Nanogold bioconjugates for direct and sensitive multiplexed immunosensing.

The use of nanogold bioconjugates for direct detection of the antibody-antigen immunoreaction is addressed. The integration of gold nanoparticles tracers as signal generators in microarray immunosensing and compact disc detection technique show important advantages to reach sensitive, selective, high throughput, reliable and cost-effective assays. For that, a thorough study of the performances of the size of spherical nanogold particles and coating density was developed. The size of the nanoparticle determines the optimal antibody dilution, being the smaller particles the best performing ones. Enhancement effect of lower size is also studied. The gold labeling method do not affects the recognition capability of the labeled proteins. As a proof of concept, the nanoconjugates were used for the simultaneous and direct determination of small molecules. Employing nanogold bioconjugates as recognition labels resulted in robust and reliable assays, reaching a sensitivity of 0.03 and 1.3µg/L for sulfasalazine and atrazine, respectively. This shows that the use of nanogold bioconjugates for direct immunosensing is very competitive, achieving highly sensitive and reproducible assays (RSD<10%). This approach would simultaneously determine both small and large molecular size targets, in different formats, using the same detection mode what paves the way for many other applications in different scenarios.

Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis.

Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR-ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA-ELISA combination is proposed for amplification at a low, constant temperature (40°C) in a short time (40 min), for the hybridisation of labelled products to specific 5'-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA-ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA-ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings.

Detection of chemical residues in tangerine juices by a duplex immunoassay.

A rapid duplex ELISA for the simultaneous determination of two of the most widely used organophosphorous insecticides in tangerine juices is described. To accomplish this aim, two individual enzyme-linked immunosorbent assays for chlorpyrifos and fenthion pesticides were integrated into one ELISA test. The strategy uses 96-well plates with specific wells coated with the corresponding haptenized conjugate. The optimized duplex ELISA was accomplished within 40 min achieving a detection limit of 0.20±0.04 µg/L and 0.50±0.06 µg/L, for chlorpyrifos and fenthion, respectively in tangerine juice samples. The determination of residues of both pesticides was carried out by simple sample dilution, without any extra sample clean-up procedure. Results of testing precision, stability, and selectivity demonstrated that the assay provided reliable analytical performances for the simultaneous determination of residues of chlorpyrifos and fenthion in fruit juice samples below the established European maximum residue limits (MRL). In addition, the accuracy and reliability of this duplex bioanalytical method is demonstrated by analyzing blind spiked juice samples and the results, correlated well with those achieved using a well-established GC/MS method (recoveries between 95% and 106%).

Detection of food-borne pathogens with DNA arrays on disk.

A DNA oligonucleotide array for duplex pathogen detection on a DVD platform is developed. The assay involves hybridization of PCR products and optical detection using compact disc technology. Different DNA array constructions for attachment of synthetic oligonucleotides on to DVD surface are evaluated, finding that streptavidin-biotin coupling method yielded the highest sensitivity in combination with enzymatic signal amplification. Issues of importance for the DNA array construction such immobilized probes design, PCR product labeling strategy and composition of the hybridization buffer were addressed. The methodology was proved scoring single nucleotide polymorphisms with high selectivity. The assay capability was also demonstrated by the identification of two pathogenic microorganisms in powder milk samples. In fifty minutes, the DVD-array system identifies Salmonella spp. and Cronobacter spp. (previously named Enterobacter sakazakii) precise and simultaneously with a sensitivity of 10(0) and 10(2) cfu/mL, respectively, in infant milk. Results were in good agreement with those obtained by quantitative real-time PCR.

Real-time observation of antigen-antibody association using a low-cost biosensing system based on photonic bandgap structures.

In this letter, we present experimental results of antibody detection using a biosensor based on photonic bandgap structures, which are interrogated using a power-based readout technique. This interrogation method allows a real-time monitoring of the association process between the antigen probes and the target antibodies, allowing the instantaneous observation of any interaction event between molecules. because etunable lasers and optical spectrum analyzers are avoided for the readout, a drastic reduction of the final cost of the platform is obtained. Furthermore, the performance of the biosensing system is significantly enhanced due to the large number of data values obtained per second.

