Antibody coating approach involving gamma globulins from non-immunized animal and second antibody antiserum

An alternative protocol for immobilization of antibodies onto plastic solid supports is presented. According to the proposed protocol, tubes are first coated with γ-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 γ- 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

A monolithic silicon optoelectronic transducer as a real- time affinity biosensor

An optical real-time affinity biosensor, which is based on a monolithic silicon optoelectronic transducer and a microfluidic module, is described. The transducer monolithically integrates silicon avalanche diodes as light sources, silicon nitride optical fibers, and p/n junction detectors and efficiently intercouples these elements through a self-alignment technique. The transducer surface is hydrophilized by oxygen plasma treatment, silanized with (3-aminopropyl)triethoxysilane and bioactivated through adsorption of the biomolecular probes. The use of a microfluidic module allows real-time monitoring of the binding reaction of the gold nanoparticle-labeled analytes with the immobilized probes. Their binding within the evanescent field at the surface of the optical fiber causes attenuated total reflection of the waveguided modes and reduction of the detector photocurrent. The biotin-streptavidin model assay was used for the evaluation of the analytical potentials of the device developed. Detection limits of 3.8 and 13 pM in terms of gold nanoparticle-labeled streptavidin were achieved for continuous- and stopped-flow assay modes, respectively. The detection sensitivity was improved by silver plating of the immolilized gold nanoparticles, and a detection limit of 20 fM was obtained after 20-min of silver plating. In addition, two different analytes, streptavidin and anti-mouse IgG, were simultaneously assayed on the same chip demonstrating the multianalyte potential of the sensor developed

Poly-L-histidine coated microfluidic devices for bacterial DNA purification without chaotropic solutions

We present a disposable polymeric microfluidic device capable of reversibly binding and purifying Salmonella DNA through solid phase extraction (SPE). The microfluidic channels are first oxygen plasma treated and simultaneously micro-nanotextured, and then functionalized with amine groups via modification with L-histidine or poly-L-histidine. L-Histidine and poly-L-histidine bind on the plasma treated chip surface, and are not detached when rinsing with DNA purification protocol buffers. A pH-dependent protocol is applied on-chip to purify Salmonella DNA, which is first bound on the protonated amines at a pH (5.0) lower than their pKa of surface amine-groups which is 6.0 and then released at a pH higher than the pKa value (10.5). It was found that modification with poly-L-histidine resulted in higher surface density of amine groups onto microfluidic channel. Using the chip modified with poly-L-histidine, high recovery efficiency of at least 550 ng of isolated Salmonella DNA as well as DNA purification from Salmonella cell lysates corresponding to less than 5000 cells or 0.026 ng of Salmonella DNA was achieved. The protocol developed does not require ethanol or chaotropic solutions typically used in DNA purification, which are known inhibitors for downstream operations such as polymerase chain reactions (PCR) and which can also attack some polymeric microfluidic materials. Therefore, the microfluidic device and the related protocol hold promise for facile incorporation in microfluidics and Lab-on-a-chip (LOC) platforms for pathogen detection or in general for DNA purification. © 2020, Springer Science+Business Media, LLC, part of Springer Nature

Capillary waveguide fluoroimmunosensor with improved repeatability and detection sensitivity

An optical capillary waveguide fluoroimmunosensor based on glass capillaries internally coated with an ultrathin poly(dimethylsiloxane) (PDMS) film is presented. The evaluation of the capillaries developed was done in comparison with aminosilanized [3-(aminopropyl)triethoxysilane, APTES] glass and poly(methylpentene) (PMP) capillaries by immobilizing rabbit γ-globulins on the internal capillary wall. Following reaction with (R)-phycoerythrin- labelled antibody, the capillary was scanned with a laser beam and the fluorescence waveguided through the capillary wall was detected by a photomultiplier placed at one of its ends. The capillaries developed provided considerably improved protein coating homogeneity (intracapillary coefficients of variation 2.9-6.6%) and repeatability (intercapillary coefficients of variation 2.1-5.0%) compared with APTES-treated ones (7.9-13.4 and 8.5-15.2%, respectively). With use of these capillaries in a sandwich-type immunosensor for the determination of rabbit γ-globulins, the assay detection limit was improved eightfold (4.4 ng/mL) compared with that obtained using PMP capillaries (35.3 ng/mL), whereas the assay repeatability was improved threefold (intra-assay coefficients of variation 5.9-13.1%) compared with APTES-treated capillaries (15.6-36%). [Figure not available: see fulltext.] © 2008 Springer-Verlag

Ultra-thin poly(dimethylsiloxane) film-coated glass capillaries for fluoroimmunosensing applications

