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We describe the design, fabrication, and successful demonstration of a sample preparation module comprising bacteria cell capture and thermal lysis on-chip with potential applications in food sample pathogen analysis. Plasma nanotexturing of the polymeric substrate allows increase of the surface area of the chip and the antibody binding capacity. Three different anti-Salmonella antibodies were directly and covalently linked to plasma treated chips without any additional linker chemistry or other treatment. Then, the Ab-modified chips were tested for their capacity to bind bacteria in the concentration range of 10(2)-10(8) cells per mL; the module exhibited 100% efficiency in Salmonella enterica serovar Typhimurium bacteria capture for cell suspensions below 10(5) cells per mL (10(4) cells injected with a 100 μL sample volume) and efficiency higher than 50% for 10(7) cells per mL. Moreover, thermal lysis achieved on-chip from as low as 10 captured cells was demonstrated and shown to compare well with off-chip lysis. Excellent selectivity (over 1 : 300) was obtained in a sample containing, in addition to S. Typhimurium and E. coli bacteria.

Protein detection and characterization based on Broad-band Mach-Zehnder Interferometry is analytically outlined and demonstrated through a monolithic silicon microphotonic transducer. Arrays of silicon light emitting diodes and monomodal silicon nitride waveguides forming Mach-Zehnder interferometers were integrated on a silicon chip. Broad-band light enters the interferometers and exits sinusoidally modulated with two distinct spectral frequencies characteristic of the two polarizations. Deconvolution in the Fourier transform domain makes possible the separation of the two polarizations and the simultaneous monitoring of the TE and the TM signals. The dual polarization analysis over a broad spectral band makes possible the refractive index calculation of the binding adlayers as well as the distinction of effective medium changes into cover medium or adlayer ones. At the same time, multi-analyte detection at concentrations in the pM range is demonstrated.

A new method for direct covalent immobilization of protein molecules (including antibodies) on organic polymers with plasma-induced random micronanoscale topography and stable-in-time chemical functionality is presented. This is achieved using a short (1-5 min) plasma etching and simultaneous micronanotexturing process, followed by a fast thermal annealing step, which induces accelerated hydrophobic recovery while preserving important chemical functionality created by the plasma. Surface-bound biomolecules resist harsh washing with sodium dodecyl sulfate and other detergents even at elevated temperatures, losing less than 40% of the biomolecules bound even at the harshest washing conditions. X-ray photoelectron spectroscopy, secondary-ion mass spectrometry, and electron paramagnetic resonance are used to unveil the chemical modification of the plasma-treated and stabilized surfaces. The nanotextured and chemically stabilized surfaces are used as substrates for the development of immunochemical assays for the sensitive detection of C-reactive protein and salmonella lipopolysaccharides through immobilization of the respective analyte-specific antibodies onto them. Such substrates are stable for a period of 1 year with ambient storage.

A novel immobilization approach involving binding of preformed streptavidin/biotinylated oligonucleotide conjugates onto surfaces coated with biotinylated bovine serum albumin is presented. Microarrays prepared according to the proposed method were compared, in terms of detection sensitivity and specificity, with other immobilization schemes employing coupling of biotinylated oligonucleotides onto directly adsorbed surface streptavidin, or sequential coupling of streptavidin and biotinylated oligonucleotides onto a layer of adsorbed biotinylated bovine serum albumin. A comparison was performed employing biotinylated oligonucleotides corresponding to wild- and mutant-type sequences of seven single point mutations of the BRCA1 gene. With respect to the other immobilization protocols, the proposed oligonucleotide immobilization approach offered the highest hybridization signals (at least 5 times higher) and permitted more elaborative washings, thus providing considerably higher discrimination between complimentary and non-complementary DNA sequences for all mutations tested. In addition, the hybridization kinetics were significantly enhanced compared to two other immobilization protocols, permitting PCR sample analysis in less than 40 min. Thus, the proposed oligonucleotide immobilization approach offered improved detection sensitivity and discrimination ability along with considerably reduced analysis time, and it is expected to find wide application in DNA mutation detection., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A complete Mach-Zehnder interferometer monolithically integrated on silicon is presented and employed as a refractive index and bio-chemical sensor. The device consists of broad-band light sources optically coupled to photodetectors through monomodal waveguides forming arrays of Mach-Zehnder interferometers, all components being monolithically integrated on silicon through mainstream silicon technology. The interferometer is photonically engineered in a way that the phase difference of light travelling through the sensing and reference arms is approximately wavelength independent. Consequently, upon effective medium changes, it becomes feasible even with a broad-band source to induce sinusoidal-type of detector photocurrents similar to the classical monochromatic counterparts. The device is completed with its fluidic and interconnect components so that on chip interferometric measurements can be performed. Examples of refractive index and protein sensing are presented to establish the potential of the proposed device for real-time in situ monitoring applications. This is the only silicon device that has achieved complete on-chip interferometry.

