oberved microwave spectra of the inert gas diatomic ions He+2, HeAr+, HeAr+ and NeAr+. The spectra arise from transitions between levels which are within a few cm-1 of the lowest dissociation limit. We use mass-selected ion beams which pass first through a waveguide cell, and then through an electric field lens in which selective fragmentation of weakly bound levels occurs. Fragment ions produced in the lens are separated by an electrostatic analyser and detected; spectra are observed by measuring changes in the fragment ion currents. The results are discussed in terms of a theoretical model which relates the high-lying vibration-rotation levels of the diatomic ions to their atomic dissociation limits. © 1993.