Multi-analyte homogenous immunoassay based on quenching of quantum dots by functionalized graphene.

We propose a homogenous multi-analyte immunoassay based on the quenching of quantum dot (QD) fluorescence by means of graphene. Two QDs with emission maxima at 636 and 607 nm were bound to antibodies selective for mouse or chicken immunoglobulins, respectively, and graphene functionalized with carboxylic moieties was employed to covalently link the respective antigen. The antibody-antigen interaction led graphene close enough to QDs to quench the QD fluorescence by resonance energy transfer. The addition of free antigens that competed with those linked to graphene acted as a "turn-on" effect on QD fluorescence. Fluorescence emitted by the two QDs could be recorded simultaneously since the QDs emitted light at different wavelengths while being excited at the same wavelength and proved to be linearly correlated with free antigen concentration. The developed assay allows measuring both antigens over 2-3 orders of magnitude and showed estimated limits of detection in the nanomolar range. This approach is thus a promising universal strategy to develop homogenous immunoassays for diverse antigens (cells, proteins, low-molecular-mass analytes) in a multi-analyte configuration.

Increased sensitivity of lateral flow immunoassay for ochratoxin A through silver enhancement.

Silver nucleation on gold has been exploited for signal amplification and has found application in several qualitative and quantitative bio-sensing techniques, thanks to the simplicity of the method and the high sensitivity achieved. Very recently, this technique has been tentatively applied to improve the performance of gold-based immunoassays. In this work, the exploitation of the signal amplification due to silver deposition on gold nanoparticles has been first applied to a competitive lateral flow immunoassay (LFIA). The signal enhancement due to silver allowed us to strongly reduce the amount of the competitor and of specific antibodies employed to build an LF device for measuring ochratoxin A (OTA), thus permitting the attainment of a highly sensitive assessment of OTA contamination, with a sensitivity gain of more than 10-fold compared to the gold-based LFIA that used the same immunoreagents and to all previously reported LFIA for measuring OTA. In addition, a less sensitive "quantitative" LFIA could be established, by suitably tuning competitor and antibody amounts, which was characterized by reproducible and accurate OTA determinations (RSD% 6-12%, recovery% 82-117%). The quantitative system allowed a reliable OTA quantification in wines and grape musts at the microgram per liter level requested by the European legislation, as demonstrated by a highly results obtained through the quantitative silver-enhanced LFIA and a reference HPLC-FLD on 30 samples.

A novel approach for a non competitive capillary electrophoresis immunoassay with laser-induced fluorescence detection for the determination of human serum albumin.

A new non competitive capillary electrophoresis immunoassay format based on a separation into a capillary modified by analyte immobilisation is described. The injection of an excess of labelled antibody off-line preincubated with the analyte allows the surface capture of the free antibody and the immunocomplex detection. It was developed using the human serum albumin (HSA) as analyte, two FITC-labelled antibodies and a HSA covalently linked capillary. Two calibration curves with good run-to-run reproducibility and LOD--respectively 14.0 nM for the FITC-polyclonal antibody and 9.0 nM for the FITC-monoclonal antibody--were achieved. The assay was applied to HSA determination in spiked samples of human urine with acceptable recoveries.

Solid-phase extraction of ochratoxin A from wine based on a binding hexapeptide prepared by combinatorial synthesis.

In this paper we describe the preparation of a hexapeptide library by combinatorial synthesis and the identification of a peptide with sequence Ser-Asn-Leu-His-Pro-Lys, which showed good affinity (K(eq)=3.4 x 10(4) M(-1)) towards the mycotoxin ochratoxin A (OTA). An immunoaffinity-like stationary phase supporting such a hexapeptide was used to develop a solid-phase extraction method for the quantification of OTA in wine samples at concentration levels down to 0.10 microg l(-1). Several different wine samples fortified with OTA at 2 and 4 microg l(-1) levels showed recovery of 94.7% and 98.4% at 2.0 and 4.0 microg l(-1), respectively, without any effect on the extraction efficiency of the matrix. The efficacy of this approach was successfully tested by comparison with an immunoaffinity extraction performed on commercial cartridges.

Validation of a qualitative immunochromatographic test for the noninvasive assessment of stress in dogs.