Selective immobilization of proteins in well-defined patterns on substrates has recently attracted considerable attention as an enabling technology for applications ranging from biosensors and BioMEMS to tissue engineering. In this work, a method is reported for low-cost, large scale and high throughput, selective immobilization of proteins on nanopatterned Si, based on colloidal lithography and plasma processing to define the areas (<300 nm) where proteins are selectively immobilized. A close-packed monolayer of PS microparticles is deposited on oxidized Si and, either after microparticle size reduction or alternatively after metal deposition through the PS close-packed monolayer, is used as etching mask to define SiO2 nanoislands (on Si). C4F8 plasma was used to selectively etch and modify the SiO2 nanoislands while depositing a fluorocarbon layer on the Si surface. The plasma-treated surfaces were chemically characterized in terms of functional group identification through XPS analysis and reaction with specific molecules. Highly selective protein immobilization mainly through physical adsorption on SiO2 nanoislands and not on surrounding Si was observed after C4F8 plasma-induced chemical modification of the substrate. The thickness of the immobilized protein monolayer was estimated by means of AFM image analysis. The method reported herein constitutes a cost-efficient route toward rapid, large surface, and high-density patterning of biomolecules on solid supports that can be easily applied in BioMEMS or microanalytical systems.

Commercially available polystyrene (PS) slides were plasma nanotextured (nano-roughened) through treatment in oxygen plasma discharges to create substrates with increased surface area for microarray applications. Conditions of plasma treatment were determined for maximum and uniform oligonucleotide immobilization on these nanotextured PS slides. Oligonucleotides were immobilized onto the surface in the form of biotinylated oligonucleotide/streptavidin conjugates to take advantage of increased protein binding capacity of the substrate. It was found that the amount of oligonucleotides that could be immobilized was increased up to ten times on plasma treated as compared with untreated slides. The sensitivity of detection of labelled hybridized probes was improved by a factor of 20. Optimized nanotextured PS slides were subsequently used to develop a microarray for the detection of three deleterious BRCA1 gene mutations by immobilizing oligonucleotides corresponding to wild and mutant-type sequences. The microarray developed on the nanotextured PS slides provided higher specific hybridization signal and discrimination ratios as compared with flat untreated PS slides.

