In this work we discuss a new label-free biosensing device based on indium tin oxide (ITO) overlaid section of a multimode optical fiber fused silica core. The sensor has been used to optical measurements also simultaneously interrogated electrochemically (EC). Due to optimized thickness and optical properties of ITO film, a lossy-mode resonance (LMR) could be observed in the optical domain, where electrical properties of the film allowed for application of the sensor as a working electrode in an EC setup. It has been confirmed that the LMR response depends on optical properties of the external medium, as well as potential applied to the electrode during cyclic voltammetry. After the ITO surface functionalization with amine groups and covalently attached biotin, the device has been applied for label-free biosensing of avidin in both the domains simultaneously. On the example of biotin-avidin detection system it was demonstrated that when avidin concentration increases a decrease in current and increase in LMR wavelength shift were recorded in EC and optical domain, respectively. Both optical and EC responses follow the protein interaction process, and thus can be used as cross-verification of the readouts. Moreover, an extended information has been achieved comparing to solely EC interrogation, i.e., the grafting process of biotin and avidin was directly monitored optically displaying individual steps of an incubation procedure.
- Klíčová slova
- Electrochemistry, Indium tin oxide (ITO), Label-free biosensing, Lossy-mode resonance, Multi-domain sensing, Optical fiber sensor, Thin film,
- MeSH
- avidin chemie izolace a purifikace MeSH
- biosenzitivní techniky * MeSH
- biotin chemie izolace a purifikace MeSH
- elektrochemické techniky * MeSH
- elektrody MeSH
- optika a fotonika MeSH
- sloučeniny cínu chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- avidin MeSH
- biotin MeSH
- indium tin oxide MeSH Prohlížeč
- sloučeniny cínu MeSH
Custom immuno-magnetic devices are desirable tools for biomedical and biotechnological applications. Herein, surface active maghemite nanoparticles (SAMNs) are proposed as a versatile platform for developing tailored immuno-magnetic nano-carriers by simple wet reactions. Two examples for conjugating native and biotinylated antibodies were presented along with their successful applications in the recognition of specific foodborne pathogens. Nanoparticles were functionalized with rhodamine B isothiocyanate (RITC), leading to a fluorescent nano-conjugate, and used for binding anti-Campylobacter fetus antibodies (SAMN@RITC@Anti-Cf). The microorganism was selectively captured in the presence of two other Campylobacter species (C. jejuni and C. coli), as verified by PCR. Alternatively, SAMNs were modified with avidin, forming a biotin-specific magnetic nano-carrier and used for the immobilization of biotinylated anti-Listeria monocytogenes antibodies (SAMN@avidin@Anti-Lm). This immuno-magnetic carrier was integrated in piezoelectric quartz crystal microbalance (QCM) sensor for the detection of L. monocytogenes in milk, showing a detection limit of 3 bacterial cells. The present work presents a new category of customized immuno-magnetic nano-carriers as a competitive option for suiting specific applications. Graphical abstract ᅟ.
- Klíčová slova
- Antibody conjugation, Campylobacter fetus, Immuno-magnetic separation, Listeria monocytogenes, Magnetic nano-carrier, Pathogen recognition,
- MeSH
- adjuvancia imunologická chemie MeSH
- avidin chemie MeSH
- Listeria imunologie MeSH
- magnetismus * MeSH
- mikrorovnovážné techniky křemenného krystalu metody MeSH
- monoklonální protilátky chemie MeSH
- nanočástice chemie MeSH
- povrchové vlastnosti MeSH
- transmisní elektronová mikroskopie MeSH
- železité sloučeniny chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- adjuvancia imunologická MeSH
- avidin MeSH
- ferric oxide MeSH Prohlížeč
- monoklonální protilátky MeSH
- železité sloučeniny MeSH
Avidin-biotin technology was used for the implementation of an enzyme-linked immunosorbent assay (AB-ELISA) as a sensitive method for the detection of anabolic androgenic steroids (AAS) present in dietary supplements. Using click chemistry, novel haptens (linker-optimized biotinylated nandrolone (NT) and testosterone (T) at positions C-3 and C-17, respectively) were designed and synthesized to be then applied as four different immobilized competitors in a proposed set of four indirect competitive AB-ELISAs. Four rabbit polyclonal antibodies of various specificities were prepared using four different immunogens synthesized from C-3 and C-17 carboxymethyloxime and hemisuccinate derivatives of NT and T, respectively. Assembled AB-ELISAs were characterized to establish method parameters such as a half-maximum inhibition concentration (0.18-12.99 ng/mL), limit of detection (0.004-0.032 ng/mL) and linear working range (the best with 0.02-1.38 ng/mL). The stability of the set simulating storage in different conditions was demonstrated. Cross reactivity (CR) was tested for 59 steroids including both endogenous and synthetic analogues in four assembled AB-systems. The focus was placed on the practical use of the method in detection of various AAS in 49 samples of counterfeit dietary supplements. The concordance between ultra high performance liquid chromatography-mass spectrometry (UHPLC-MS) and the CR corrected data from AB-ELISA indicated the potential of this method even to quantification of T propionate, NT phenyl propionate, and NT decanoate in such a complex matter. Copyright © 2016 John Wiley & Sons, Ltd.