Salivary cortisol is regarded as a reliable parameter for the noninvasive assessment of the welfare of animals, because it is strictly related to stress levels. Several methods are available for salivary cortisol measurement in mammals, however rapid diagnostic test for detecting salivary cortisol are confined to humans. The availability of such non invasive diagnostic tools operable in situ would facilitate monitoring of animal welfare. The Cortisol stress™ test provides a simple and rapid tool to discriminate cortisol levels in canine saliva above or below 4ng/ml, which has been suggested as the cut-off value for distinguishing unstressed dogs from those experiencing stress. The test is based on a competitive immunochromatographic assay (ICT) using gold nanoparticles as probes, in which the color intensity of the Test line is inversely correlated to the salivary cortisol level. The qualitative result is obtained by the visual observation of the color formed on the Test line compared to that of the Control line We evaluated the accuracy of the test by determining salivary cortisol in 85 samples of canine saliva belonging to dogs with very variable age, sex, breed, and life history, and comparing the qualitative results to those obtained by a reference ELISA kit. Agreeing results were obtained through the two methods, and the ICT showed high diagnostic sensitivity, specificity and efficiency (100%, 98.4%, and 98.8%, respectively). Furthermore, we evaluated the precision of the test by an experimental design approach, which combines errors due to within-day and between-day variation with the biological variability, and demonstrated that the test could be reliably applied for correctly classifying canine samples, according to their salivary cortisol level. Moreover, we studied the shelf-life of the device in three experimental conditions. We confirmed the stability of the ICT at 4°C and 25°C for at least six months and observed similar results for an accelerated stability study conducted for 7days at 37°C, which suggest that the stability of ICT device could be estimated by the accelerated experiment alternatively to the real-time study.

A fluorescent immunochromatographic strip test using Quantum Dots for fumonisins detection.

A fluorescent immunochromatographic strip test (ICST) based on the use of Quantum Dots (QD) was developed and applied to detect fumonisins in maize samples. A limit of detection for fumonisin B1 of 2.8 µg L(-1) was achieved, with an analytical working range of 3-350 µg L(-1), corresponding to 30-3500 µg kg(-1) in maize flour samples, according with the extraction procedure. The time required to perform the analysis was 22 min, including sample preparation. Recovery values in the range from 91.4% to 105.4% with coefficients of variation not exceeding 5% were obtained for fortified and naturally contaminated maize flour samples. To evaluate the possible improvements due to the use of QD for ICST technology, we performed a direct comparison of the proposed QD-ICST to a gold nanoparticles- and a chemiluminescent-ICST previously developed for fumonisins detection, in which the same immunoreagents were employed.

Optimization of the cyclodextrin-assisted capillary electrophoresis separation of the enantiomers of phenoxyacid herbicides.

An ethylcarbonate derivative of beta-cyclodextrin (beta-CD) with three substituents per molecule, hydroxypropyl-beta-CD and native alpha-CD have been tested as resolving agents in the capillary zone electrophoresis (CZE) separation of the four enantiomers of the herbicides mecoprop and dichlorprop. The performances of the three compounds have been quantified by means of two-levels full factorial design and the inclusion constants were calculated from CZE migration time data. Possible structure of inclusion complexes have been proposed, on the basis of molecular mechanics simulations.

Molecularly imprinted solid-phase extraction sorbent for the clean-up of chlorinated phenoxyacids from aqueous samples.

A molecularly imprinted polymer (MIP) was synthesized using the herbicide 2,4,5-trichlorophenoxyacetic acid as a template, 4-vinylpyridine as an interacting monomer, ethylendimethacrylate as a cross-linker and a methanol-water mixture as a porogen. The binding properties and the selectivity of the polymer towards the template were investigated by frontal and zonal liquid chromatography. The polymer was used as a solid-phase extraction material for the clean-up of the template molecule and some related herbicides (2,4-dichlorophenoxyacetic acid, fenoprop, dichlorprop) from river water samples at a concentration level of ng/ml with quantitative recoveries comparable with those obtained with a traditional C18 reversed-phase column when analyzed by capillary electrophoresis. The results obtained show that the MIP-based approach to the solid-phase extraction is comparable with the more traditional solid-phase extraction with C18 reversed-phase columns in terms of recovery, but it is superior in terms of sample clean-up.

Chromatographic characterization of molecularly imprinted polymers binding the herbicide 2,4,5-trichlorophenoxyacetic acid.

Two polymers binding the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) were prepared by utilising the technique of the non-covalent molecular imprinting polymerisation in an aqueous medium. The polymers obtained were packed in HPLC columns and the effects of the mobile phase composition on the retention of the imprinting molecule and the selectivity of the stationary phases towards several analogous structures were studied by liquid chromatography. The columns showed a good level of selectivity towards the template and strictly related molecules. It was found that the molecular recognition mechanism acting on the columns was dependent on a combination of ion pair and hydrophobic interactions.

Multivariate analysis of the selectivity for a pentachlorophenol-imprinted polymer.

