The analysis of low-abundance protein molecules in human serum is reported based on counting of the individual affinity-captured analyte on a solid sensor surface, yielding a readout format similar to digital assays. In this approach, a sandwich immunoassay with rolling circle amplification (RCA) is used for single molecule detection (SMD) through associating the target analyte with spatially distinct bright spots observed by fluorescence microscopy. The unspecific interaction of the target analyte and other immunoassay constituents with the sensor surface is of particular interest in this work, as it ultimately limits the performance of this assay. It is minimized by the design of the respective biointerface and thiol self-assembled monolayer with oligoethylene (OEG) head groups, and a poly[oligo(ethylene glycol) methacrylate] (pHOEGMA) antifouling polymer brush was used for the immobilization of the capture antibody (cAb) on the sensor surface. The assay relying on fluorescent postlabeling of long single-stranded DNA that are grafted from the detection antibody (dAb) by RCA was established with the help of combined surface plasmon resonance and surface plasmon-enhanced fluorescence monitoring of reaction kinetics. These techniques were employed for in situ measurements of conjugating of cAb to the sensor surface, tagging of short single-stranded DNA to dAb, affinity capture of the target analyte from the analyzed liquid sample, and the fluorescence readout of the RCA product. Through mitigation of adsorption of nontarget molecules on the sensor surface by tailoring of the antifouling biointerface, optimizing conjugation chemistry, and by implementing weak Coulombic repelling between dAb and the sensor surface, the limit of detection (LOD) of the assay was substantially improved. For the chosen interleukin-6 biomarker, SMD assay with LOD at a concentration of 4.3 fM was achieved for model (spiked) samples, and validation of the ability of detection of standard human serum samples is demonstrated.

• Keywords
• antifouling biointerface, biomarker, digital readout of assay, rolling circle amplification, single molecule detection, surface plasmon resonance, surface plasmon-enhanced fluorescence,
• MeSH
• DNA, Single-Stranded * MeSH
• Humans MeSH
• Surface Plasmon Resonance * methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Single-Stranded * MeSH

Antifouling polymer layers containing extracellular matrix-derived peptide motifs offer promising new options for biomimetic surface engineering. In this contribution, we report the design of antifouling vascular grafts bearing biofunctional peptide motifs for tissue regeneration applications based on hierarchical polymer brushes. Hierarchical diblock poly(methyl ether oligo(ethylene glycol) methacrylate-block-glycidyl methacrylate) brushes bearing azide groups (poly(MeOEGMA-block-GMA-N3)) were grown by surface-initiated atom transfer radical polymerization (SI-ATRP) and functionalized with biomimetic RGD peptide sequences. Varying the conditions of copper-catalyzed alkyne-azide "click" reaction allowed for the immobilization of RGD peptides in a wide surface concentration range. The synthesized hierarchical polymer brushes bearing peptide motifs were characterized in detail using various surface sensitive physicochemical methods. The hierarchical brushes presenting the RGD sequences provided excellent cell adhesion properties and at the same time remained resistant to fouling from blood plasma. The synthesis of anti-fouling hierarchical brushes bearing 1.2 × 103 nmol/cm2 RGD biomimetic sequences has been adapted for the surface modification of commercially available grafts of woven polyethylene terephthalate (PET) fibers. The fiber mesh was endowed with polymerization initiator groups via aminolysis and acylation reactions optimized for the material. The obtained bioactive antifouling vascular grafts promoted the specific adhesion and growth of endothelial cells, thus providing a potential avenue for endothelialization of artificial conduits.

• Keywords
• RGD peptide, X-ray photoelectron spectroscopy, biomimetic surface, hierarchical bioactive polymer brushes, vascular graft, “click”-chemistry,
• MeSH
• Adsorption MeSH
• Amino Acid Motifs MeSH
• Azides chemistry   MeSH
• Coated Materials, Biocompatible * MeSH
• Biomimetic Materials * MeSH
• Cell Adhesion MeSH
• Cell Division MeSH
• Endothelium, Vascular physiology   MeSH
• Blood Vessel Prosthesis * MeSH
• Click Chemistry MeSH
• Human Umbilical Vein Endothelial Cells MeSH
• Immobilized Proteins MeSH
• Silicon MeSH
• Plasma MeSH
• Blood Proteins MeSH
• Humans MeSH
• Oligopeptides chemistry   MeSH
• Polyethylene Terephthalates chemistry   MeSH
• Polymerization * MeSH
• Surface Properties MeSH
• Guided Tissue Regeneration instrumentation   MeSH
• Glass MeSH
• Materials Testing MeSH
• Thrombosis prevention & control   MeSH
• Gold MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• arginyl-glycyl-aspartic acid MeSH Browser
• Azides MeSH
• Coated Materials, Biocompatible * MeSH
• Immobilized Proteins MeSH
• Silicon MeSH
• Blood Proteins MeSH
• Oligopeptides MeSH
• Polyethylene Terephthalates MeSH
• Gold MeSH