- Klíčová slova
- ELISA, anabolics, avidin, biotin, dietary supplements, nandrolone, testosterone,
- MeSH
- anabolika analýza MeSH
- avidin chemie MeSH
- biotin chemie MeSH
- ELISA metody MeSH
- králíci MeSH
- limita detekce MeSH
- nandrolon analýza MeSH
- potravní doplňky analýza MeSH
- testosteron analýza MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- Názvy látek
- anabolika MeSH
- avidin MeSH
- biotin MeSH
- nandrolon MeSH
- testosteron MeSH
This paper presents a synthesis of a novel nanoparticle label with selective biorecognition properties based on a biotinylated silver-dendrimer nanocomposite (AgDNC). Two types of labels, a biotin-AgDNC (bio-AgDNC) and a biotinylated AgDNC with a poly(ethylene)glycol spacer (bio-PEG-AgDNC), were synthesized from a generation 7 (G7) hydroxyl-terminated ethylenediamine-core-type (2-carbon core) PAMAM dendrimer (DDM) by an N,N'-dicyclohexylcarbodiimide (DDC) biotin coupling and a NaBH(4) silver reduction method. Synthesized conjugates were characterized by several analytical methods, such as UV-vis, FTIR, AFM, TEM, ELISA, HABA assay and SPR. The results show that stable biotinylated nanocomposites can be formed either with internalized silver nanoparticles (AgNPs) in a DMM polymer backbone ('type I') or as externally protected ('type E'), depending on the molar ratio of the silver/DMM conjugate and type of conjugate. Furthermore, the selective biorecognition function of the biotin is not affected by the AgNPs' synthesis step, which allows a potential application of silver nanocomposite conjugates as biospecific labels in various bioanalytical assays, or potentially as fluorescence cell biomarkers. An exploitation of the presented label in the development of electrochemical immunosensors is anticipated.
- MeSH
- avidin metabolismus MeSH
- barvení a značení metody MeSH
- biotin chemie metabolismus MeSH
- dendrimery MeSH
- ELISA MeSH
- kinetika MeSH
- mikroskopie atomárních sil MeSH
- molekulární modely MeSH
- nanokompozity chemie MeSH
- polyaminy chemická syntéza chemie MeSH
- povrchová plasmonová rezonance MeSH
- spektrofotometrie ultrafialová MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- stříbro chemie MeSH
- transmisní elektronová mikroskopie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- avidin MeSH
- biotin MeSH
- dendrimery MeSH
- PAMAM Starburst MeSH Prohlížeč
- polyaminy MeSH
- stříbro MeSH
A complex of osmium tetroxide with 2,2'-bipyridine (Os,bipy) has been applied as a chemical probe of DNA structure as well as an electroactive DNA label. The Os,bipy has been known to form covalent adducts with pyrimidine DNA bases. Besides the pyrimidines, electrochemically active covalent adducts with Os,bipy are formed also by tryptophan (W) residues in peptides and proteins. In this paper we show that Os,bipy-treated proteins possessing W residues (such as avidin, streptavidin, or lysozyme) yield at the pyrolytic graphite electrode (PGE) a specific signal (peak alphaW) the potential of which differs from the potentials of signals produced by free Os,bipy or by Os,bipy-modified DNA. No such signal is observed with proteins lacking W (such as ribonuclease A or alpha-synuclein). Subpicomole amounts of W-containing proteins modified with Os,bipy can easily be detected using adsorptive transfer stripping voltammetry with the PGE. Binding of biotin to avidin interferes with Os,bipy modification of the protein, in agreement with the location of W residues within the biotin-binding site of avidin. These Ws are accessible for modification in the absence of biotin but hidden (protected from modification) in the avidin-biotin complex. The Os,bipy-modified avidin is unable to bind biotin, and its quarternary structure is disrupted. Analogous effects were observed with another biotin-binding protein, streptavidin. Our results demonstrate that modification of proteins with Os,bipy under conditions close to physiological, followed by a simple electrochemical analysis, can be applied in the microanalysis of protein structure and interactions.
