The design and application of an inkjet-printed electrochemically reduced graphene oxide microelectrode for HT-2 mycotoxin immunoenzymatic biosensing is reported. A water-based graphene oxide ink was first formulated and single-drop line working microelectrodes were inkjet-printed onto poly(ethylene 2,6-naphthalate) substrates, with dimensions of 78 μm in width and 30 nm in height after solvent evaporation. The printed graphene oxide microelectrodes were electrochemically reduced and characterized by Raman and X-ray photoelectron spectroscopies in addition to microscopies. Through optimization of the electrochemical reduction parameters, differential pulse voltammetry were performed to examine the sensing of 1-naphthol (1-N), where it was revealed that reduction times had significant effects on electrode performance. The developed microelectrodes were then used as an immunoenzymatic biosensor for the detection of HT-2 mycotoxin based on carbodiimide linking of the microelectrode surface and HT-2 toxin antigen binding fragment of antibody (anti-HT2 (10) Fab). The HT-2 toxin and anti-HT2 (10) Fab reaction was reported by anti-HT2 immune complex single-chain variable fragment of antibody fused with alkaline phosphatase (anti-IC-HT2 scFv-ALP) which is able to produce an electroactive reporter - 1-N. The biosensor showed detection limit of 1.6 ng ∙ mL-1 and a linear dynamic range of 6.3 - 100.0 ng ∙ mL-1 within a 5 min incubation with 1-naphthyl phosphate (1-NP) substrate.

• MeSH
• biosenzitivní techniky přístrojové vybavení   MeSH
• design vybavení MeSH
• elektrochemické techniky přístrojové vybavení   MeSH
• grafit chemie   MeSH
• imobilizační protilátky chemie   MeSH
• imunoenzymatické techniky přístrojové vybavení   MeSH
• mikroelektrody MeSH
• oxidace-redukce MeSH
• reagenční papírky analýza   MeSH
• T-2 toxin analogy a deriváty   analýza   MeSH
• Publikační typ
• časopisecké články MeSH

In this paper several advances were implemented for glycoprofiling of prostate specific antigen (PSA), what can be applied for better prostate cancer (PCa) diagnostics in the future: 1) application of Au nanoshells with a magnetic core (MP@silica@Au); 2) use of surface plasmons of Au nanoshells with a magnetic core for spontaneous immobilization of zwitterionic molecules via diazonium salt grafting; 3) a double anti-fouling strategy with integration of zwitterionic molecules on Au surface and on MP@silica@Au particles was implemented to resist non-specific protein binding; 4) application of anti-PSA antibody modified Au nanoshells with a magnetic core for enrichment of PSA from a complex matrix of a human serum; 5) direct incubation of anti-PSA modified MP@silica@Au with affinity bound PSA to the lectin modified electrode surface. The electrochemical impedance spectroscopy (EIS) signal was enhanced 43 times integrating Au nanoshells with a magnetic core compared to the biosensor without them. This proof-of-concept study shows that the biosensor could detect PSA down to 1.2 fM and at the same time to glycoprofile such low PSA concentration using a lectin patterned biosensor device. The biosensor offers a recovery index of 108%, when serum sample was spiked with a physiological concentration of PSA (3.5 ng mL-1).

• MeSH
• biosenzitivní techniky * MeSH
• imobilizační protilátky chemie   imunologie   MeSH
• impedanční spektroskopie metody   MeSH
• lidé MeSH
• nádory prostaty diagnóza   patologie   MeSH
• nanoslupky chemie   MeSH
• prostata patologie   MeSH
• prostatický specifický antigen chemie   izolace a purifikace   MeSH
• zlato chemie   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Coxiella burnetii (C. burnetii) is the etiological agent of a Q fever-the re-emerging disease with considerable economic impact. Due to many similar symptoms with commonly occurring infections, its clinical diagnosis is very difficult. Thus, a strong effort should be taken to raise the awareness and develop a robust strategy for an accurate diagnosis. The identification of specific C. burnetii biomarkers could be valuable for a sensitive and selective diagnosis of the disease. Herein, we described a workflow to identify immunoreactive proteins of C. burnetii with a high confidence. It is based on immunocapturing of bacterial antigens by biofunctionalized magnetic microspheres, followed by tandem mass spectrometry (MS/MS) identification. We detected dozens of previously reported antigens and proposed 15 novel biomarkers, which specificity was confirmed by in silico epitope analysis. Among them, the cardiolipin synthetase participating in the synthesis of cardiolipin was recognized. This biomarker could play a critical role in the early management of acute Q fever and prevention of Q fever endocarditis.