A pentachlorophenol (PCP)-imprinted polymer (MIP) was obtained by thermal polymerization of a mixture of template, 4-vinylpyridine and ethylene glycol dimethacrylate with molar ratio 1 +3 + 27, using as porogenic solvent methanol-water ( 3 + 1(v/v)). The polymer was packed in an HPLC column and selectivity towards 52 PCP-related phenols (22-chloro-, 21-alkyl-, 4-aryl-, 3-methoxy- and 6-polyphenols) was measured using acetonitrile-acetic acid (99 + 1(v/v)) as mobile phase. The same was made for a reference polymer obtained without pentachlorophenol (NIP). The molecular recognition properties of the imprinted polymer were expressed in terms of selectivity index (SI), calculated for each phenol as k(NIP)/k(MIP). Sixteen molecular descriptors were calculated for each molecule: qO, the partial charge of the phenolic oxygen atom; qH, the partial charge of the phenolic hydrogen atom; Deltaq, the absolute value of the difference qO - qH; HOMO, the highest occupied molecular orbital; LUMO, the lowest unoccupied molecular orbital; Deltaorb, absolute value of the difference HOMO - LUMO; micro(2), the square of total dipole moment; MW, the molecular weight; SAS, the solvent-accessible molecular surface area; hSAS, the hydrophobic solvent-accessible molecular surface area; Svdw, the van der Waals molecular surface area; hSvdw, the hydrophobic part of Svdw; MOv, the molecular ovality; RG, the radius of gyration; logP, the logarithm of n-octanol-water partition coefficient; pK, the phenolic dissociation constant. Correlations between selectivity index and these descriptors were searched utilizing multivariate principal component analysis (PCA). The multivariate model obtained by regression on the principal components correlate collectively several of the calculated descriptors with the polymer selectivity. The magnitude of the model's parameters shows that selectivity is strongly influenced by molecular descriptors having structural character, such as MW, hSvdw and logP, while the effect of molecular descriptors having electronic character, such as qO and pK, is much less marked.

Catalytic and spectroscopic characterisation of a copper-substituted alcohol dehydrogenase from yeast.

Yeast alcohol dehydrogenase (Y-ADH) is widely studied for its biotechnological importance and various attempts to improve its catalytic properties have been made. In this paper, a catalytically active metal-substituted Y-ADH was prepared in vitro by substituting one zinc atom with copper. EPR and Raman spectroscopy suggest that copper maintains the same co-ordination geometry as zinc in native Y-ADH. The active Cu-ADH shows lower substrate affinity and lower specific activity (SA) than native ADH, but greater than a previously obtained Co-ADH. Furthermore, Cu-ADH maintains its catalytic efficiency in a wider pH range than native enzyme.

The complexation of mercury (II) and organomercurial compounds by 8-hydroxyquinoline-bovine serum albumin conjugates.

The complexing properties of conjugates between 8-hydroxyquinoline and bovine serum albumin (Ox-BSA) towards inorganic and organic mercury were studied. Two Ox-BSA conjugates (different substitution ratio) were prepared and their complexing properties were studied. Through the use of titration curves with mercury (II), methylmercury and ethylmercury an evaluation of the complex stoichiometry and stability was obtained, showing that Ox-BSA has good affinity for all investigated mercuric compounds and that the stability increases in the order: Hg (II) < CH3Hg+ < C2H5Hg+, whatever conjugate is considered. Complexes show a stoichiometry of 1:1 between mercury and 8-hydroxyquinoline residues, except with the high substituted conjugate and Hg2+ ion. The skill of the high substituted conjugate to bind inorganic and organic mercury in the presence of NaCl was also studied. Organic mercuric complexes do not show significant modification due to NaCl. Nevertheless, considering inorganic mercury, the number of retained metal ions per protein molecule increases if the NaCl concentration becomes higher than 0.1 M, probably because at high NaCl concentrations 1:1 complexes between mercury and 8-hydroxyquinoline are preferred to 1:2 complexes.

Functionalized biopolymers as soluble macromolecular chelating agents.

Two different conjugates of bovine serum albumin (BSA) with lysine and a derivative of imidazole have been synthesized to obtain watersoluble macromolecules with binding properties against bivalent transition metal ions. Syntheses have been carried out using the 60 aminogroups or the 99 carboxylic groups on BSA for the coupling reactions, with such molar ratios able to produce highly substituted BSA. The skill of each conjugate to bind metal ions in aqueous medium was studied through the use of titration curves with some metal ions, characterized by a good affinity for the free ligand. Both the conjugates allow us to recover a high number of metal ions per protein molecule, close to the number of ligand molecules on the BSA surface in the case of the lysine conjugate, whereas in the case of the imidazole conjugate M3L complexes are performed.

