Disk-based one-dimensional photonic crystal slabs for label-free immunosensing.

One-dimensional photonic crystal slabs are periodic optical nanostructures that produce guided-mode resonance. They couple part of the incident light into the waveguide generating bandgaps in the transmittance spectrum, whose position is sensitive to refractive index variations on their surface. In this study, we present one-dimensional photonic crystal slab biosensors based on the internal nanogrooved structure of Blu-ray disks for label-free immunosensing. We demonstrated that this polycarbonate structure coated with a critical thickness of TiO2 generates guided-mode resonance. Its optical behavior was established comparing it with other compact disk structures. The results were theoretically calculated and experimentally demonstrated, all them being in agreement. The bioanalytical performance of these photonic crystals was experimentally demonstrated in a model assay to quantify IgGs as well as in two immunoassays to determine the biomarkers C-reactive protein and lactate dehydrogenase (detection limits of 0.1, 87, and 13 nM, respectively). The results are promising towards the development of new low-cost, portable, and label-free optical biosensors that join these photonic crystals with dedicated bioanalytical scanners based on compact disk drives.

Gold, carbon, and aluminum low-reflectivity compact discs as microassaying platforms.

Compact disc (CD) surface modification with different reflective materials offers new working perspectives as bioanalytical platforms. The potential of gold, carbon, and aluminum low-reflectivity compact discs as a substrate for microassaying is presented. CD polycarbonate bases were coated with these reflecting materials, maintaining optical properties. Probes immobilization onto reflective layers was studied by both passive adsorption and covalent linking. Different chemical modifications were made on substrates to provide functional groups capable of anchoring probes with primary amines covalently. Thus, self-assembled monolayers were performed by chemisorption of 11-mercaptoundecanoic acid on gold surfaces, and silanization with N-(trimethoxysilylpropyl)ethylenediamine triacetic acid on aluminum to provide a carboxylic acid functional group. Carbon oxidation with oxygen plasma afforded similar functionalization on the discs coated with this material. Performance of the studied materials as reflective layers was evaluated, and as proof of concept, a microimmunoassay for a neurotoxic compound was studied on the three surfaces. The results show the possibility of doing assays on this new supports with good analytical performances while maintaining discs' optical and mechanical properties to be read by a CD player.

Aluminum oxide nanoparticles as carriers and adjuvants for eliciting antibodies from non-immunogenic haptens.

Functionalized nanomaterials have applications yet to be discovered, especially in the biological field and, in particular, the in vivo production of antibodies. In this paper, we show that aluminum oxide nanoparticles that are covalently coupled to haptens (small molecular mass compounds) activate the immune system, eliciting antibodies (polyclonal and monoclonal) specific for the herbicide atrazine, the antibiotic sulfasalazine, and the vitamin biotin in mice and rabbits. The particles play the role of carrier and adjuvant, with the immune response being dependent on size and crystallinity. The affinity constants of the antibodies are similar to those reached with traditional immunization strategies based on the use of carrier proteins. This approach has not been previously described, being of scientific and practical interest, as it can lead to immunogens safer than the conventional ones, potentially applicable to human vaccination. In addition, the useful advantages of this technique include the stability of the metallic particles, the synthesis of immunogens in organic media, the versatility of particle derivatization, the ease of purification, the full chemical characterization of immunogens, the lack of a requirement for an adjuvant, the reduction of the cross reactivity, and the low cost of materials. Also, the analytical performances of the immunoassays developed using these antibodies are comparable to those obtained by the standard protocols. Analytical applications of the developed ELISAs were fully demonstrated. Characterization (IgG nature, affinity constants, etc.) of the immunoreagents were also fully assessed.

Antibody-capped mesoporous nanoscopic materials: design of a probe for the selective chromo-fluorogenic detection of finasteride.

The synthesis of capped mesoporous silica nanoparticles (MSN) conjugated with an antibody (AB) as a gatekeeper has been carried out in order to obtain a delivery system able to release an entrapped cargo (dye) in the presence of a target molecule (antigen) to which the conjugated antibody binds selectively. In particular, MSN loaded with rhodamine B and functionalized on the external surface with a suitable derivative of N-(t-butyl)-3-oxo-(5α,17ß)-4-aza-androst-1-ene-17-carboxamide (finasteride) have been prepared (S1). The addition of polyclonal antibodies against finasteride induced capping of the pores due to the interaction with the anchored hapten-like finasteride derivative to give a MSN-hapten-AB nanoparticle S1-AB. It was found that the addition of capped material S1-AB to water solutions containing finasteride resulted in displacement of the antibody, pore uncapping and entrapped-dye release. The response of the gated material is highly selective, and only finasteride, among other steroids, was able to induce a significant uncapping process. Compared with finasteride, the finasteride metabolite was able to release 17 % of the dye, whereas the exogen steroids testosterone, metenolone and 16-ß-hydroxystanozolol only induced very little release of rhodamine B (lower than 10 %) from aqueous suspensions containing sensing solid S1-AB. A detection limit as low as 20 ppb was found for the fluorimetric detection of finasteride. In order to evaluate a possible application of the material for label-free detection of finasteride, the capped material was isolated and stored to give final sensing solid S1-AB-i. It was found to display a similar behavior towards finasteride as to that shown by freshly prepared S1-AB; even after a period of two months, no significant loss of selectivity or sensitivity was noted. Moreover, to study the application for the detection of finasteride in biological samples, this "aged" material, S1-AB-i, was tested using commercially available blank urine as matrix. Samples containing 70 and 90 % blank urine were spiked with a defined amount of finasteride, and the concentration was determined using capped S1-AB-i. Recovery ranges from 94 % to 118 % were reached.

