Nanoparticles functionalized with specific biological recognition molecules play a major role for sensor response enhancement in surface plasmon resonance (SPR) based biosensors. The functionalization procedure of such nanoparticles is crucial, since it influences their interactions with the environment and determines their applicability to biomolecular detection in complex matrices. In this work we show how the ζ-potential (Zpot) of bio-functionalized gold spherical NPs (Bio-NPs) is related to the SPR sensor response enhancement of an immune-sandwich-assay for the detection of the carcinoembryonic antigen (CEA), a cancer marker for colorectal carcinomas. In particular, we prepare bio-functional nanoparticles by varying the amount of peptide (either streptavidin or antibody against CEA) bound on their surface. Specific and non-specific sensor responses, reproducibility, and colloidal stability of those bio-functional nanoparticles are measured via SPR and compared to ζ-potential values. Those parameters are first measured in buffer solution, then measured again when the surface of the biosensor is exposed to blood plasma, and finally when the nanoparticles are immersed in blood plasma and flowed overnight on the biosensor. We found that ζ-potential values can guide the design of bio-functional NPs with improved binding efficiency and reduced non-specific sensor response, suitable reproducibility and colloidal stability, even in complex matrixes like blood plasma.

• Klíčová slova
• SPR, biosensor, blood, functionalization, gold nanoparticles, immuno-assay, peptide, ζ-potential,
• Publikační typ
• časopisecké články MeSH

Engineered combinatorial libraries derived from small protein scaffolds represent a powerful tool for generating novel binders with high affinity, required specificity and designed inhibitory function. This work was aimed to generate a collection of recombinant binders of human interleukin-23 receptor (IL-23R), which is a key element of proinflammatory IL-23-mediated signaling. A library of variants derived from the three-helix bundle scaffold of the albumin-binding domain (ABD) of streptococcal protein G and ribosome display were used to select for high-affinity binders of recombinant extracellular IL-23R. A collection of 34 IL-23R-binding proteins (called REX binders), corresponding to 18 different sequence variants, was used to identify a group of ligands that inhibited binding of the recombinant p19 subunit of IL-23, or the biologically active human IL-23 cytokine, to the recombinant IL-23R or soluble IL-23R-IgG chimera. The strongest competitors for IL-23R binding in ELISA were confirmed to recognize human IL-23R-IgG in surface plasmon resonance experiments, estimating the binding affinity in the sub- to nanomolar range. We further demonstrated that several REX variants bind to human leukemic cell lines K-562, THP-1 and Jurkat, and this binding correlated with IL-23R cell-surface expression. The REX125, REX009 and REX128 variants competed with the p19 protein for binding to THP-1 cells. Moreover, the presence of REX125, REX009 and REX115 variants significantly inhibited the IL-23-driven expansion of IL-17-producing primary human CD4(+) T-cells. Thus, we conclude that unique IL-23R antagonists derived from the ABD scaffold were generated that might be useful in designing novel anti-inflammatory biologicals.

• Klíčová slova
• combinatorial library, cytokine, engineered binding protein, protein scaffold, psoriasis, ribosome display,
• MeSH
• antiflogistika chemie   farmakologie   MeSH
• bakteriální proteiny chemie   MeSH
• buňky K562 MeSH
• buňky Th17 účinky léků   metabolismus   MeSH
• imunologické faktory chemie   farmakologie   MeSH
• interleukin-23 chemie   fyziologie   MeSH
• Jurkat buňky MeSH
• kompetitivní vazba MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• peptidové fragmenty chemie   farmakologie   MeSH
• preklinické hodnocení léčiv MeSH
• receptory interleukinů antagonisté a inhibitory   fyziologie   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antiflogistika MeSH
• bakteriální proteiny MeSH
• IgG Fc-binding protein, Streptococcus MeSH Prohlížeč
• IL23R protein, human MeSH Prohlížeč
• imunologické faktory MeSH
• interleukin-23 MeSH
• peptidové fragmenty MeSH
• receptory interleukinů MeSH

Solid-phase hybridization, i.e. the process of recognition between DNA probes immobilized on a solid surface and complementary targets in a solution is a central process in DNA microarray and biosensor technologies. In this work, we investigate the simultaneous effect of monovalent and divalent cations on the hybridization of fully complementary or partly mismatched DNA targets to DNA probes immobilized on the surface of a surface plasmon resonance sensor. Our results demonstrate that the hybridization process is substantially influenced by the cation shielding effect and that this effect differs substantially for solid-phase hybridization, due to the high surface density of negatively charged probes, and hybridization in a solution. In our study divalent magnesium is found to be much more efficient in duplex stabilization than monovalent sodium (15 mM Mg2+ in buffer led to significantly higher hybridization than even 1 M Na+). This trend is opposite to that established for oligonucleotides in a solution. It is also shown that solid-phase duplex destabilization substantially increases with the length of the involved oligonucleotides. Moreover, it is demonstrated that the use of a buffer with the appropriate cation composition can improve the discrimination of complementary and point mismatched DNA targets.

• MeSH
• chybné párování bází MeSH
• DNA sondy chemie   MeSH
• hořčík chemie   MeSH
• hybridizace nukleových kyselin * MeSH
• kationty dvojmocné chemie   MeSH
• kationty jednomocné chemie   MeSH
• povrchová plasmonová rezonance * MeSH
• sodík chemie   MeSH
• spektrofotometrie ultrafialová MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA sondy MeSH
• hořčík MeSH
• kationty dvojmocné MeSH
• kationty jednomocné MeSH
• sodík MeSH