Molecular recognition of polycyclic aromatic hydrocarbons by pyrene-imprinted microspheres.

Pyrene-imprinted microbeads that display molecular recognition towards polyaromatic hydrocarbons (PAHs) were obtained by the aqueous suspension thermopolymerization of a mixture of template, 4-vinylpyridine and divinylbenzene in the molar ratio of 1:8:40. The microbeads were packed into an HPLC column and the retention behaviour of pyrene in the presence of eluents of increasing polarity was investigated by measuring the binding capacity and the imprinting factor. Selectivity was evaluated by eluting pyrene and 22 other related PAHs in the HPLC column when equilibrated with acetonitrile-dichloromethane 4:1 (v/v). Twelve molecular descriptors were calculated for each PAH molecule: MW, the molecular weight; SAS, the solvent-accessible molecular surface area; Svdw, the van der Waals molecular surface area; Vol, the van der Waals molecular volume; MOv, the molecular ovality; RG, the radius of gyration; B/L, the breadth-to-length ratio; micro(2), the square of the total dipole moment; HOMO, the highest occupied molecular orbital; LUMO, the lowest unoccupied molecular orbital; Deltaorb, the absolute value of the difference between the HOMO and LUMO; log P, the logarithm of the n-octanol-water partition coefficient. Quantitative structure-retention relationships between the logarithm of the capacity factors and these descriptors were searched for using a multiple linear regression (MLR) method. The best regression models obtained showed that the capacity factor correlated well with those molecular descriptors which had structural character, such as logP, while the effect of the molecular descriptors with electronic character was negligible. The results obtained indicate that the molecular recognition of PAHs by the imprinted polymer is controlled by the shape and dimension of the binding sites through hydrophobic interactions.

Binding properties of 2,4,5-trichlorophenoxyacetic acid-imprinted polymers prepared with different molar ratios between template and functional monomer.

Several molecularly-imprinted polymers binding the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) were prepared with a molar ratio between the functional monomer and the template molecule in the pre-polymerisation mixture set between 1+2 and 20+1. The functional monomer used was 4-vinylpyridine (4-VP), the cross-linker was ethylene dimethacrylate, and the porogenic solvent was a mixture of methanol-water 3+1 (v/v). The polymers obtained were grinded, sieved and packed in 100 mm x3.9 mm HPLC columns. The effects of the mobile phase composition were evaluated by eluting the columns with acetonitrile-water mixtures. The results obtained indicate that column capacity, selectivity factor and the imprinting effect are controlled by ion-pair and hydrophobic interactions between the analyte and the stationary phase. In the full range of ratios considered, column capacity, selectivity factor and imprinting effect are inversely proportional to the molar ratio between the template molecule and the functional monomer.

A general method to perform a noncompetitive immunoassay for small molecules.

A new general method to perform a noncompetitive immunoassay for low-molecular-mass analytes (less than 6000 Da) is described and checked using cortisol as a model system. The method is based on the use of a "polydentate ligand" (cortisol-poly(L-lysine) conjugate) able to block the antibody sites unoccupied by the analyte, followed by the replacement of an antibody-bound analyte by an enzyme-labeled analyte (cortisol-horseradish peroxidase), and permits the direct measurement of the analyte bound sites. The observed signal shows a near-linear correlation with the analyte concentration. The characteristics of interactions between the analyte and polydentate ligand with the specific antibody were studied to perform a preliminary evaluation of the noncompetitive immunoassay for cortisol. The noncompetitive assay was compared with a competitive immunoassay obtained under the same conditions and using the same reagents. The results of the experiments showed a lower detection limit for the noncompetitive model (0.15 ng mL-1 rather than 0.72 ng mL-1), emphasizing that the model is successful. Moreover, as the polydentate ligand is prepared from the same hapten used for the immunogen synthesis, this type of noncompetitive immunoassay appears generally applicable to all small molecules for which antibodies have been obtained.

Chromatographic characterization of a molecular imprinted polymer binding cortisol.

A cortisol-binding polymer was prepared by utilising a non-covalent molecular imprinting polymerisation technique. The obtained polymer was packed in a high-performance liquid chromatography (HPLC) column; the selectivity was studied by liquid chromatography, eluting cortisol, cortisone, corticosterone, progesterone, 11-ketoprogesterone, 11alpha-hydroxyprogesterone, 17alpha-hydroxyprogesterone, cortisol 21-hemisuccinate, and cortisol 21-acetate with chloroform, containing 0.5% (v/v) acetic acid, as mobile phase. The mechanism of molecular imprinting was confirmed and a good selectivity for cortisol, with limited recognition for cortisone and 11alpha-hydroxyprogesterone, was found.