• MeSH
• antigeny bakteriální analýza   krev   imunologie   MeSH
• bakteriální proteiny analýza   krev   imunologie   MeSH
• chromatografie kapalinová MeSH
• Coxiella burnetii imunologie   MeSH
• imobilizační protilátky krev   chemie   imunologie   MeSH
• lidé MeSH
• proteomika MeSH
• protilátky bakteriální krev   chemie   imunologie   MeSH
• Q-horečka krev   diagnóza   imunologie   MeSH
• tandemová hmotnostní spektrometrie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Functional gold nanoparticles (AuNPs) are commonly used to enhance the response of optical affinity biosensors. In this work, we investigated the effect of preparation conditions on functional properties of AuNPs functionalized with antibody (Ab-AuNPs), specifically AuNPs with antibody against carcinoembryonic antigen (CEA) covalently attached via carboxy-terminated oligo-ethylene thiolate linker layer. The following parameters of preparation of Ab-AuNP have been found to have a significant effect on Ab-AuNP performance in affinity biosensors: the time of reaction of activated AuNPs with antibody, concentrations of antibody and amino-coupling reagents, and composition of immobilization buffer (molarity and salt content). In contrast, pH of immobilization buffer has been demonstrated to have only a minor influence. Our experiments showed that the Ab-AuNPs prepared under optimum conditions offered a binding efficiency of Ab-AuNPs to CEA as high as 63%, which is more than 4 times better than the best efficiencies reported for similar functional AuNPs so far. We employed these Ab-AuNPs with a surface plasmon resonance (SPR) biosensor for the detection of CEA and showed that the Ab-AuNPs enhanced the sensor response to CEA by a factor of 1000. We also demonstrated that the Ab-AuNPs allow the biosensor to detect CEA at concentrations as low as 12 and 40 pg/mL in buffer and 50% blood plasma, respectively.

• MeSH
• imobilizační protilátky chemie   MeSH
• karcinoembryonální antigen analýza   krev   MeSH
• koncentrace vodíkových iontů MeSH
• kovové nanočástice chemie   MeSH
• lidé MeSH
• limita detekce MeSH
• povrchová plasmonová rezonance metody   MeSH
• pufry MeSH
• zlato chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH

Brassinosteroids (BRs) are plant-specific steroid hormones that play essential roles in the regulation of many important physiological processes in plant life. Their extremely low concentrations (~pmoles/g FW) in plant tissue and huge differences in polarity of individual members within the BR family hamper their detection and quantification. To address this problem, an immunoaffinity sorbent with broad specificity and high capacity for different BR metabolites containing a monoclonal antibody (mAb) against a BR spacer (20S)-2α,3α-dihydroxy-7-oxa-7α-homo-5α-pregnane-6-one-20 carboxylic acid (BR4812) was used for the rapid and highly selective isolation of endogenous BRs containing a 2α,3α-diol in ring A from minute plant samples. This enrichment procedure was successfully applied as a sample preparation method prior to quantitative analysis of BRs in real plant tissues by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Use of immunoaffinity chromatography (IAC) increased the sensitivity of the UHPLC-MS/MS analysis owing to improvements in the BR signal-to-noise ratio (S/N) and matrix factor (MF). Although MF values of BRs analyzed in classical samples ranged from 8.9% to 47.4%, MF values for the IAC purified samples reached 44.5-96.6%. Thus, the developed IAC-UHPLC-MS/MS approach was shown to be a simple, robust, effective and extremely fast procedure requiring minute amounts of plant samples suitable for the quantitative profiling of many BR metabolites, helping to overcome the major problems associated with their determination in very complex plant matrices.