- MeSH
- 2,2'-dipyridyl chemie MeSH
- avidin chemie MeSH
- biotin chemie MeSH
- elektrochemie MeSH
- elektrody MeSH
- elektrony * MeSH
- molekulární struktura MeSH
- oxid osmičelý analýza chemie MeSH
- proteiny analýza chemie MeSH
- reagencia zkříženě vázaná chemie MeSH
- tryptofan analýza chemie MeSH
- uhlík chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 2,2'-dipyridyl MeSH
- avidin MeSH
- biotin MeSH
- oxid osmičelý MeSH
- proteiny MeSH
- reagencia zkříženě vázaná MeSH
- tryptofan MeSH
- uhlík MeSH
Four series of macroporous hydrogels based on crosslinked copolymers of 2-hydroxyethyl methacrylate (HEMA)-sodium methacrylate (MANa), copolymer HEMA-[2-(methacryloyloxy)ethyl]trimethylammonium chloride (MOETACl), terpolymer HEMA-MANa-MOETACl and on a polyelectrolyte complex were used as carriers for immobilization of proteins, chicken egg white albumin and avidin. The adsorption capacity of the hydrogels for the two proteins, kinetics and pH dependence of albumin adsorption and desorption were studied. The morphology of the hydrogels with and without immobilized albumin was studied by low-vacuum scanning electron microscopy.
- MeSH
- adsorpce MeSH
- albuminy analýza chemie ultrastruktura MeSH
- avidin analýza chemie ultrastruktura MeSH
- biokompatibilní potahované materiály analýza chemie MeSH
- hydrogely analýza chemie MeSH
- kinetika MeSH
- methakryláty analýza chemie MeSH
- poréznost MeSH
- povrchové vlastnosti MeSH
- testování materiálů MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- albuminy MeSH
- avidin MeSH
- biokompatibilní potahované materiály MeSH
- hydrogely MeSH
- hydroxyethyl methacrylate MeSH Prohlížeč
- methakryláty MeSH
The electroanalytical determination of avidin in solution, in a carbon paste, and in a transgenic maize extract was performed in acidic medium at a carbon paste electrode (CPE). The oxidative voltammetric signal resulting from the presence of tyrosine and tryptophan in avidin was observed using square-wave voltammetry. The process could be used to determine avidin concentrations up to 3 fM (100 amol in 3 microl drop) in solution, 700 fM (174 fmol in 250 microl solution) in an avidin-modified electrode, and 174 nM in a maize seed extract. In the case of the avidin-modified CPE, several parameters were studied in order to optimize the measurements, such as electrode accumulation time, composition of the avidin-modified CPE, and the elution time of avidin. In addition, the avidin-modified electrode was used to detect biotin in solution (the detection limit was 7.6 pmol in a 6 mul drop) and to detect biotin in a pharmaceutical drug after various solvent extraction procedures. Comparable studies for the detection of biotin were developed using HPLC with diode array detection (HPLC-DAD) and flow injection analysis with electrochemical detection, which allowed biotin to be detected at levels as low as 614 pM and 6.6 nM, respectively. The effects of applied potential, acetonitrile content, and flow rate of the mobile phase on the FIA-ED signal were also studied.
- MeSH
- avidin analýza chemie MeSH
- biotin analýza chemie MeSH
- elektrochemie metody MeSH
- elektrody MeSH
- lékové interakce * MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- uhlík chemie MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- avidin MeSH
- biotin MeSH
- uhlík MeSH
The proteins streptavidin and avidin were electrochemically detected in solution by adsorptive transfer stripping square wave voltammetry (AdTS SWV) at a carbon paste electrode (CPE). AdTS SWV was used to quantify biotinylated oligonucleotides, DNA hybridizations, and avidin in extracts of transgenic avidin maize. The detection limits of denatured and native streptavidin were 6 pM and 120 nM, respectively. The results demonstrated that streptavidin/avidin AdTS SWV is a sensitive and specific method for quantifying DNA and proteins in biological samples such as foods and tissue extracts, including genetically modified crops (avidin maize) and other plants in neighboring fields.