Methods for homogeneous coverage of surfaces with biomolecules are critical for the development of bioanalytical microsystems. Here we present a simple method for coating glass capillaries with an ultra-thin poly(dimethylsiloxane) (PDMS) film in order to be used as optical waveguide fluoroimmunosensors. A model rabbit γ-globulin binding assay was employed for the evaluation of modified capillaries in terms of protein binding capacity and protein coating homogeneity. It was found that the PDMS-modified capillaries provided excellent protein coating homogeneity inside each one capillary (CVs 2.9-6.6%) and between-capillaries repeatability (CVs 2.1-5.0%) compared to aminosilanized ones. Furthermore, the waveguiding properties of the glass were not affected by the proposed surface modification. © 2008 Elsevier B.V. All rights reserved

Dual-cardiac marker capillary waveguide fluoroimmunosensor based on tyramide signal amplification

The early diagnosis of acute myocardial infarction requires the determination of several markers in serum shortly after its incidence. The markers most widely employed are the isoenzyme MB of creatine kinase (CK-MB) and the cardiac troponin I (cTnI). In the present work, a capillary waveguide fluoroimmunosensor for fast and highly sensitive simultaneous determination of these markers in serum samples is demonstrated. The dual-analyte immunosensor was realized using glass capillaries internally modified with an ultrathin poly(dimethylsiloxane) film by creating discrete bands of analyte-specific antibodies. The capillary was then filled with a mixture of sample and biotinylated detection antibodies followed by reaction with streptavidin- horseradish peroxidase and incubation with a fluorescently labeled tyramide derivative to accumulate fluorescent labels onto immunoreaction bands. Upon scanning the capillary with a laser beam, part of the emitted fluorescence is trapped and waveguided through the capillary wall to a photomultiplier placed on one of its ends. The employment of tyramide signal amplification provided detection limits of 0.2 and 0.5 ng/mL for cTnI and CK-MB, respectively, in a total assay time of 30 min compared to 0.8 and 0.6 ng/mL obtained for the corresponding assays when the conventional fluorescent label R-phycoerythrin was used in a 65-min assay. In addition, the proposed immunosensor provided accurate and repeatable measurements (intra-assay and interassay coefficients of variation lower than 10%), and the values determined in serum samples were in good agreement with those obtained with commercially available enzyme immunoassays. Thus, the proposed capillary waveguide fluoroimmunosensor has all the required characteristics for fast and reliable diagnosis of acute myocardial infarction. © 2009 Springer-Verlag

Fast, sensitive and selective determination of herbicide glyphosate in water samples with a White Light Reflectance Spectroscopy immunosensor

An optical immunosensor based on White Light Reflectance Spectroscopy is described for the determination of the herbicide glyphosate in drinking water samples. The biosensor allows for the label-free real-time monitoring of biomolecular interactions taking place onto a SiO2/Si chip by transforming the shift in the reflected interference spectrum caused by the immunoreaction to effective biomolecular adlayer thickness. Glyphosate determination is accomplished by functionalizing the chip with a protein conjugate of the herbicide followed by a competitive immunoassay format. Prior to the assay, glyphosate derivatization in the calibrators and/or the samples was performed through reaction with succinic anhydride. Under the optimized assay protocol, a detection limit of 10 pg mL−1 was achieved. Recovery values ranging from 90.0 to 110% were determined in spiked bottled and tap water samples, demonstrating the accuracy of the method. In addition, the sensor could be regenerated and re-used for at least 14 times without statistically significant effect on the assay sensitivity and accuracy. The excellent analytical performance and short analysis time (approx. 25 min), combined with the small sensor size, should be helpful for the fast on-site determination of glyphosate in drinking water samples. © 2020 Elsevier B.V

Orthogonal patterning of multiple biomolecules using an organic fluorinated resist and imprint lithography

The ability to spatially deposit multiple biomolecules onto a single surface with high-resolution while retaining biomolecule stability and integrity is critical to the development of micro- and nanoscale biodevices. While conventional lithographic patterning methods are attractive for this application, they typically require the use of UV exposure and/or harsh solvents and imaging materials, which may be damaging to fragile biomolecules. Here, we report the development of a new patterning process based on a fluorinated patterning material that is soluble in hydrofluoroether solvents, which we show to be benign to biomolecules, including proteins and DNA. We demonstrate the implementation of these materials into an orthogonal processing system for patterning multibiomolecule arrays by imprint lithography at room temperature. We further showcase this method's capacity for fabricating patterns of receptor-specific ligands for fundamental cell studies. © 2013 American Chemical Society

Improved DNA microarray detection sensitivity through immobilization of preformed in solution streptavidin/biotinylated oligonucleotide conjugates

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. © 2015 Elsevier B.V