Herein, we report a novel concept of low-cost flexible platform for fluorescence-based biosensor. The surface of polyethylene naphthalate (PEN) foil was exposed to KrF excimer laser through a photolitographic contact mask. Laser initiated surface modification resulted in micro-patterned areas with surface functional groups available for localized covalent immobilization of biotin. High affinity binding protein (albumin-binding domain (ABD) of protein G, Streptococcus G148) recognizing human serum albumin (HSA), genetically fused with streptavidin (SA-ABDwt), was immobilized on the micro-patterned surface through biotin-streptavidin coupling. Fluorescently labelled HSA analyte was detected in several blocking environments, in 1% bovine serum albumin (BSA) and 6% fetal serum albumin (FBS), respectively. We conclude that the presented novel concept enabled us to micropattern functional biosensing layers on the surface of PEN foil in a fast and easy way. It brings all necessary aspects for continuous roll-to-roll fabrication of low-cost optical bioanalytical devices.
- MeSH
- biotin metabolismus MeSH
- fotoelektronová spektroskopie MeSH
- lidé MeSH
- mikrotechnologie metody MeSH
- naftaleny chemie MeSH
- optické jevy * MeSH
- polyethyleny chemie MeSH
- povrchové vlastnosti MeSH
- sérový albumin metabolismus MeSH
- streptavidin metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Molecular diagnostics may provide tailored and cost efficient treatment for infectious disease and cancer. Rolling circle amplification (RCA) of padlock probes guarantees high specificity to identify nucleic acid targets down to single nucleotide resolution in a multiplex fashion. This makes the assay suitable for molecular analysis of various diseases, and interesting to integrate into automated devices for point-of-care analysis. A critical prerequisite for many molecular assays is (i) target-specific isolation from complex clinical samples and (ii) removal of reagents, inhibitors and contaminants between reaction steps. Efficient solid supports are therefore essential to enable multi-step, multi-analyte protocols. Superparamagnetic micro- and nanoparticles, with large surface area and rapid liquid-phase kinetics, are attractive for multi-step protocols. Recently, streptavidin-modified magnetic monodispersed poly(2-hydroxyethyl methacrylate) (STV-mag.PHEMA) microspheres were developed by multiple swelling polymerization. They are easily separated by a magnet and exhibit low non-specific protein sorption. In this study, the performance and the binding efficiency of STV-mag.PHEMA was addressed by circle-to-circle amplification (C2CA). A lower number of RCA products were detected as compared to the gold standard Dynabeads. Nevertheless, this study was the first to successfully adapt STV-mag.PHEMA microspheres as solid support in a DNA-based protocol, which is an important finding. The STV-mag.PHEMA microspheres were larger with about 16 times less surface area as compared to the Dynabeads, which might partly explain the lower rolling circle product (RCP) count obtained. Further research is currently ongoing comparing particles of similar sizes and optimizing reaction conditions to establish their full utility in the field. Ultimately, low cost and versatile particles are a great resource to facilitate future clinical molecular diagnostics.
- MeSH
- DNA chemie metabolismus MeSH
- imobilizované proteiny chemie metabolismus MeSH
- magnetismus MeSH
- mikrosféry * MeSH
- mikroskopie elektronová rastrovací MeSH
- polyhydroxyethylmethakrylát chemie MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- streptavidin chemie metabolismus MeSH
- techniky amplifikace nukleových kyselin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Currently, the influenza virus infects millions of individuals every year. Since the influenza virus represents one of the greatest threats, it is necessary to develop a diagnostic technique that can quickly, inexpensively, and accurately detect the virus to effectively treat and control seasonal and pandemic strains. This study presents an alternative to current detection methods. The flow-injection analysis-based biosensor, which can rapidly and economically analyze a wide panel of influenza virus strains by using paramagnetic particles modified with glycan, can selectively bind to specific viral A/H5N1/Vietnam/1203/2004 protein-labeled quantum dots. Optimized detection of cadmium sulfide quantum dots (CdS QDs)-protein complexes connected to paramagnetic microbeads was performed using differential pulse voltammetry on the surface of a hanging mercury drop electrode (HMDE) and/or glassy carbon electrode (GCE). Detection limit (3 S/N) estimations based on cadmium(II) ions quantification were 0.1 μg/mL or 10 μg/mL viral protein at HMDE or GCE, respectively. Viral protein detection was directly determined using differential pulse voltammetry Brdicka reaction. The limit detection (3 S/N) of viral protein was estimated as 0.1 μg/mL. Streptavidin-modified paramagnetic particles were mixed with biotinylated selective glycan to modify their surfaces. Under optimized conditions (250 μg/mL of glycan, 30-min long interaction with viral protein, 25°C and 400 rpm), the viral protein labeled with quantum dots was selectively isolated and its cadmium(II) content was determined. Cadmium was present in detectable amounts of 10 ng per mg of protein. Using this method, submicrogram concentrations of viral proteins can be identified.
- MeSH
- biosenzitivní techniky přístrojové vybavení metody MeSH
- biotin chemie metabolismus MeSH
- elektrochemické techniky přístrojové vybavení metody MeSH
- elektrody MeSH
- hemaglutininové glykoproteiny viru chřipky analýza metabolismus MeSH
- kadmium analýza MeSH
- kvantové tečky MeSH
- limita detekce MeSH
- lineární modely MeSH
- magnetické nanočástice chemie MeSH
- průtoková injekční analýza přístrojové vybavení metody MeSH
- rtuť chemie MeSH
- sloučeniny kadmia chemie MeSH
- streptavidin chemie metabolismus MeSH
- sulfidy chemie MeSH
- uhlík chemie MeSH
- virus chřipky A, podtyp H5N1 chemie izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The choice of tools that enable efficient targeting of exogenous antigens (Ag) for processing and presentation by professional Ag-presenting cells (APC) remains limited. This represents, indeed, a bottleneck in development of vaccines inducing specific T-cell responses. Here, we describe a novel strategy of Ag delivery into APCs. The Ag of choice is fused to the N- or C-terminus of streptavidin (SA) and tetrameric Ag-SA or SA-Ag fusion proteins are produced in E. coli and purified by 2-Iminobiotin-Agarose affinity chromatography. Alternatively, Ag-SA proteins are purified from urea extracts of E. coli inclusion bodies and refolded in vitro into functional tetramers. Complexes with biotinylated antibodies targeting cell surface receptors are formed and used to deliver the Ags of choice for processing and presentation by APCs and induction of Ag-specific CD4+ and CD8+ T-cell responses in vitro and in vivo.
- MeSH
- antigeny CD11c imunologie metabolismus MeSH
- antigeny chemie genetika imunologie metabolismus MeSH
- biotinylace MeSH
- buněčné linie MeSH
- dendritické buňky imunologie metabolismus MeSH
- kur domácí MeSH
- molekulární sekvence - údaje MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- ovalbumin chemie genetika imunologie metabolismus MeSH
- receptory buněčného povrchu imunologie metabolismus MeSH
- rekombinantní fúzní proteiny chemie genetika imunologie metabolismus MeSH
- rozpustnost MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- streptavidin chemie genetika imunologie metabolismus MeSH
- T-lymfocyty imunologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