Bio-Photonic Sensing Cells over transparent substrates for anti-gestrinone antibodies biosensing.

In a previous work we introduced the term Bio-Photonic Sensing Cells (BICELLs), referred to periodic networks of nano-pillar suitable for biosensing when are vertically interrogated. In this article, we demonstrate the biosensing capabilities of a type of micrometric size BICELLs made of SU-8 nano-pillars fabricated over transparent substrates. We verify the biochips functionality comparing the theoretical simulations with the experimental results when are optically interrogated in transmission. We also demonstrate a sensitivity enhancement by reducing the pitch among nano-pillars from 800 to 700 nm. Thus, the Limit of Detection achievable in these types of BICELLs is in the order of 64 pg/mL for 700 nm in pitch among nano-pillars in comparison with 292 pg/mL for 800 nm in pitch when are interrogated by Fourier Transform Visible and Infrared Spectrometry. The experiments exhibited a good reproducibility with a relative standard deviation of 0.29% measured within 8 days for a specific concentration. Finally, BICELLs functionality was tested in real conditions with unpurified rabbit serum for detecting anti-gestrinone antibodies, demonstrating the high performance of this type of BICELLs to detect specific antibodies having immobilized the suitable bioreceptors onto the sensing surface.

Detection of sulphathiazole in honey samples using a lateral flow immunoassay.

A lateral flow immunoassay (LFIA) was developed in the competitive reaction format and applied to test sulphathiazole (STZ) residues in honey samples. To prepare the assay test, a hapten conjugate and goat antirabbit antiserum as capture and control reagent, respectively, were dispensed on nitrocellulose membrane. Polyclonal antiserum against sulphathiazole was conjugated to colloidal gold nanoparticles and used as the detection reagent. The visual limit of detection (cut-off value) of the sulphathiazole LFIA was 15ng/g, reaching qualitative results within 10min. The assay was evaluated with STZ spiked honey samples from different geographical origins (n=25). The results were in good agreement with those obtained from liquid chromatography separation and mass spectroscopy detection (LC-MS), indicating that the LFIA test might be used as a qualitative method for the determination of sulphathiazole residues without expensive equipment. The test was also highly specific, showing no cross-reactivity to other chemically similar antibiotics. To our knowledge, this is the only work where a development of LFIA tests for the detection of sulphathiazole residues is performed.

Microfluidic and transducer technologies for lab on a chip applications.

Point-of-care diagnostic devices typically require six distinct qualities: they must deliver at least the same sensitivity and selectivity, and for a cost per assay no greater than that of today's central lab technologies, deliver results in a short period of time (〈15 min at GP; 〈2h in hospital), be portable or at least small in scale, and require no or extremely little sample preparation. State-of-the-art devices deliver information of several markers in the same measurement.

Single-strand DNA detection using a planar photonic-crystal-waveguide-based sensor.

We report an experimental demonstration of single-strand DNA (ssDNA) detection at room temperature using a photonic-crystal-waveguide-based optical sensor. The sensor surface was previously biofunctionalized with ssDNA probes to be used as specific target receptors. Our experiments showed that it is possible to detect these hybridization events using planar photonic-crystal structures, reaching an estimated detection limit as low as 19.8 nM for the detection of the complementary DNA strand.

Label-free biosensing by means of periodic lattices of high aspect ratio SU-8 nano-pillars.

