Polymerization-based signal amplification under ambient conditions with thirty-five second reaction times

Abstract

Although polymerization-based amplification (PBA) has demonstrated promise as an inexpensive technique for use in molecular diagnostics, oxygen inhibition of radical photopolymerization has hindered its implementation in point-of-care devices. The addition of 0.3-0.7 μM eosin to an aqueous acrylate monomer solution containing a tertiary amine allows an interfacial polymerization reaction to proceed in air only near regions of a test surface where additional eosin initiators coupled to proteins have been localized as a function of molecular recognition events. The dose of light required for the reaction is inversely related to eosin concentration. This system achieves sensitivities comparable to those reported for inert gas-purged systems and requires significantly shorter reaction times. We provide several comparisons of this system with other implementations of polymerization-based amplification.

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

“…With an average of three eosin molecules coupled to each binding event, detection limits have been established in several test systems. [9,13,18] In the present work, we investigate whether increasing the number of eosin molecules localized per binding event improves the detection limit, as expected on the basis of our previous observations in the fluorescein/tertiary amine co-initiation system. [19] To this end, we have synthesized a set of dual-functional polymers, termed macroinitiators, bearing a protein for molecular recognition and a variable number of eosin substituents for the ability to couple initiation of radical polymerization to molecular recognition.…”

“…Detailed information on the test surface, including density of biotin and spot size, was obtained by fluorescence techniques as previously described. [9,19] fluorescence intensity in the target spots than the other macroinitiators; for example, the average intensities were 20800 (± 1100) for 6, 34000 (± 1100) for 7 (see Figure S1), 52400 (± 240) for 8, and 30600 (± 580) for 9 in the first row (Figure 2b-d). On the other hand, the fluorescence intensity of the background is proportional to the number of eosin molecules per the copolymer, where the average intensities were 1450 (± 460) for 6, 2420 (± 680) for 7 (see Figure S1), 25900 (± 3100) for 8, and 26800 (± 350) for 9.…”

“…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.…”

“…[1][2][3][4] Cost, ease-of-use, and performance are each of major importance for wide adoption of detection platforms and techniques. [5] With these criteria in mind, we and others have developed polymerization-based signal amplification [6][7][8][9] as a method that provides rapid (< 1 min) and unambiguous visualization of results in RDTs using low-cost chemistry that is currently used in a wide variety of inexpensive consumer products [10][11][12] . 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.…”

“…[6,7,19] Of these, the eosin/tertiary amine co-initiation system provides the best performance in an oxygen environment in terms of detection sensitivity accompanied by a rapid reaction rate (< 1 min). [8,9,19] The limit of detection occurs when the local initiator density in the vicinity of binding events of interest falls below a threshold level required for the polymerization reaction to overcome inhibition by molecular oxygen. With an average of three eosin molecules coupled to each binding event, detection limits have been established in several test systems.…”