Enzyme-linked immunosorbent assays for the synthetic steroid gestrinone.

Gestrinone is a synthetic steroid hormone with anti-estrogenic and anti-progesterone properties. It is used to treat endometriosis, shrink uterine fibroids and reduce menorrhagia; besides, it has been investigated for use as contraceptive. Also, due to its anabolic effects, it has been included in the banned list of performance enhanced drugs in sport. Polyclonal antibodies raised against bovine serum albumin coupled to gestrinone 3-carboxymethyloxime (3OCMO-G) were used to develop two highly sensitive and specific enzyme-linked immunosorbent assays for gestrinone. One of them, based on direct format, shows a detection limit (LD) of 0.09+/-0.03 ng L(-1). The second assay, hapten-protein coating format, can detect until (LD) 0.14+/-0.05 ng L(-1). Both immunoassays were also highly specific, showing negligible or no cross-reactivity to other anabolic steroids. The developed ELISAs detected lower amounts of gestrinone than those determined by the reference chromatographic HPLC/MS/MS methods. The direct format was applied to quantify this steroid in spiked human urine without sample pre-treatment, with recovery values between 76 and 122%.

Enzyme-linked immunosorbent assays for doping control of 5alpha-reductase inhibitors finasteride and dutasteride.

Finasteride and dutasteride are 5alpha-reductase inhibitors included in the World Anti-Doping Agency's list of banned substances. Two highly sensitive and selective ELISA assays were developed for these compounds. Polyclonal rabbit antibodies were raised using synthesized haptens and other commercial products. The best immunoassay obtained, based on an antibody-coated format, showed a limit of detection of 0.01 microg L(-1) and an IC(50) of 0.75 microg L(-1) for finasteride (cross-reactivity with dutasteride<4%). The second assay allowed finasteride and dutasteride determination, with limits of detection of 0.013 and 0.021 microg L(-1), and IC(50) values 0.18 and 1.18 microg L(-1), respectively. Both assays were highly selective to a set of anabolic steroids, but they showed 37% and 30% cross-reactivity with the major urinary metabolite of finasteride, allowing its determination. The developed ELISA had better sensitivity than HPLC/MS/MS method and was applied as a screening technique to quantify dutasteride, finasteride, and its main metabolite in human urine without sample pre-treatment. Moreover, the analysis of dutasteride's excretion urines by ELISA was used to obtain its human excretion rate, essential to improve the analytical strategies about this type of drugs (permitted as medicines and prohibited in sport) and to establish an effective anti-doping policy.

Development of immunoassays to determinate sulfamethoxazole residues in wastewaters.

Different immunoassays have been developed to monitor sulfamethoxazole (SMX), an antibiotic frequently detected in water. The immunoassays were developed and optimized in two different formats: ELISA in polystyrene 96-well plate and microarray on compact disc (CD) support. Competitive microimmunoassays were performed by direct adsorption of immunoreagents on the polycarbonate surface of a low-reflectivity CD. Results were displayed using nanogold-labeled immunoglobulins and silver staining developer. High sensitivity was achieved with both formats: LD 0.001 ng mL(-1) in the ELISA-plate, and 0.09 ng mL(-1) in the CD format. The novel CD methodology presents advantages such as simplicity, sensitivity, portability, analytical capacity (2560 spots per disc), in addition to reductions in immunoreagents required, costs and time for analysis. Both proposed methods were applied to determine SMX at nanograms per litre level in wastewater samples without previous preconcentration. Finally, the determination of several fortified wastewater samples using the CD format protocol showed a mean recovery of 87%, which confirms that this assay format is a good screening tool to detect sulfamethoxazole in water samples rapidly and reliably.

Selective enzyme-linked immunosorbent assay for triclosan. Application to wastewater treatment plant effluents.