• MeSH
• Brassica napus chemie   MeSH
• brassinosteroidy analýza   izolace a purifikace   MeSH
• chromatografie afinitní metody   MeSH
• imobilizační protilátky chemie   MeSH
• imunosorbenty chemie   MeSH
• regulátory růstu rostlin analýza   izolace a purifikace   MeSH
• rostlinné extrakty chemie   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH

Recent outbreaks of foodborne illnesses have shown that foodborne bacterial pathogens present a significant threat to public health, resulting in an increased need for technologies capable of fast and reliable screening of food commodities. The optimal method of pathogen detection in foods should: (i) be rapid, specific, and sensitive; (ii) require minimum sample preparation; and (iii) be robust and cost-effective, thus enabling use in the field. Here we report the use of a SPR biosensor based on ultra-low fouling and functionalizable poly(carboxybetaine acrylamide) (pCBAA) brushes for the rapid and sensitive detection of bacterial pathogens in crude food samples utilizing a three-step detection assay. We studied both the surface resistance to fouling and the functional capabilities of these brushes with respect to each step of the assay, namely: (I) incubation of the sensor with crude food samples, resulting in the capture of bacteria by antibodies immobilized to the pCBAA coating, (II) binding of secondary biotinylated antibody (Ab2) to previously captured bacteria, and (III) binding of streptavidin-coated gold nanoparticles to the biotinylated Ab2 in order to enhance the sensor response. We also investigated the effects of the brush thickness on the biorecognition capabilities of the gold-grafted functionalized pCBAA coatings. We demonstrate that pCBAA-compared to standard low-fouling OEG-based alkanethiolate self-assemabled monolayers-exhibits superior surface resistance regarding both fouling from complex food samples as well as the non-specific binding of S-AuNPs. We further demonstrate that a SPR biosensor based on a pCBAA brush with a thickness as low as 20 nm was capable of detecting E. coli O157:H7 and Salmonella sp. in complex hamburger and cucumber samples with extraordinary sensitivity and specificity. The limits of detection for the two bacteria in cucumber and hamburger extracts were determined to be 57 CFU/mL and 17 CFU/mL for E. coli and 7.4 × 10(3) CFU/mL and 11.7 × 10(3)CFU/mL for Salmonella sp., respectively. In addition, we demonstrate the simultaneous detection of E. coli and Salmonella sp. in hamburger sample using a multichannel SPR biosensor having appropriate functional coatings.

• MeSH
• biosenzitivní techniky * MeSH
• Escherichia coli O157 izolace a purifikace   patogenita   MeSH
• imobilizační protilátky chemie   MeSH
• kontaminace potravin MeSH
• lidé MeSH
• limita detekce MeSH
• nemoci přenášené potravou diagnóza   mikrobiologie   MeSH
• potravinářská mikrobiologie * MeSH
• povrchová plasmonová rezonance MeSH
• zlato chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A novel microfluidic label-free bead-based metallothionein immunosensors was designed. To the surface of superparamagnetic agarose beads coated with protein A, polyclonal chicken IgY specifically recognizing metallothionein (MT) were immobilized via rabbit IgG. The Brdicka reaction was used for metallothionein detection in a microfluidic printed 3D chip. The assembled chip consisted of a single copper wire coated with a thin layer of amalgam as working electrode. Optimization of MT detection using designed microfluidic chip was performed in stationary system as well as in the flow arrangement at various flow rates (0-1800 μL/min). In stationary arrangement it is possible to detect MT concentrations up to 30 ng/mL level, flow arrangement allows reliable detection of even lower concentration (12.5 ng/mL). The assembled miniature flow chip was subsequently tested for the detection of MT elevated levels (at approx. level 100 μg/mL) in samples of patients with cancer. The stability of constructed device for metallothionein detection in flow arrangement was found to be several days without any maintenance needed.

• MeSH
• design vybavení MeSH
• elektrochemické techniky přístrojové vybavení   metody   MeSH
• elektrody MeSH
• imobilizační protilátky chemie   metabolismus   MeSH
• imunoglobulin G chemie   metabolismus   MeSH
• imunoglobuliny chemie   metabolismus   MeSH
• imunomagnetická separace přístrojové vybavení   metody   MeSH
• králíci MeSH
• kur domácí MeSH
• lidé středního věku MeSH
• lidé MeSH
• metalothionein krev   MeSH
• nádory hlavy a krku krev   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Screen-printed platinum electrodes as transducer and magnetic beads as solid phase were combined to develop a particle-based electrochemical immunosensor for monitoring the serious food allergen ovalbumin. The standard arrangement of enzyme-linked immunosorbent assay became the basis for designing the immunosensor. A sandwich-type immunocomplex was formed between magnetic particles functionalized with specific anti-ovalbumin immunoglobulin G and captured ovalbumin molecules, and secondary anti-ovalbumin antibodies conjugated with the enzyme horseradish peroxidase were subsequently added as label tag. The electrochemical signal proportional to the enzymatic reaction of horseradish peroxidase during the reduction of hydrogen peroxide with thionine as electron mediator was measured by linear sweep voltammetry. The newly established method of ovalbumin detection exhibits high sensitivity suitable for quantification in the range of 11 to 222nM and a detection limit of 5nM. Magnetic beads-based assay format using external magnets for rapid and simple separation has been proven to be an excellent basis for electrochemical detection and quantification of food allergens in highly complex sample matrices.