We demonstrate a method to create high density protein microarrays with excellent spot uniformity using photolithography and plasma processing on low cost commercially available microscope glass slides. Protein deposition and fluorescence signal evaluation on these substrates are performed by standard arrayers and scanners. To this end, spots of commercial photoresists (AZ5214, SU8 and Ormocomp(®)) were defined through lithography on glass substrates followed by short SF(6) plasma treatment and selective protein adsorption on these spots with respect to glass (spot to background fluorescence signal ratios 30:1 to 40:1) was demonstrated using model protein binding assays. Among the photoresists tested, Ormocomp was selected since it provided the highest protein binding capacity. No ageing of Ormocomp/glass substrates in terms of protein binding capacity was observed for at least two months. Besides to protein microarrays, DNA microarrays were also developed by spotting streptavidin-biotinylated oligonucleotide conjugates corresponding to wild- and mutant-type sequences of four deleterious BRCA1 gene mutations. For all of the examined mutations, higher specific hybridization signals (1.5-4 times) and improved discrimination ratios between wild- and mutant-type sequences as well as higher spot uniformity and repeatability were demonstrated on Ormocomp/glass substrates with intra- and inter-spot CVs of 8.0% and 4.5%, respectively, compared to commercial polystyrene (intra- and inter-spot CVs 36% and 18%) and epoxy-coated glass (intra- and inter-spot CVs 26% and 20%) slides. Thus, the proposed substrates can be readily applied to protein and DNA microarrays fabrication and, moreover, the described method for selective protein adsorption can be advantageously implemented in various analytical microdevices for multi-analyte detection., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Poly(methyl methacrylate) (PMMA) substrates were nanotextured through treatment in oxygen plasma to create substrates with increased surface area for protein microarray applications. Conditions of plasma treatment were found for maximum uniform protein adsorption on these nanotextured PMMA surfaces. Similar results were obtained using both a high-density plasma (HDP) and a low-density reactive ion etcher (RIE), suggesting independence from the plasma reactor type. The protein binding was evaluated by studying the adsorption of two model proteins, namely, biotinylated bovine serum albumin (b-BSA) and rabbit gamma-globulins (RgG). The immobilization of these proteins onto the surfaces was quantitatively determined through reaction with fluorescently labeled binding molecules. It was found that the adsorption of both proteins was increased up to 6-fold with plasma treatment compared to untreated surfaces and up to 4-fold compared to epoxy-coated glass slides. The sensitivity of detection was improved by 2 orders of magnitude. Moreover, highly homogeneous protein spots were created on optimized plasma-nanotextured surfaces through deposition with an automated microarray spotter, revealing the potential of plasma-nanotextured surfaces as protein microarray substrates.

In this work we present the development of a multi-analyte immunosensor for the determination of follitropin, human chorionic gonadotropin and prolactin in human serum. The immunosensor is based on plastic capillaries. According to the methodology, discrete areas of the internal capillary surface are coated with different antibodies, which are highly specific for each one of the analytes to be determined. The sample that will be analyzed along with a mixture of analyte-specific biotinylated antibodies is introduced into the capillary. The coated and the detection antibodies react with different epitopes of the analytes in the sample to form a 'sandwich'. The detection is based on reaction of the immobilized biotinylated antibody with streptavidin labeled with R-phycoerythrin. The fluorescent areas formed were quantified by scanning the capillary with a light beam of appropriate wavelength. A light sensor placed at the end of the capillary detects the emitted photons, that are trapped and waveguided into the capillary walls. The multi-analyte immunosensor assays were characterized by high specificity and short analysis time. In addition, the results obtained by the multi-analyte optical capillary immunosensor were comparable to those obtained by immunofluorimetric assays performed in microtitration wells. Potential applications of the proposed immunosensor include determination of several analyte panels in a broad spectrum of disciplines such as endocrinology, hematology, and oncology.

A complete antibody coating protocol for the preparation of dry antibody coated tubes is presented. This protocol is based on a recently described antibody immobilization principle. We modify this immobilization principle in order to improve and simplify the coating procedure. In addition, we propose a drying procedure that provides long-term storage stability of the antibody coated tubes. According to the modified protocol, polystyrene plastic tubes are first coated with rabbit gamma-globulins. The tubes are incubated with a sheep anti-rabbitIgG antiserum dilution. After incubation, antigen-specific antibody antiserum raised in rabbits is added directly into the tubes containing the sheep anti-rabbit IgG antiserum solution (difference from the original protocol). Finally, the tubes are washed, blocked, and dried following the drying procedure developed. The suitability of the modified protocol for the development of immunoassays requiring high loading of antibody was exemplified through the development of a RIA for total thyroxin. The estimated assay characteristics (detection limit 4 microg/L, dynamic range up to 210 microg/L, within-run CV 2.7-5.7%, between-run CV 5.1-7.3%, recovery 84.4-112%, cross-reactivity for T3 1.9%) were comparable with those provided by commercially available RIA kits for the determination of thyroxin.