- MeSH
- adsorpce MeSH
- avidin analýza chemie genetika MeSH
- biotin chemie MeSH
- DNA metabolismus MeSH
- elektrochemie metody MeSH
- elektrody MeSH
- geneticky modifikované rostliny chemie genetika MeSH
- hybridizace nukleových kyselin metody MeSH
- kukuřice setá chemie genetika MeSH
- streptavidin analýza chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- avidin MeSH
- biotin MeSH
- DNA MeSH
- streptavidin MeSH
Boar seminal plasma was separated into five protein fractions (I-V) (>100, 55, 45, 30, 5-15 kDa) by gel filtration chromatography on Sephadex G-75 SF at pH 7.4. RP HPLC of protein fractions I-V and N-terminal sequencing of their individual components revealed that high-molecular-weight aggregates consisted mainly of DQH sperm surface protein and AQN, AWN, PSP II spermadhesins, while fraction IV consisted of heterodimers of PSP spermadhesins only. Spermadhesins as monomers were present in seminal plasma in a very low amount. Biotinylated fractions I-IV containing AWN, AQN, DQH, and PSP proteins were bound to boar epididymal and ejaculated spermatozoa with the same efficiency. Aggregates containing AWN, AQN, DQH, PSP II proteins (fractions I-III) and their HPLC-separated monomeric forms interacted with phosphorylcholine. Aggregates containing the DQH protein and AWN spermadhesins as well as their separated monomeric proteins interacted strongly with acidic polysaccharides. PSP II interacted with some acidic polysaccharides, while the fraction IV corresponding to heterodimer PSP IPSP II did not show any binding to acidic polysaccharides and zona pellucida. Fractions I-III showed affinity to cholesterol. The strongest interaction was observed between biotinylated glycoproteins of porcine zona pellucida and AWN 1-containing aggregates and separated proteins. AQN 1 spermadhesin effectively blocked the sperm binding to oocytes. These results suggest that under physiological conditions, the aggregated forms of seminal plasma proteins (DQH, AQN, AWN, PSP II) rather than the individual proteins might take part in coating the sperm surface, in sperm capacitation and in primary binding of spermatozoa to zona pellucida of the ovum.
- MeSH
- akrosin antagonisté a inhibitory MeSH
- avidin MeSH
- biotinylace MeSH
- cholesterol analogy a deriváty metabolismus MeSH
- chondroitin sulfáty chemie farmakologie MeSH
- fluorescenční barviva MeSH
- gelová chromatografie MeSH
- glykoproteiny chemie metabolismus MeSH
- heparin analogy a deriváty metabolismus MeSH
- interakce spermie a vajíčka MeSH
- membránové glykoproteiny chemie metabolismus MeSH
- prasata * MeSH
- proteiny semenné plazmy * MeSH
- sekreční proteiny semenných váčků * MeSH
- sperma chemie MeSH
- spermie chemie metabolismus MeSH
- transportní proteiny chemie metabolismus MeSH
- vazba proteinů MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- zona pellucida chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- akrosin MeSH
- avidin MeSH
- cholesterol MeSH
- chondroitin sulfáty MeSH
- DQH sperm surface protein, Sus scrofa MeSH Prohlížeč
- fluorescenční barviva MeSH
- glykoproteiny MeSH
- heparin MeSH
- membránové glykoproteiny MeSH
- proteiny semenné plazmy * MeSH
- sekreční proteiny semenných váčků * MeSH
- seminal vesicle secretory protein 109, porcine MeSH Prohlížeč
- spermadhesin MeSH Prohlížeč
- transportní proteiny MeSH
An enzyme avidin-biotin assay for the detection of anti-HBcAg antibody in human sera was developed. The assay uses genetically engineered HBcAg. HBcAg is immobilized on the surface of the wells of microtitre plates and the test serum sample, biotin-labelled HBcAg and streptavidin-labelled horseradish peroxidase are added. The assay was found to be specific and was compared with a commercial radioimmunoassay kit for sensitivity by testing 96 human clinical sera for anti-HBcAg antibody. Both assays gave identical results.
- MeSH
- avidin * MeSH
- biotin * MeSH
- ELISA MeSH
- Escherichia coli MeSH
- hepatitida B - antigeny jádrové genetika imunologie MeSH
- hepatitida B - protilátky analýza MeSH
- hepatitida B diagnóza MeSH
- klonování DNA MeSH
- lidé MeSH
- radioimunoanalýza MeSH
- senzitivita a specificita MeSH
- virus hepatitidy B imunologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
- Názvy látek
- avidin * MeSH
- biotin * MeSH
- hepatitida B - antigeny jádrové MeSH
- hepatitida B - protilátky MeSH