We developed biophotonic sensing arrays of 60x60 microm(2) made of periodic lattices of high aspect ratio SU-8 nano-pillars in order to demonstrate their capability for label-free molecule detection, as well as the sensitivity enhancement in comparison with a single layer of SU-8. The biophotonic sensing arrays, that we call BICELLs (Biophotonic sensing cells), are interrogated vertically by using micron spot size Fourier transform visible and IR spectrometry (FT-VIS-IR). We monitored the surface immobilization of bovine serum albumin (BSA) antigen and anti-BSA antibody (aBSA) recognition. The bioassay exhibits a limit of detection (LOD) in the order of 2 ng/ml limited by the wavenumber uncertainty during the interrogation process. We also estimated and compared the theoretical biolayer thickness with previous results.

Label-free optical biosensing with slot-waveguides.

We demonstrate label-free molecule detection by using an integrated biosensor based on a Si(3)N(4)/SiO(2) slot-waveguide microring resonator. Bovine serum albumin (BSA) and anti-BSA molecular binding events on the sensor surface are monitored through the measurement of resonant wavelength shifts with varying biomolecule concentrations. The biosensor exhibited sensitivities of 1.8 and 3.2 nm/(ng/mm(2)) for the detection of anti-BSA and BSA, respectively. The estimated detection limits are 28 and 16 pg/mm(2) for anti-BSA and BSA, respectively, limited by wavelength resolution.

Optical immunosensors for environmental monitoring: how far have we come?

Immunosensing has proved to be a very interesting research area. This review discusses what has actually been achieved in the field of optical immunosensing for environmental screening, and what still needs to be done. The review is presented from a practical point of view. In terms of the basic design of the immunosensor, there is a trend towards decreasing assay time; indeed, this has been reduced from 15-20 minutes to less than 5 minutes. Another goal is to simplify the manifold, and label-free approaches combining indirect assay formats and the detection of antibody binding are popular. Rapid displacement assays have also been investigated thoroughly. In terms of some important features of immunosensing devices, the reusability of the sensing element has been studied in great depth, and working lifetimes of more than five hundred assays can now be found for all assay formats. Multianalyte assays are now being investigated, and current systems are able to monitor 2-3 target compounds, although this number is set to increase greatly (to >30) in the near future. In this sense, an increasing number of publications can be found on microarrays intended for multianalyte determinations. The application of immunosensing to real situations is the main challenge. Immunosensors are barely commercialized and are yet to be established as research or routine tools, due to a lack of validated protocols for a wide range of sample matrices. Regarding compounds considered as analytes, some significant pollutants such as dioxins or pharmaceuticals are rarely chosen as targets, although the current tendency is towards a broader spectrum of analytes. New immunoreagents should be raised for these compounds, for use in immunosensors that can be used as screening tools.

Development of a simple extraction procedure for chlorpyrifos determination in food samples by immunoassay.

The suitability of immunoassay methodology for rapid and accurate determination of chlorpyrifos in vegetables was tested. The optimised ELISA detection limit was 0.32ng/ml, with a working range from 0.69 to 6.21ng/ml and an immunoassay test-mid point (IC(50)) of 2.08ng/ml. A rapid sample preparation procedure considering different parameters such as the amount of sample, volume of extractant, extraction time and dilution factor was optimised. The developed direct extraction (DE) and multiresidue (ME) standard procedures were performed in different fortified fresh and processed vegetable samples (tomato, bonnet pepper, bean, pea, asparagus, broccoli, watermelon, melon, lettuce, cucumber, celery and red pepper). Recoveries were in all cases in the whole range 85.2-108.9% for both DE and ME extracts. Also, the comparison of the results obtained by both immunochemical and chromatographic methods for spiked fruits and vegetables were good with a correlation coefficient (r) of 0.97.

Immunosensors for pollutants working in organic media. Study of performances of different tracers with luminescent detection.