A heterogeneous fluoroimmunoassay approach with measurement of the fluorescence signal directly onto the solid support is described. Fluorescein was employed as fluorescent label whereas plastic microtitration wells were used as solid carriers. The influence of the type of the plastic wells on the proposed approach was investigated. Different types of commercially available plastic microtitration wells (white-opaque, black, and transparent) were tested. The solid supports were judged on the grounds of the maximum fluorescence signal, the precision, and the sensitivity they provided in a model non-competitive assay for mouse IgG. Among the solid supports tested, the white-opaque wells provided the most promising results with respect to the parameters in question. This support was also used for the development of a competitive fluoroimmunoassay for the determination of total thyroxin in human serum samples in order to assess the validity of the proposed approach under real immunoassay conditions. The assay developed had appropriate analytical characteristics for the determination of thyroxin and the serum sample values obtained were well correlated with those determined by a commercial solid-phase radioimmunoassay.

An alternative protocol for immobilization of antibodies onto plastic solid supports is presented. According to the proposed protocol, tubes are first coated with gamma-globulins from non-immunized animal of the same species as that from which the antigen-specific antibody has been developed. Then, an excess of second antibody is added to the tubes and the anti-species specific antibodies present in the antiserum are immunoadsorbed on the immobilized gamma-globulins. Finally, the antigen specific antibody is immunoadsorbed on the immobilized second antibody. We found that the coating protocol developed allows the use of antigen-specific and second antibody antisera dilutions, thus avoiding the need for affinity purification of antibodies. Additionally, it provides solid-phase second antibody with increased binding capacity compared to the directly adsorbed onto the solid second antibody. The advantages of the proposed coating protocol were demonstrated through the development of a solid-phase radioimmunoassay for the determination of total triiodothyronine in human serum samples.

In the present work we propose a new optical immunosensor based on capillary geometry and capable of multianalyte determinations. The device is made of a polystyrene capillary tube. The inner walls of the capillary are segmented into distinct bands which are coated with appropriate binding molecules. Following excitation, some of the fluorescent photons emitted by the label are trapped and waveguided into the capillary walls provided they are launched towards the walls and within the critical angle. Here, Europium-labeled streptavidin reacted with different amounts of biotinylated bovine serum albumin immobilized onto each one of the bands. Due to the small inner volume of the capillary and the multianalyte feature we expect that the proposed device can be used for fast and inexpensive assays.

A simple, one-step protocol for synthesizing biotinylmono[125I]iodotyramine and biotinyldi[125I]iodotyramine, which are used as tracer substrates in biotinidase radioassays, is presented. This synthetic protocol uses reversed-phase HPLC for the isolation of the biotinylamide analogues from the reaction mixture. The HPLC method developed can potentially be applied to a scaled-up synthetic protocol for non-radioactive biotinylmono- and diiodotyramine. In addition, it can be used for the identification of the above-mentioned compounds.

We describe new colorimetric methods for the direct determination of total solid-supported sulfydryl, aldehydo, hydrazido, and N-hydroxysuccinimido carboxylate groups as well as of immobilized cysteine, tyrosine, and thyroxin using only the commercially available bicinchoninic acid/copper protein assay reagent. The method is based on the ability of these groups to reduce Cu2+ to Cu+, which forms a chelate complex with bicinchoninic acid absorbing at 562 nm. Each assay requires only one incubation step of the solids with the reagent for 1 h at 60 degrees C. The quantitation of the different groups is finally carried out through standard curves of appropriate substances. Using the assays developed we determined the amount of the above-mentioned functional groups and ligands onto several commercially available solid supports. The values obtained were in agreement with those provided by relative literature methods and/or by the manufacturers. The assays were found to be accurate, precise (interassay CV less than 3%), and very sensitive, allowing the determination of nmol quantities of functional groups per assay tube.