A sensitive and highly selective enzyme-linked immunosorbent assay was developed for triclosan, one of the most common bactericides in personal care products, which is also considered as an emerging contaminant, given its presence in the effluents of sewage and wastewater treatment plants. From synthesized haptens, polyclonal rabbit antibodies against triclosan were raised. The best ELISA immunoassay was based on an antibody-coated format, yielding a detection limit of 0.03 microg L(-1), an I50 of 3.85 microg L(-1), and a dynamic range from 0.22 to 42.16 microg L(-1), with little or no cross-reactivity (< 10%) to similarly structured compounds, including its metabolite methyltriclosan (CR < 6%). The assay was applied as a screening method to quantify triclosan in surface water and in wastewaters. After C18 solid-phase extraction, nanogram per liter concentrations were determined in effluents of urban wastewater treatment plants. The satisfactory recoveries achieved as well as the agreement between immunochemical and chromatographic methods (GC-MS) indicate its potential for either screening or laboratory quantification.

Highly sensitive enzyme-linked immunosorbent assay for chlorpyrifos. Application to olive oil analysis.

Highly sensitive enzyme-linked immunoassays for chlorpyrifos, one of the most applied insecticides, are presented. Several haptens were synthesized for immunoreagent production and ELISA development. The best immunoassays obtained are based on an indirect coated-plate immunoassay format. Two assays were optimized; one shows a limit of detection of 0.3 ng L(-1), an I50 of 271 ng L(-1), and a dynamic range between 4 and 16 474 ng L(-1). The other one has a limit of detection of 0.07 ng L(-1), an I50 of 7 ng L(-1), and a dynamic range between 0.4 and 302 ng L(-1). The assays were used to quantify chlorpyrifos in olive oil using a simple and rapid sample extraction procedure. The good recoveries achieved in both assays (109% mean value) and the agreement with values given by the GC reference method (110% mean value) indicate their potential for either screening or laboratory quantification.

Evaluation of a novel malathion immunoassay for groundwater and surface water analysis.

Malathion is an organophosphorus insecticide commonly used in crops and indoor applications. Negative effects of malathion on human health and ecosystems are of growing concern. In this work, novel malathion haptens are synthesized to develop an ELISA screening method. The immunoassay is based on a conjugate-coated format and shows a limit of detection of 0.11 ng/mL, an IC50 of 1.58 ng/mL, a dynamic range between 0.23 and 10.94 ng/ mL, and a cross-reactivity of <2% with structurally related compounds. The developed ELISA has been used to quantify malathion in groundwater and surface water samples. The good recoveries achieved and the agreement with those given by the GC-MS reference method indicate the potential of the assay for environmental monitoring of malathion in natural waters without purification or preconcentration steps. The effect of dissolved organic matter (humic acids) on the ELISA was evaluated, resulting in the conclusion that the immunoassay can be successfully performed in surface water samples with a humic acid content up to 10 mg/L, without sample pretreatment. Samples with a high humic acid content can be analyzed through ELISA after solid-phase extraction, eluting with acetone or acetonitrile.

Assessment of novel diazinon immunoassays for water analysis.

Diazinon is a broad organophosphate insecticide used in agricultural and other treatments, resulting in widespread water contamination. The development of easy-to-use screening immunoanalytical methods is an interesting tool to study environmental pollution impact. Two novel strategies for diazinon hapten synthesis are addressed. One of them attaches the spacer arm to the oxygen atom of the diazinon aromatic ring. The other one retains the diazinon basic structure linking the spacer to an aromatic carbon. A total of eight diazinon haptens were synthesized, demonstrating that they are suitable for immunoreagent (protein conjugates and polyclonal antibodies) production. The optimized ELISA is based on conjugate-coated format and had a detection limit of 0.40 microg/L, showing little or no cross-reactivity to similar tested compounds. The immunoassays were used as a tool to quantify diazinon in natural waters. Results are in agreement with those given by GC-MS reference method. Mean recoveries ranging between 99% and 105% confirm the potential of our approach to determine diazinon in samples without purification or preconcentration steps, being applied as a screening method for field monitoring of diazinon in river waters.

Enzyme-linked immunosorbent assay for the organophosphorus insecticide fenthion. Influence of hapten structure.

Novel procedures for fenthion hapten synthesis are described following three different strategies. The first one attaches the spacer arm to the oxygen atom of the aromatic fenthion ring. The second one binds it through the thiophosphate moiety and the third strategy consists on the attachment of the spacer arm to the sulfur atom of the molecule. A total of nine fenthion haptens have been synthesized and used for immunoreagent production (protein conjugates and polyclonal antibodies). The developed conjugate-coated format ELISA exhibited a detection limit of 0.03 ng/ml, an IC50 of 0.05 ng/ml and a dynamic range between 0.03 and 1 ng/ml. There was little or no cross-reactivity to similar tested compounds. The ELISA was used to determine fenthion residues in white wine samples without any purification or preconcentration steps. Recoveries ranged between 81% and 113%.