Impact of Dissociation Constant on the Detection Sensitivity of Polymerization-Based Signal Amplification Reactions

Abstract

Many studies have demonstrated the concept of using free-radical polymerization reactions to provide signal amplification so that molecular recognition events indicative of disease states may be detected in a simple and low-cost manner. We provide the first systematic study of how the dissociation constant impacts detection sensitivity in these assays, having chosen a range of dissociation constants (nanomolar to picomolar) that is typical of those encountered in molecular diagnostic applications that detect protein-protein binding events. In addition, we use experimental results to validate a mass-action kinetic model that may be used to predict assay performance as an alternative or supplement to the empirical approach to developing new polymerization-based amplification assays that has characterized the field to date.

Balancing the Initiation and Molecular Recognition Capabilities of Eosin Macroinitiators of Polymerization‐Based Signal Amplification Reactions

“…Figure 4 presents the original image (left), the original with a mask for automated image analysis (middle), and the software-calculated mean intensity from the spots in each row (right). As detailed in earlier work [9,18] , interfacial polymerization coupled were below the threshold for amplification and polymerization was not observed in the 10-250 sec range. With macroinitiator 9, the overall initiator density localized on the surface was higher than observed with 6 and 7, but the difference in initiator densities inside the features and outside the features was not great enough to enable a photoreaction time that resulted in polymerization only in regions where molecular recognition occurred.…”

“…In polymerizationbased amplification (PBA), initiators of radical polymerization reactions are localized as a function of molecular recognition events, and upon activation using light, a cross-linked hydrogel forms from an aqueous monomer solution. The resulting micron-scale hydrogel [7,9] is easily visualized without instrumentation, and the technique has been used to detect DNA hybridization [8,13,14] , antibody-antigen recognition [15][16][17] , and other protein-protein binding events [18] . PBA tolerates complex sample matrices including serum [13] , cell lysates [9] , and fixed and permeabilized cells.…”

A quantitative analysis of peroxy-mediated cyclic regeneration of eosin under oxygen-rich photopolymerization conditions

“…a protein, nucleic acid or other biological molecule), in a solution that also includes many other components, specifically binds to a surface that has been prefunctionalized with a binding partner for the analyte [12,20,31]. The other components are then washed away, after which a solution containing eosin-conjugated binding molecules is added.…”

“…Air-tolerant polymerization reactions simplify PBA procedures; however, the use of AGET ATRP for PBA is not ideal as it requires hours to achieve nanometer-thick films that cannot be easily detected [28,29], making it unsuitable for diagnostic tests that require rapid response times. Conversely, eosin, a photoreducible xanthene dye, has been shown to initiate polymerization reactions that result in micron-scale hydrogel films in 35 s to 2 min in air in this context [12,20,31], making it a suitable choice as a photoinitiator for PBA when rapid response times are required.…”

“…Recent experimental evidence shows that inclusion of sub-micromolar concentrations of eosin in the monomer solution allows the reaction to proceed without purging using inert gases and in the presence of a constant influx of oxygen from air [20,31], but theoretical contributions towards the elucidation of the mechanism behind eosin's ability to initiate polymerization in the presence of a large excess of oxygen are lacking. Previous numerical models [35,36] incorporating the eosin-tertiary amine initiation system assumed oxygen was absent and estimated the unknown rate constants associated with initiation by using them collectively as a fit parameter to match model results with experimentally measured conversion profiles.…”