A simple and sensitive method for the quantitative determination of solid-supported primary and/or secondary amino groups using commercially available reagents is described. The solid supports are treated in an aqueous environment with either 2-iminothiolane (ITL) or sulpho-succinimidyl-3-(4-hydroxyphenyl)propionate (sulpho-SHPP), which introduce one sulphydryl or one hydroxyphenyl group per amino group reacted, respectively. These groups are capable of reducing Cu2+ to Cu+ in alkaline medium. Thus, after removal of the excess reagents through washing, subsequent incubation of the solids with 2,2'-bicinchoninic acid (BCA) copper protein reagent results in production of Cu+ in the solution, which forms a chelate complex with BCA absorbing at 562 nm. The quantitation of the groups introduced on the surfaces, and therefore of the reacted amino groups, is carried out through standard curves of cysteine solutions for ITL, or tyrosine solutions for sulpho-SHPP-treated solids. Using ITL, only the primary amino groups are determined, whereas sulpho-SHPP provided the primary and secondary reactive amino groups. The method is versatile and can be used for the estimation of amino groups onto several biomedical solid matrices, and should provide useful information for the covalent immobilization of ligands (e.g. drugs, antibodies).

We synthesized biotinylated mono- and di-iodotyramine and their radioactive counterparts and used these substances as substrates to estimate serum biotinidase activity in a radioassay system. The Km values determined for mono- and di-iodobiotinyl derivatives were 15.8 and 25.9 microM, respectively, whereas, the maximum velocities of the enzymatic reaction were 27.0 and 8.7 nmol.min-1.mL-1, respectively. Both substrates competed with biocytin for the same active site of the enzyme and the Ki values were 7.30 and 9.56 microM for the mono- and di-iodinated substrate, respectively. Higher assay sensitivity was obtained using [125I]biotinyl-monoiodotyramine as substrate, and the values obtained were directly related with those determined with the well-established colorimetric method (r = 0.9377, n = 31). However, for routine use, the assay may be accomplished by diluting the radiotracer with biocytin instead of its "cold" counterpart, because it is a commercially available reagent. The values obtained in this case were very well correlated with those determined by the colorimetric assay as well (r = 0.9289, n = 31).

We developed a direct competitive-type immunoassay for progesterone in serum that combines the advantages of the biotin-streptavidin system with the antibody-immunobilization approach. We synthesized biotinylated progesterone derivatives of five different proteins and, after initial evaluation of the conjugates, selected biotinylated bovine IgG-progesterone as the most suitable tracer. Progesterone released from binding proteins with danazol competes with the biotinylated tracer conjugate for binding to a limited amount of a mouse anti-progesterone monoclonal antibody in microtitration wells coated with a goat anti-mouse IgG antibody. The binding of the biotinylated tracer is then monitored by reaction with a streptavidin-based universal detection reagent developed for time-resolved fluorometry. The assay demonstrated typical performance characteristics with respect to the dynamic range, detection limit, and precision. Recovery averaged 99.3% (SD 8.3%) and dilution experiments showed good linearity. Measurements correlated well with those from three commercially available direct immunoassays for progesterone.

We describe a new multianalyte immunoassay principle and apply it to the simultaneous immunoassay of lutropin, follitropin, choriogonadotropin, and prolactin in serum. The method is based on the coating of distinct areas of polystyrene with analyte-specific antibodies. These antibodies react with the analyte and immobilize it in a specific area while another biotinylated antibody also reacts with the analyte to form a sandwich. After addition of streptavidin labeled with the fluorescent europium chelate of 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid, fluorescent areas are formed, the intensity of which is related to the amount of each analyte present in the sample. The fluorescent areas are quantified on the dry solid phase with laser-excited time-resolved fluorometric measurements. The assays developed are highly sensitive, precise, and accurate. We believe that this system shows potential for multianalyte immunoassay of diverse groups of compounds in disciplines such as endocrinology, infectious disease, hematology, and oncology.