Cells are continuously sensing their microenvironment and subsequently respond to different physicochemical cues by the activation or inhibition of different signaling pathways. To study a very complex cellular response, it is necessary to diminish background environmental influences and highlight the particular event. However, surface-driven nonspecific interactions of the abundant biomolecules from the environment influence the targeted cell response significantly. Yes-associated protein (YAP) translocation may serve as a marker of human hepatocellular carcinoma (Huh7) cell responses to the extracellular matrix and surface-mediated stresses. Here, we propose a platform of tunable functionable antifouling poly(carboxybetain) (pCB)-based brushes to achieve a molecularly clean background for studying arginine, glycine, and aspartic acid (RGD)-induced YAP-connected mechanotransduction. Using two different sets of RGD-functionalized zwitterionic antifouling coatings with varying compositions of the antifouling layer, a clear correlation of YAP distribution with RGD functionalization concentrations was observed. On the other hand, commonly used surface passivation by the oligo(ethylene glycol)-based self-assembled monolayer (SAM) shows no potential to induce dependency of the YAP distribution on RGD concentrations. The results indicate that the antifouling background is a crucial component of surface-based cellular response studies, and pCB-based zwitterionic antifouling brush architectures may serve as a potential next-generation easily functionable surface platform for the monitoring and quantification of cellular processes.

• Keywords
• antifouling polymer brushes, cell mechanotransduction, cell signaling, functional biointerfaces, surface modification, zwitterionic material,
• MeSH
• Acrylamides chemistry   MeSH
• Coated Materials, Biocompatible chemistry   MeSH
• Biofouling prevention & control   MeSH
• Mechanotransduction, Cellular * MeSH
• Extracellular Matrix metabolism   MeSH
• Humans MeSH
• Stress, Mechanical MeSH
• Cell Line, Tumor MeSH
• Oligopeptides chemistry   MeSH
• Proto-Oncogene Proteins c-yes metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Acrylamides MeSH
• arginyl-glycyl-aspartic acid MeSH Browser
• Coated Materials, Biocompatible MeSH
• Oligopeptides MeSH
• Proto-Oncogene Proteins c-yes MeSH
• YES1 protein, human MeSH Browser
• zwitterion carboxybetaine acrylamide MeSH Browser

A biosensor for the detection of hepatitis B antibodies in clinical saliva was developed. Compared to conventional analysis of blood serum, it offers the advantage of noninvasive collection of samples. Detection of biomarkers in saliva imposes two major challenges associated with the low analyte concentration and increased surface fouling. The detection of minute amounts of hepatitis B antibodies was performed by plasmonically amplified fluorescence sandwich immunoassay. To have access to specific detection, we prevented the nonspecific adsorption of biomolecules present in saliva by brushes of poly[(N-(2-hydroxypropyl) methacrylamide)-co-(carboxybetaine methacrylamide)] grafted from the gold sensor surface and post modified with hepatitis B surface antigen. Obtained results were validated against the response measured with ELISA at a certified laboratory using serum from the same patients.

• MeSH
• Biomarkers analysis   MeSH
• Biosensing Techniques methods   MeSH
• Spectrometry, Fluorescence MeSH
• Hepatitis B Surface Antigens chemistry   immunology   MeSH
• Hepatitis B Antibodies analysis   blood   immunology   MeSH
• Immobilized Proteins chemistry   immunology   MeSH
• Immunoassay MeSH
• Humans MeSH
• Polymers chemistry   MeSH
• Surface Plasmon Resonance MeSH
• Saliva metabolism   MeSH
• Gold chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Biomarkers MeSH
• Hepatitis B Surface Antigens MeSH
• Hepatitis B Antibodies MeSH
• Immobilized Proteins MeSH
• Polymers MeSH
• Gold MeSH