A comparative study of enzymatic and non-enzymatic labels combined with luminescence detection, developed for immunosensing of pesticide residues (carbaryl, 1-naphthol, irgarol 1051) in organic media, is presented. Peroxidase and alkaline phosphatase enzymes with fluorogenic (3-p-hydroxyphenylpropanoic acid) and luminogenic (AMPPD derivative) substrates, respectively, were assessed as enzymatic markers. As an alternative, terbium(III) chelate, with time-resolved fluorescence detection, was evaluated as a non-enzymatic label. The best sensitivity was achieved by use of alkaline phosphatase in an immunocomplex capture assay format (I (50) values 0.06, 0.27, and 7.45 microg L(-1) in buffer, 1:1 methanol-buffer, and methanol, respectively). Results were also good (I (50) 1.00 and 6.30 microg L(-1) for water and aqueous-organic mixture, respectively) for Tb(III) chelate in an immobilized conjugate assay format. Use of alkaline phosphatase label to measure carbaryl (100 ng L(-1)) in different spiked river water samples, after solid-phase extraction and analyte elution with an ethyl acetate-methanol mixture, resulted in recoveries ranging from 81 to 98%, with acceptable precision (CV 4-14%, n=4).

Determination of atrazine and carbaryl pesticide residues in vegetable samples using a multianalyte dipstick immunoassay format.

A dipstick assay for the simultaneous determination of atrazine and carbaryl in vegetable samples was developed. The analytical method involved a fast extraction procedure followed by a multi-strip membrane test. The assay took 10 min, with a detection limit (naked eye) of 10 and 200 micro g l(-1) for atrazine and carbaryl, respectively. The cross-reactivities to related compounds tested were negligible except for propazine. Quantification of the pesticides was carried out by measuring the dot colour with a spectrophotometer. IC(50) values (inhibition constant at 50% of the maximum binding) of 2.04 microg l(-1) for atrazine and 92.8 microg l(-1) for carbaryl were achieved by this approach. Vegetable samples were extracted with MeOH. The proposed methodology was used to analyse atrazine and carbaryl and check for compliance with European Union maximum residue levels for vegetables. Recoveries (75-105%) were in agreement with those obtained by GC/MS or HPLC. Standard curves using 25% methanol/75% TBS were used for food sample assays using a multi-analyte dipstick. IC(50) values were 9.2 microg kg(-1) for atrazine and 179.2 microg kg(-1) for cabaryl.

Evaluation of enzyme-linked immunoassays for the determination of chloroacetanilides in water and soils.

Reliable and sensitive indirect ELISAs for the quantitative determination of metolachlor, alachlor, and acetochlor were developed. Each herbicide was conjugated to a carrier protein via thioether linkage, and the product was used either as an immunogen or to prepare coating conjugates. The suitability of using the same chemical strategy to raise polyclonal antibodies against chloroacetanilides structurally related compounds and their metabolites is discussed. Under best conditions, detection limits of 0.06, 0.3, and 0.4 microg/L for metolachlor, alachlor, and acetochlor were reached, respectively. The optimized ELISAs were also highly specific, showing little or no cross-reactivity to other similar compounds. Immunoassays were used as a toolto determine critical chloroacetanilide herbicides in water and soil samples without purification steps. The excellent recoveries obtained (mean value ranging between 90% and 98%) confirm the potential of this approach to control these herbicides in the environment being applied as a "screening" method either for field monitoring or laboratory.

Determination of L-ascorbic acid in Lycopersicon fruits by capillary zone electrophoresis.

This study shows an improved method for the determination of L-ascorbic acid (l-AA) in fruits of Lycopersicon by capillary zone electrophoresis (CZE). Two backgrounds electrolytes (BGEs) have been tested: (i) 400 mM borate at pH 8.0 and 1 x 10(-2)% hexadimethrine bromide, for the separation of Eulycopersicon subgenus species; and (ii) as in BGE(i) but supplemented with 20% (v/v) acetonitrile, for the separation of species of the Eriopersicon subgenus. The present procedures were compared with two routine methods-enzymatic assay and potentiometric titration with 2,6-dichlorophenol-indophenol. While these routine methods presented some difficulties in quantifying l-AA in several Lycopersicon fruits, CZE was successfully applied in all the analyzed samples. The proposed CZE protocols give lower detection limits (<0.4 microg ml(-1)); are cheaper, quicker, and highly reproducible; and can be applied to analyze large series of samples (ca. 50 samples per day) which is utmost importance, not only in screening trials for internal quality and tomato breeding programs, but also in systematic and routine characterization of Lycopersicon fruits.