A radioassay for determining biotinidase activity in human serum was developed, using N-[beta-(4-OH-3-125I-phenyl)ethyl]-biotinamide in combination with biocytin as the substrate, avidin as a binding protein, and polyethylene glycol as a separation reagent. The gamma-emitting 125I-biotinamide (= tracer) was synthesized by coupling (pH 8.5, 20-22 degrees C, 90 min) N-hydroxysuccinimidobiotin to 125I-tyramine. Using polyethylene glycol as a separation reagent, it was possible to eliminate several problems that were encountered when other separation reagents were used. Biotinidase activity was evaluated following the cleavage of the 125I-biotinamide and expressed in fmol of tracer cleaved.min-1.ml-1 in the presence of 9 nmol of biocytin. Under the conditions used, the time response of the assay was linear up to 3 h. The method is simple to perform, more sensitive than the previously described methods, and reproducible (intra- and interassay CVs of 4.9 and 10.2%, respectively) and allows the simultaneous handling of more than 100 samples in less than 3 h.

Here we describe a method for isolating mono- and di-iodine 125 tyramines from a crude tyramine radio-iodination reaction mixture. Tyramine was radio-iodinated according to a modified chloramine-T method. Mono- and diforms were separated from each other, as well as from free 125I and unreacted tyramine, by means of paper electrophoresis or thin-layer chromatography. Both 125I-tyramine derivatives finally obtained were pure and had high specific activities, which averaged 2500 Ci/mmol for mono-125I-tyramine and 5000 Ci/mmol for di-125I-tyramine. Due to their characteristics, these derivatives can be used for labelling various substances indirectly and preparing in vivo and in vitro tracers of high purity and specific activity, when conjugation labelling is required.

A radioimmunoassay method for the measurement of diethylstilbestrol directly on 50 microliters of human serum was established using a tritium-labeled radioligand and a double antibody as a separation reagent. The assay was sensitive (less than 0.17 micrograms/L), reproducible (intra-assay and interassay coefficient of variation values, 8.14% and 8.25%, respectively), and accurate (recovery up to 95%). Factors influencing the assay are identified and discussed.

We present a solid-phase immobilization method for radioligand assays, using an immunoadsorption coating procedure of anti-triiodothyronine rabbit IgG (anti-T3 IgG) onto second antibody (sheep anti-rabbit IgG) covalently bound to Amino-Dylark beads. The second antibody was in excess, compared with the first antibody, thus eliminating reproducibility problems between immunoadsorptions. Beads coated with second antibody can be used to immobilize a variety of antigen-specific first antibodies. The amount of anti-T3 antibody required for solid-phase T3 radioimmunoassay (RIA) was only 10% more, per assay tube, than that utilized in liquid-phase T3 RIA, in which polyethylene glycol solution was the separation reagent; characteristics of assay performance were comparable. The immobilization procedure requires high-titer antisera or antigen-specific IgG and seems advantageous because of the decrease in antibody requirements without significant modification of antibody functionality.

The immunoreactivity of anti-triiodothyronine (anti-T3) IgG, pretreated at acidic pH and adsorbed onto polystyrene beads, was significantly greater than that determined for native anti-T3 IgG immobilized at optimum pH 3.5. Acidic-pH-pretreated IgG, adsorbed at pH 7.0 onto ethanol-treated beads, had less immunoreactivity than that on untreated beads and gave values similar to those of native IgG adsorbed onto activated beads at pH 9.6. The rate of immobilization onto treated beads was significantly greater than onto untreated ones, and the binding was reproducible (intra- and inter-batch coating CV, 3.7-4.6% and 7.2%, respectively) and very resistant to successive washings. A post-washing incubation in 10 mg/L bovine serum albumin solution was required to eliminate the decreased immobilized immunoreactivity caused by some washing reagents. For solid-phase radioimmunoassays of T3, 40% less antibody was needed when acidic-pH-pretreated IgG was adsorbed onto untreated beads or when native IgG was adsorbed onto ethanol-treated beads, in comparison with native IgG adsorbed onto untreated beads. Results and assay characteristics with such beads were comparable with results for solid-phase and liquid-phase polyethylene glycol assay of T3.