Sandwich Immuno-RCA Assay with Single Molecule Counting Readout: The Importance of Biointerface Design.

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.

Mass-Spectrometric Identification of Proteins and Pathways Responsible for Fouling on Poly(ethylene glycol) Methacrylate Polymer Brushes.

Prevention of fouling from proteins in blood plasma attracts significant efforts, and great progress is made in identifying surface coatings that display antifouling properties. In particular, poly(ethylene glycol) (PEG) is widely used and dense PEG-like cylindrical brushes of poly[oligo(ethylene glycol) methacrylate] (poly(OEGMA)) can drastically reduce blood plasma fouling. Herein, a comprehensive study of the variation of blood plasma fouling on this surface, including the analysis of the composition of protein deposits on poly(OEGMA) coatings after contact with blood plasma from many different donors, is reported. Correlation between the plasma fouling behavior and protein deposit composition points to the activation of the complement system as the main culprit of dramatically increased and accelerated deposition of blood plasma proteins on this type of antifouling coating, specifically through the classical pathway. These findings are consistent with observations on PEGylated drug carriers and highlight the importance of understanding the potential interactions between antifouling coatings and their environment.