Comparison of multianalyte immunosensor formats for on-line determination of organic compounds.

Studies leading to the development of multianalyte immunosensing approaches are presented herein. Competitive capture formats are developed for carbaryl, atrazine, and irgarol 1051 as target compounds. Three proposals have been tested: sequential, additive, and simultaneous formats. For individual determinations, the best format is the sequential mode; the additive mode is useful only for qualitative analyses; and the simultaneous mode is preferable for screening purposes. The proposed systems show to be advantageous over single-analyte sensors and other multianalyte approaches. In all cases, the sensitivity reached (limit of detection) is high enough for the analysis of samples containing levels of each pesticide lower than 0.1 microg/L, and sensor reusability is very good (>600 determinations). The applicability of the multianalyte immunosensors to on-line pollution surveillance in natural waters, as well as their advantages and limitations, is discussed.

Development of immunosensors for the analysis of 1-naphthol in organic media.

Immunosensor systems have been developed for the rapid determination of 1-naphthol. In this work, the comparison of performance of immunosensors working in aqueous and organic media was done. Direct, indirect and capture formats were studied. Immunoreagents were immobilized on controlled pore glass (CPG), hidroxysuccinimide agarose gel or on azlactone Protein A/G supports. The Protein A/G-based sensor showed the best performance. In aqueous media, a LOD of 16.2 microg l(-1) and a DR of 33.7-586.6 microg l(-1) were achieved employing Tween 20 at a concentration ranging from 0.01 to 0.05% v/v. Maximum sensitivity was reached with 0.025% of surfactant. Binary mixtures of methanol or acetonitrile with aqueous buffer and ternary mixtures of methanol/isopropanol or ethyl acetate/methanol with the same buffer were studied as organic media. The mixture 50% MeOH-50% 20 mM sodium phosphate, pH 8, with 0.05% (v/v) Tween 20 resulted to be the best. A detection limit of 12.0 microg l(-1) and a dynamic range of 53.6-17,756.0 microg l(-1) were reached. The recycling of Protein A/G-based sensor working in this media was about 300 assays. Preconcentration factors around 250 were achieved using methanol as extracting solvent. It has been demonstrated that the technique can be successful in carrying out the analysis of low solubility in water analytes, such as 1-naphthol. The sensors developed can use higher concentrations of organic solvent (up to 50% methanol) compared to ELISA. On the other hand, the advantage of preconcentration can also be taken for the use of the same procedure as recommended for standard sample treatments.

Current trends in immunoassay-based kits for pesticide analysis.

Detection of pesticides and their metabolites in food and environmental samples in real time is the goal of many industries. Immunoassay technology has several attributes that make it a useful tool for screening purposes (e.g., selectivity, sensitivity, portability, and rapid turnaround time). Approximately 90% of the developed immunoassays for the pesticide residue analysis use the ELISA technique. Commercial kits are tailored to target different analytes, thus eliminating in some cases the need for clean-up steps. The manageability of the immunoassay test kit, together with its accuracy and speed of analysis, allows the rapid determination in situ of many samples simultaneously. This article gives an overview on the applications of the immunokits for pesticide analysis in drinking water and foods, as well as examples of different immunoassay formats commonly used. Special attention is given to sample extraction and clean-up procedures. Application to the determination of common pesticides and their detection limit are summarized. Immunoassay kits offer many practical advantages, and the acceptance of these methods depends on several factors, including the demonstration of quality and validity compared with reference methods. Although the advantages of the technique and their applications to food industry quality control are scarcely referred to in the literature.