• MeSH
• biosenzitivní techniky přístrojové vybavení   metody   MeSH
• elektrická vodivost MeSH
• elektrochemie MeSH
• elektrody MeSH
• imobilizační protilátky chemie   imunologie   MeSH
• imunoanalýza přístrojové vybavení   metody   MeSH
• limita detekce MeSH
• magnety chemie   MeSH
• mikrosféry * MeSH
• ovalbumin škodlivé účinky   analýza   MeSH
• platina chemie   MeSH
• potravinová alergie metabolismus   MeSH
• transport elektronů MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In this study, we describe a particular step in developing a microfluidic device for capture and detection of circulating tumor cells-specifically the preparation of an immunosorbent for implementation into the separation chip. We highlight some of the most important specifics connected with superparamegnetic microspheres for microfluidic purposes. Factors such as nonspecific adsorption on microfluidic channels, interactions with model cell lines, and tendency to aggregation were investigated. Poly(glycidyl methacrylate) microspheres with carboxyl groups were employed for this purpose. To address the aforementioned challenges, the microspheres were coated with hydrazide-PEG-hydrazide, and subsequently anti-epithelial cell adhesion molecule (EpCAM) antibody was immobilized. The prepared anti-EpCAM immunosorbent was pretested using model cell lines with differing EpCAM density (MCF7, SKBR3, A549, and Raji) in a batchwise arrangement. Finally, the entire system was implemented and studied in an Ephesia chip and an evaluation was performed by the MCF7 cell line.

• MeSH
• antigeny nádorové metabolismus   MeSH
• imobilizační protilátky chemie   metabolismus   MeSH
• imunomagnetická separace přístrojové vybavení   metody   MeSH
• kyseliny polymethakrylové chemie   MeSH
• lidé MeSH
• magnety * MeSH
• mikrofluidní analytické techniky přístrojové vybavení   MeSH
• mikrosféry MeSH
• molekuly buněčné adheze metabolismus   MeSH
• monoklonální protilátky chemie   metabolismus   MeSH
• nádorové cirkulující buňky * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Lactoferrin (LF) is approximately 80 kDa iron-binding protein, which is important part of saliva and other body fluids. Due to its ability to bind metal ions, it has many biologically important functions. In this study, a method for the isolation of LF from a biological sample using robotically prepared antibody-modified paramagnetic particles was developed using robotic pipetting station. The method consisted of the following optimised steps. Protein G was bound on the paramagnetic particles, on which goat antibody (10 μg) was linked. LF was subsequently added to microtitration plate, which had affinity to goat antibody and the interaction lasted for 30 min. We found that the highest signals were obtained using the combination of goat antibody 1:3000, murine antibody 1:5000 and conjugate 1:1500. Horseradish peroxidase reducing 3,3,5,5-tetramethylbenzidine (TMB) was linked to the merged complex. The resulted product of this reaction was subsequently analysed spectrometrically with detection limit (3 S/N) as 5 ng/mL. In addition, we also determined TMB by stopped flow injection analysis with electrochemical detection. The limit of detection (3 S/N) was estimated as 0.1 μg/mL. To compare spectrometric and electrochemical approach for detection of TMB, calibration range of bead-LF-antibodies complex was prepared and was determined using a least-squares correlation with coefficient R² higher than 0.95, indicating a very good agreement of the results obtained.

• MeSH
• design vybavení MeSH
• dospělí MeSH
• elektrochemické techniky přístrojové vybavení   MeSH
• imobilizační protilátky chemie   MeSH
• laktoferrin analýza   izolace a purifikace   MeSH
• lidé MeSH
• limita detekce MeSH
• magnety chemie   MeSH
• průtoková injekční analýza přístrojové vybavení   MeSH
• sliny chemie   MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH