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. 2025 Nov 19;10(47):57657–57665. doi: 10.1021/acsomega.5c09072

Systematic Optimization of Fluorogenic ARGET ATRP toward Rapid and Oxygen-Tolerant Analyte Detection

Jordan B McMurry †, Jose Ricardo dos Remedios †, Nicholas L Cipolla †, Max Chamoun †, Christina B Cooley †,*
PMCID: PMC12676337  PMID: 41358131

Abstract

Sensitive analyte detection requires the rapid amplification of small molecular interactions into observable macro readouts. Fluorogenic activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) afford real-time, visible fluorescence generation as a function of polymerization initiator (or analyte) concentration, as dark, nonfluorescent monomers reveal their fluorescence upon incorporation into the growing polymer chain. Though encouraging, the practicality of the initially developed fluorogenic ARGET ATRP platform is severely limited by long reaction times and the sensitivity to oxygen, precluding its development for bioanalyte detection applications. This paper describes a systematic optimization study to probe the effect of several fluorogenic ARGET ATRP reaction variables, evaluating the effects of surfactant, catalyst loading, halide salt, and reaction solvent on fluorescence generation. Combining the effects of optimized individual reaction components affords a significant increase in both the rate and magnitude of fluorescence generation and renders the optimized platform tolerant to ambient oxygen, able to reliably detect initiator or model analyte when conducted in open air. Taken together, these dramatic strides in the optimization of fluorogenic ARGET ATRP open the door to applications for rapid bioanalyte detection.

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Introduction

The ability to detect and quantify the presence of chemical and biological analytes is fundamental to many areas of science and industry, including research, medicine, manufacturing, and quality control. The high-impact area of disease diagnostics particularly requires the detection of low concentrations of proteins or other disease-associated antigens for early diagnosis and intervention. Though many sensitive and effective diagnostic assays have been developed, these tests are often expensive and time-consuming and require skilled individuals to conduct. , Thus, there is a need for a method of analyte detection and signal amplification that does not require expensive, specialized equipment while still maintaining or improving upon the sensitivity, sample throughput, and versatility of existing detection assays.

Radical chain polymerization has recently emerged as a promising chemical method for the simple, low-cost, and robust amplification and detection of bioanalytes. − Controlled radical polymerization methods such as atom transfer radical polymerization (ATRP) have been demonstrated to quantitatively detect bioanalytes, including DNA and proteins. − Our group has developed a real-time fluorescence method for amplifying the initiator signal by fluorogenic activators regenerated by electron transfer (ARGET) ATRP, utilizing nonfluorescent “dark” monomers that become visibly fluorescent under UV irradiation only when polymerized. This technique allows for the qualitative and quantitative detection of polymerization initiator spanning orders of magnitude of concentrations (from pM to mM) and has been applied to the detection of model protein bioanalytes in both isolated and heterogeneous biological systems.

Though promising, major drawbacks to our initially developed fluorogenic ARGET ATRP technique limit its potential for biodetection applications; namely, long reaction times (∼24 h) and oxygen sensitivity. The originally developed fluorogenic ARGET ATRP copolymerization method applied standard aqueous ARGET ATRP PEG methacrylate 3 controlled polymerization conditions modified by the addition of the fluorogenic polycyclic aromatic hydrocarbon anthracene monomer 2 and added surfactant for fluorogenic monomer solubilization (Scheme ). Multiple components of this aqueous ARGET ATRP system are known to impact polymerization kinetics, including surfactant, catalyst loading, , salt, , and reducing agent. Further, our fluorogenic ARGET ATRP systemwith one water-soluble monomer and one hydrophobic monomer solubilized by a surfactantis unique; to our knowledge, there are no other reported copolymerizations with a similar composition of monomers.

1. Original Fluorogenic ARGET ATRP Reaction Conditions as the Starting Point for Optimization.

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Toward the goal of faster, brighter fluorescence generation for downstream biodetection applications, we describe herein a systematic approach to optimization of our fluorogenic ARGET ATRP platform by exploring the individual and collective impact of surfactant, catalyst, reducing agent, halide, and solvent on polymer fluorescence and reaction kinetics. Varied reaction components were compared to initially reported conditions (Scheme ) in the presence and absence of initiator as the model analyte. Ultimately, optimized fluorogenic ATRP conditions were identified that drastically increase the fluorescence generation and polymerization kinetics without significant background fluorescence. Optimized conditions further enable oxygen tolerance, allowing for fluorogenic ARGET ATRP reactions to be conducted in the open air.

Results and Discussion

Effect of Surfactant Type

Surfactant addition is required for fluorogenic aqueous ATRP due to the hydrophobic nature of fluorogenic monomer 2 (Scheme ). Original reaction conditions incorporated the anionic surfactant sodium dodecyl sulfate (SDS), which facilitated fluorogenic monomer solubilization; however, fluorescence was particularly sensitive to SDS concentrations as higher concentrations (>18 mM) significantly reduced polymer fluorescence. In similar aqueous ATRP reactions, the presence and type of surfactant have been further shown to significantly affect the induction period and polymerization rate as well as solubilization efficiency and polymer interaction. , As the surfactant represented a critical and underexplored variable in our fluorogenic ARGET ATRP platform, our optimization studies began by screening four alternative surfactants of varying classifications to assess their effects on fluorescence generation.

In addition to the anionic surfactant SDS, anionic sodium dodecyl benzenesulfonate (SDBS), cationic cetylpyridinium chloride (CPC), and neutral t-ocylphenoxy polyethoxyethanol (TX-100) and polyoxyethylene (20) cetyl ether (Brij-58) were chosen for their range of properties and anthracene solubilization efficiencies. The minimum amount of each surfactant needed to solubilize fluorogenic anthracene monomer 2 to the same degree as 18 mM SDS was determined by visually comparing stock solutions of the monomer in water as increasing amounts of each surfactant were added (Figure S1, Table S1).

The concentrations determined for each surfactant were then used to conduct fluorogenic polymerization reactions, as shown in Scheme , replacing SDS with the alternate surfactants tested. Figure exhibits the most representative results from several trials comparing the relative fluorescence of each polymerization at 24 h. TX-100 and Brij-58, both nonionic surfactants, led to the brightest fluorescence at 24 h, with SDBS and SDS (both anionic surfactants) exhibiting approximately equal but lower fluorescence than the neutral surfactants. CPC, the cationic surfactant, led to significantly lower fluorescence. Of the two neutral surfactants, TX-100 demonstrated more consistency in reaction fluorescence and more quickly solubilized the fluorogenic monomer when added to the stock solution. Thus, TX-100 was chosen as the preferred surfactant for continued optimization. Several concentrations of TX-100 were also tested, but no significant improvements in fluorescence were achieved beyond the original concentration of 0.43 mM.

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Fluorogenic ARGET ATRP with different surfactants. Fluorescence emission spectra of fluorogenic ARGET ATRP (ex 371 nm), as shown in Scheme with surfactants SDBS (red), SDS (orange), CPC (green), TX-100 (blue), and Brij-58 (purple) following 24 h of reaction time.

When compared to the critical micelle concentration (CMC) for each surfactant in water, the concentration of each surfactant needed to fully solubilize fluorogenic anthracene monomer 2 in the stock solution always exceeded the CMC for that surfactant (Table S1). This correlation suggests that the surfactant must be concentrated enough to form micelles before the fluorogenic monomer (at a concentration of 5 mM) can be fully solubilized. When used in polymerization, however, the only surfactants meeting or exceeding their CMCs were SDS, Brij-58, and TX-100. Surfactant concentration relative to CMC is known to affect polymerization kinetics, with emulsion polymerization becoming possible at approximately 2–3 times the CMC for a given surfactant. However, the ratio of CMC to the concentration of surfactant in polymerization did not appear to correlate with the relative success of each surfactant at achieving maximum fluorescence in a 24 h fluorogenic ARGET ATRP reaction. As the CMC can change readily under different conditions, the threshold for emulsion polymerization may or may not have been reached for the surfactants at the highest concentrations relative to their CMCs.

The maximum fluorescence at 24 h (Figure ) did appear to generally correlate with the solubilization efficiency of each surfactant for anthracene, with some exceptions. The use of SDS and SDBS led to very similar maximum fluorescence over several trials, with neither surfactant reliably surpassing the other. While SDS was anticipated to have higher solubilization ability, both surfactants are anionic and have similar structures (Figure S1b), which may explain why the difference between polymerizations using SDS and SDBS is very small. In addition, the solubilization efficiency of CPC would be expected to fall between those of SDS and TX-100, but the maximum fluorescence of a polymerization using CPC falls far below those of all other surfactants tested. This discrepancy is likely due to the fact that micelles formed by CPC are specific fluorescence quenchers of polycyclic aromatic hydrocarbons, including anthracene. Together, results suggest that improvements in fluorescence observed with alternative surfactants are most likely due to increases in the ability to fully solubilize the fluorogenic monomer.

Surfactant Effects on Polymerization Kinetics and Variability

With TX-100 determined to be the optimal surfactant in water for achieving the highest fluorescence at a given reaction time during fluorogenic polymerization, its effect on the kinetics of fluorescence generation was explored. Fluorogenic polymerization reactions were run in a sealed cuvette with either TX-100 or SDS as surfactant, with fluorescence measurements obtained each minute (Figure ). Polymerizations with both surfactants exhibited similar hyperbolic curves of increase in fluorescence over time, with the most rapid increase at the beginning of the reaction and a plateau observed in later time points. However, the polymerization using TX-100 exhibited a greater initial rate of increase in fluorescence compared to SDS, in addition to reaching a higher fluorescence emission by 24 h (Figure a). Further, this method allowed for observation of the lag time in fluorescence at the beginning of the polymerizations, with a significantly longer lag time in fluorescence generation observed in the case of SDS (Figure b). The hyperbolic curve of the fluorescence emission is consistent with the majority of the fluorogenic monomer being incorporated into polymers relatively early in the reaction. While polymerization of the PEG monomer 3 still occurs after the 24-h time point, fluorescence does not appear to significantly increase after 24 h of reaction time, indicating that either all of the fluorogenic anthracene monomer 2 has polymerized by 24 h, or the maximum fluorescence possible for the An-PEG copolymers was reached regardless of continued incorporation of fluorogenic monomer. These results demonstrate that the surfactant used can have a significant impact on the polymerization kinetics of the fluorogenic monomer.

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Fluorogenic ARGET ATRP kinetics with SDS or TX-100. Fluorescence (ex 371 nm, em 426 nm) of fluorogenic ARGET ATRP, as shown in Scheme with surfactants SDS (orange) or TX-100 (blue), measured every minute. (a) Full 24 h measurement for each trial. (b) Zoom in of first 30 min of polymerization reactions depicted in (a).

To further probe the effects of TX-100 on aspects of the polymerization reaction, the variability in the fluorescence readout over multiple trials was compared for SDS and TX-100 (Figure S2). TX-100 has a lower variability in fluorescence throughout the reaction compared to SDS, with a significantly lower coefficient of variation observed for TX-100, especially at early time points (Table S2). This decrease in variability between reactions with TX-100 compared to SDS is likely due to the unique ways in which SDS can interact with the ATRP catalyst that TX-100 does not share. SDS can form an ionic complex with the ATRP catalyst, facilitating its migration into micelles and out of the aqueous phase, and is also known to coordinate with the copper-ligand catalyst complex. , These interactions, which do not contribute to polymerization progress, could lead to increased variability in polymerizations with SDS as the surfactant, while TX-100 removes the opportunity for these side reactions and reduces variability. Taken together, TX-100 is a superior surfactant to SDS in water for fluorogenic ATRP, offering improvements in the maximum fluorescence reached and rate of fluorescence increase while reducing the polymerization lag time and variability.

Effects of Catalyst Loading and Reducing Agent

The copper catalyst/ligand complex is the component that most directly controls the rate of an ARGET ATRP reaction; the equilibrium between its active (activator) and inactive (deactivator) states determines the rate of polymerization and is affected by several other reaction components, including the halide salt and reducing agent applied for catalyst regeneration. The catalyst’s concentration is typically optimized for a balance between reaction speed and high control over rate and molecular weight. , The reducing agent concentration also must be balanced, as a minimum is required for catalyst regeneration, while excess can lead to uncontrolled polymerization and unwanted side reactions. The aqueous ARGET ATRP report that inspired our original fluorogenic ATRP reaction conditions demonstrated that higher catalyst loading can increase the rate of polymerization without dramatically impacting its controlled or “living” polymerization character, which suggests that higher concentrations of catalyst could be used in fluorogenic ARGET ATRP to increase the rate at which fluorescence develops.

To evaluate the effect of catalyst loading on fluorogenic ARGET ATRP, the CuBr2/TPMA ligand complex concentration was increased to double or higher than its original concentration. These experiments were also run with a no-initiator control to ensure that faster fluorescence rates did not lead to uncontrolled background polymerization in the absence of initiator, which would represent false positives in a downstream biodetection assay. When the concentration of catalyst/ligand complex was increased above double the initial concentration (above 0.02 mol %), significant fluorescence was observed in the no-initiator control (Figure S3). However, double catalyst loading (from 0.01 to 0.02 mol %) led to significantly higher fluorescence at all time points without observable background fluorescence (Figure a).

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Optimization of catalyst/ligand loading and reducing agent. Maximum fluorescence of fluorogenic ARGET ATRP (ex 371 nm, em 426 nm) over time with TX-100 as a surfactant. (Left) Fluorescence of polymerizations comparing the CuBr2 catalyst and TPMA ligand concentration, with standard catalyst/ligand loading (0.01 mol %) with no initiator 3 present (light purple), standard catalyst/ligand with a 2 mM initiator (dark purple), doubled catalyst/ligand concentration (0.02 mol %) with no initiator (light green), and doubled catalyst/ligand with a 2 mM initiator (dark green). (Right) Fluorescence of polymerizations with doubled catalyst/ligand loading (0.02 mol %), comparing standard ascorbic acid concentration (0.93 mM, blue) to doubled ascorbic acid concentration (1.87 mM, orange).

Active catalyst generation is dependent upon the ascorbic acid reducing agent, which is typically in a molar ratio of 19:1 with the catalyst. When the catalyst concentration was increased, the ratio between the two was reduced, potentially creating a bottleneck for catalyst regeneration due to the lower relative ascorbic acid concentration. To test whether increasing the ascorbic acid in proportion with the increase in the catalyst would also lead to an improvement in fluorescence, two polymerizations with double catalyst loading were compared: one where the concentration of ascorbic acid was doubled (from 0.93 to 1.87 mM) and one that was kept at the standard 0.93 mM concentration (Figure b). The results suggest that the lower proportion of ascorbic acid was bottlenecking the polymerization; after 1 h, the polymerization without double ascorbic acid stopped increasing in fluorescence, and its maximum fluorescence was much lower than the polymerization that doubled the ascorbic acid concentration. Taken together, these results indicated that double the initial catalyst/ligand loading and reducing agent concentration (0.02 mol % catalyst and 1.87 mM ascorbic acid) were found to be the optimal concentrations for fluorogenic ARGET ATRP.

Effects of Halide and Solvent

The halide salt additive in ARGET ATRP reactions shifts the equilibrium of the catalyst toward the deactivator state, providing stability and control of the polymerization rate. Chloride salts (Cl–) are generally preferred in aqueous ARGET ATRP, as they provide optimal control over molecular weight and dispersity, while bromide salts (Br-) are known to increase the polymerization rate. To assess whether the rate increase expected with bromide salt addition would be beneficial for fluorogenic ARGET ATRP, standard sodium chloride salt and tetraethylammonium bromide (TEABr) were tested for impact on fluorescence in the presence and absence of an initiator (Figure S4). Although the bromide salt led to higher fluorescence as expected, the high level of background fluorescence observed in the absence of initiator rendered bromide salts too active for use in fluorogenic ARGET ATRP.

While conducting fluorogenic ARGET ATRP in aqueous solvent is critical for keeping the assay compatible with biological analytes, the type of aqueous environmenti.e., pure water versus buffercould also impact fluorescence and the relationship between halide addition. Aqueous ARGET ATRP has been conducted in H2O and in phosphate-buffered saline (PBS, pH 7.1). , Both solvents were found to produce similarly well-controlled polymerization reactions; however, H2O and PBS were also found to differently affect catalyst stability upon the addition of varying halides. Thus, the optimal halide concentration was explored concurrently with the optimal solvent for the fluorogenic ARGET ATRP (Figure ). While changing salt concentrations did not dramatically affect fluorescence levels in either H2O or PBS buffer (Figure , data not shown), a substantial increase in fluorescence generation was observed when fluorogenic ATRP was conducted in PBS buffer, with the optimal conditions in PBS buffer without added halide (90 mM NaCl), a similar salt concentration as utilized in H2O (98 mM NaCl, Figure ).

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Effect of solvent and salt concentration. Maximum fluorescence of fluorogenic ARGET ATRP (ex 371 nm, em 426 nm) over time with TX-100 as a surfactant comparing polymerizations in DI water with added NaCl (blue, 98 mM Cl–), in PBS with added NaCl (orange, 188 mM Cl–), and in PBS without added salt (green, 90 mM Cl–).

The benefit of decreasing the salt concentration is most likely explained by the role of the halide salt in the polymerization; the chloride ions act as a source of stability for the catalyst, shifting the equilibrium toward the deactivator state and keeping the concentration of radicals in the solution low and decreasing the rate of polymerization. Thus, lower salt concentrations in PBS lead to improved reaction rate. The significant improvement in fluorescence properties observed in PBS was unexpected, and while not fully understood, could be explained in part by the beneficial impacts of higher pH and improved catalyst stability in PBS relative to H2O.

As reaction solvents can have a significant effect on CMC and surfactant behavior, SDS and TX-100 were also compared in polymerizations with PBS to determine whether TX-100 continued to provide optimal fluorescence parameters in the new solvent. Interestingly, in PBS, the preferred surfactant switches back to SDS, which significantly outperforms TX-100 in fluorogenic ARGET ATRP (Figure ).

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Surfactant effect in PBS as solvent. Maximum fluorescence of fluorogenic ARGET ATRP (ex 371 nm, em 426 nm) over time with TX-100 (blue) or SDS (purple) as a surfactant.

The significant improvement in the kinetics of fluorescence generation mediated by SDS in PBS conditions can most likely be attributed to the effects of salt concentration and ionic strength on the behavior of anionic surfactants, as the addition of electrolytes increases the adsorption of anionic surfactants on nonpolar surfaces lowering the CMC and increasing solubilization efficiency. Thus, while TX-100 provides superior fluorescence characteristics in aqueous fluorogenic ARGET ATRP in pure water, the anionic surfactant SDS is optimal for buffered aqueous fluorogenic ARGET ATRP.

Putting It All TogetherOptimized Conditions

Once the individual reaction components of fluorogenic ARGET ATRP had each been optimized, as described above, the conditions were combined to determine the total effects on the rate of fluorescence generation (Figure ). The combined optimized conditions are shown in Figure a, with changes from our initial fluorogenic ARGET ATRP reaction conditions of doubling the catalyst loading and reducing agent (0.02 and 4 mol %, respectively) and switching to PBS as the reaction solvent. Although these deviations from our initially reported procedure may seem rather minor, their combined impact on fluorescence rate and generation is dramatic (Figure b,c), with a comparison of the different time points demonstrating that the optimized conditions appear almost as bright by one h as the standard conditions reached by 24 h. Further, no increase in background fluorescence was observed in the sample without an initiator under optimized conditions. This suggests a sufficient level of control is maintained under optimized conditions for biodetection applications. Analysis of the synthesized polymers at the 24 h time point by size exclusion chromatography (SEC) reveals that the increase in reaction rate under optimized conditions, as observed by fluorescence, does lead to relatively larger polymers when comparing the same time points (MW for optimized versus standard conditions 115,400 versus 82,100, respectively,Figure S5). This increase in the polymerization reaction rate under optimized conditions also affords a slightly higher dispersity value (1.31), although the optimized conditions remain in a regime of controlled polymerization by ATRP.

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Optimized conditions for fluorogenic ARGET ATRP. (a) Scheme for final optimized fluorogenic ARGET ATRP reaction conditions. (b) Maximum fluorescence (ex 371 nm, em 426 nm) of standard fluorogenic ATRP reaction conditions as shown in Scheme with and without an initiator (dark vs light blue, respectively) compared to optimized fluorogenic ARGET ATRP reaction conditions in a) with and without an initiator (dark vs light purple, respectively). (c) Photographs of the reactions [left: original Scheme conditions; right: optimized (a) conditions] at indicated time points illuminated by a hand-held UV light (365 nm).

Oxygen Tolerability

The dramatically increased reaction kinetics afforded by the optimized fluorogenic ARGET ATRP reaction conditions represented a major step toward achieving our eventual goal of a robust, operationally simple, and applicable biodetection assay. However, the ability to run fluorogenic ARGET ATRP reactions in open air, as opposed to rigorous degassing techniques, would further facilitate applications of this detection platform. The standard fluorogenic ARGET ATRP conditions carried out in open air led to complete or near-complete quenching of the reaction fluorescence, with little to no observable fluorescence in 24 h. Further, in background polymerizations without an initiator, oxygen was capable of initiating the reaction, leading to background polymerization and fluorescence where none was expected, a potential false positive in the detection assay. Although oxygen is deleterious to radical polymerization in general, due to its diradical nature and ability to quench propagating radicals, oxygen can have variable effects on ARGET ATRP reactions, with the ability to interact with the catalyst and the addition of higher ascorbic acid concentrations suggesting a hint of oxygen tolerance.

Due to the increases in catalyst loading and reducing agent in optimized fluorogenic ARGET ATRP, we explored whether optimized reaction conditions afforded an improvement in the ability to tolerate oxygen and run reactions in open air (Figure ). As oxygen has been shown to cause significant variability in radical polymerization, multiple trials of old (standard) and new (optimized) fluorogenic polymerization reaction conditions were assessed in open air in the presence and absence of an initiator. While polymerization was completely quenched in the standard polymerization conditions across several trials, the optimized reaction conditions afforded high levels of fluorescence in open-air conditions, exhibiting significantly improved tolerance to oxygen (Figure ). Trace oxygen was able to initiate polymerization in the absence of initiator, providing significantly higher background fluorescence than when degassed; however, the background is kept low at early time points (t = 1 h, Figure b), allowing for clear discrimination between samples with or without initiator. Although improvements in oxygen tolerability could further reduce the background fluorescence and reaction volume, the results here indicate that the optimization of fluorogenic ARGET ATRP has greatly increased the practicality and ease of conducting fluorogenic polymerization reactions as the purging of oxygen for cuvette-scale reactions is no longer required.

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Oxygen tolerance of optimized fluorogenic ARGET ATRP. (Left) Maximum fluorescence of fluorogenic ARGET ATRP (ex 371 nm, em 426 nm) open to air under original (old) reaction conditions as shown in Scheme with and without initiator (dark vs light blue, respectively) compared to optimized (new) fluorogenic ATRP reaction conditions as shown in Figure a with and without initiator (dark vs light purple, respectively). Error bars represent the standard deviation (n = 3 trials). (Right) Photographs of the reactions at t = 1 h of reaction time illuminated by a hand-held UV light (365 nm).

Conclusions

An ideal biodetection assay requires that the signal of small concentrations of analyte can be amplified to a detectable level in a relatively short period of time. To apply fluorogenic ARGET ATRP as a viable detection platform, significant improvements in reaction speed, fluorescence, and oxygen tolerability were required. It was further critical to keep the variability and level of background fluorescence in the absence of an analyte low. With these criteria in mind, systematic optimization of each variable in fluorogenic ARGET ATRP, including surfactant, catalyst loading, reducing agent concentration, added halide, and reaction solvent, was investigated to elucidate ideal conditions for the kinetics of fluorescence generation. TX-100 was found to be the optimal surfactant for reactions conducted in water as the solvent, whereas SDS proved optimal for fluorogenic polymerization reactions in PBS. Doubling of the catalyst loading and reducing agent concentration and switching of the solvent to PBS buffer afforded high levels of fluorescence generation with no significant background fluorescence in the absence of a model analyte. Further, the optimized reaction conditions exhibited a degree of oxygen tolerance, allowing for cuvette-scale reactions to be conducted in open air for the first time. While further optimization to enable full oxygen tolerance without a detectable background signal is ongoing, the optimization efforts described here offer dramatic improvements to the initially described fluorogenic ARGET ATRP biodetection platform, opening the door for new biodetection applications.

Experimental Section

General Methods

All air- and moisture-sensitive reactions were carried out in glassware that was oven-dried (>130 °C) and cooled under nitrogen (N2) gas. Reaction vessels were sealed with rubber septa and maintained in an inert environment under a positive pressure of anhydrous N2. Stirring was accomplished via magnetic Teflon-coated stir bars. Solid reagents were measured on a Mettler Toledo MS204TS balance. Air- and moisture-sensitive liquids were transferred via a syringe under an atmosphere of N2. Reaction temperatures refer to the bath temperatures at which the reaction vessel was partially immersed. Room temperature indicates an external temperature of 20–25 °C. Elevated temperatures (30 °C) were achieved by the use of a mineral oil bath heated by a VWR 620-HPS hot plate/stirrer.

Materials

Unless otherwise noted, all commercial solvents and reagents were purchased from Millipore-Sigma USA and used as received. Anthracene methacrylamide fluorogenic monomer 2 was synthesized as described previously. Oligo­(ethylene oxide) methyl ether methacrylate (3, 99%, average molecular weight 500) was passed over a column of basic alumina (Millipore-Sigma USA) prior to use. Water was obtained from a Barnstead Nanopure Infinity water system (18 MΩ cm). Gases (N2) were of 5.0 grade supplied by Praxair and used without further purification.

Physical and Spectroscopic Measurements

Fluorescence spectra and intensities were determined with a Photon Technology International Quanta-Master model QM-1 fluorimeter with 2–3 mm slit widths. Kinetic fluorescence measurements were taken using a SpectraMax M3Microplate Reader from Molecular Devices LLC. Samples were run in quartz cuvettes (4 clear walls, path length 1 cm, 10 mm septum) purchased from Science Outlet. Photos of polymerization reactions were taken with iPhone cameras and illuminated by long-wave (365 nm) ultraviolet (UV) light using a hand-held UV lamp (UVP compact). Size exclusion chromatography was performed on a Tosoh EcoSec Elite HLC-8420 gel permeation chromatograph with TSKgel GMHHR-M and TSKgel SuperH-RC columns with a flow rate of 0.5 mL/min at 40 °C. The eluting solvent was tetrahydrofuran (THF). The GPC was calibrated using ReadyCal set Poly­(methyl methacrylate) standards Mp 500–2,700,000 from PSS Polymer Standards Service GmbH.

General Fluorogenic Aqueous ARGET ATRP Copolymerization Procedure

PEG methacrylate 3 (0.700 g, 1.4 mmol), NaCl (17.8 mg, 0.3 mmol), 5 mM stock solution of anthracene methacrylamide 2 in water with 183 mM SDS (300 μL, 1.5 μmol monomer, 55.0 μmol SDS), 100 mM 2-hydroxyethyl-2-bromoisobutyrate initiator 1 in water (60 μL, 6 μmol), and a stock solution of 25 mM CuBr2 and 200 mM tris­(2-pyridylmethyl)­amine, TPMA, in water (6 μL, 0.15 μmol CuBr2, 1.2 μmol TPMA) were dissolved in H2O (2.03 mL). The solution was sealed and purged with nitrogen for 60 min and then transferred to a sealed quartz cuvette (also purged with N2) and placed in a 30 °C oil bath with a stir bar. A 16 mM ascorbic acid solution in water was separately purged with N2 for 30–60 min, and then 0.09 mL (1.4 μmol) was added to the solution at t = 0, and again at t = 1 h to start the reaction. At various time points, the reaction was removed from the oil bath and examined for fluorescence by fluorimeter emission scans and/or photographs after irradiation by long-wave UV light (365 nm). The reaction was monitored for a total of 24 h. Modifications of the described procedure are required when changing the amounts or identities of various components as needed. When the surfactant is changed, the amounts and stock solution concentrations are given in Table S1. When changing the catalyst/ligand and/or salt concentrations, the relevant stock solutions were made with higher concentrations (double to quadruple) as indicated. In the case of no initiator reactions to investigate negative control of detection effects, the initiator stock solution was omitted, and the amount of water added increased by 60 μL to maintain a consistent reaction volume. For open-air reactions, no solutions were purged with N2 and cuvettes were left uncapped for the duration of the reaction.

Optimized Fluorogenic Aqueous ARGET ATRP Copolymerization Procedure

PEG methacrylate 3 (0.700 g, 1.4 mmol), 5 mM stock solution of anthracene methacrylamide 2 in water with 183 mM SDS (300 μL, 1.5 μmol monomer, 55.0 μmol SDS), 100 mM 2-hydroxyethyl-2-bromoisobutyrate initiator 1 in water (60 μL, 6 μmol), and a stock solution of 25 mM CuBr2 and 200 mM TPMA in water (12 μL, 0.30 μmol CuBr2, 2.4 μmol TPMA) were dissolved in phosphate buffered saline (PBS, 2.04 mL). The solution was sealed and purged with nitrogen for 60 min, then transferred to a sealed quartz cuvette (also purged with N2) and placed in a 30 °C oil bath with a stir bar. A 32 mM ascorbic acid solution in water was separately purged with N2 for 30–60 min, and then 0.09 mL (2.8 μmol) was added to the solution at t = 0, and again at t = 45 min to start the reaction. At various time points, the reaction was removed from the oil bath and examined for fluorescence by fluorimeter emission scans and/or photographs after irradiation by long-wave UV light. The reaction was monitored for a total of 24 h. Modifications of the described procedure are required when changing the amounts or identities of various components as needed. When changing the surfactant, amounts and stock solution concentrations are given in Table S1. In the case of no-initiator reactions to investigate negative control of detection effects, the initiator stock solution was omitted and the amount of PBS added increased by 60 μL to maintain a consistent reaction volume. For open-air reactions, no solutions were purged with N2 and cuvettes were left uncapped for the duration of the reaction.

Kinetic Characterization of Polymerization Fluorescence

A single polymerization reaction was prepared as described above. After the reaction was transferred to a sealed quartz cuvette, the cuvette was placed in the SpectraMax M3Microplate Reader at 33 °C, and the kinetic/fluorescence function was used to take fluorescence readings at an excitation wavelength of 371 nm and an emission wavelength of 426 nm once every minute for 24 h (PMT gain at 200 nm, auto cutoff at 420 nm). Ascorbic acid was injected into the cuvette as described in the general procedure without removing the reaction vessel from the plate reader.

Supplementary Material

ao5c09072_si_001.pdf (12.8MB, pdf)

Acknowledgments

We gratefully acknowledge the National Science Foundation (CHE-2045398), the Welch Foundation (W-1984 and W-0031), the Arnold and Mabel Beckman Foundation, and Trinity University for financial support. Further acknowledgement is made to the Donors of the American Chemical Society Petroleum Research Fund for support of this research.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c09072.

  • Solubilization of anthracene methacrylamide 2 with different surfactants, variability in fluorogenic ARGET ATRP, higher catalyst/ligand loadings lead to fluorescence in the absence of an initiator, comparison of TEABr salt to NaCl, surfactant critical micelle concentration (CMC) vs. experimental concentrations, SEC trace of original (old) versus optimized (new) fluorogenic ARGET ATRP reaction (PDF)

‡.

Department of Chemistry, Northwestern University, 2145 Sheridan Rd, Evanstone, Illinois 60208, United States

The authors declare no competing financial interest.

Published as part of ACS Omega special issue “Undergraduate Research as the Stimulus for Scientific Progress in the USA”.

References

  1. Liu Q., Jin X., Cheng J., Zhou H., Zhang Y., Dai Y.. Advances in the Application of Molecular Diagnostic Techniques for the Detection of Infectious Disease Pathogens (Review) Mol. Med. Rep. 2023;27(5):104. doi: 10.3892/mmr.2023.12991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Urdea M., Penny L. A., Olmsted S. S., Giovanni M. Y., Kaspar P., Shepherd A., Wilson P., Dahl C. A., Buchsbaum S., Moeller G., Hay Burgess D. C.. Requirements for High Impact Diagnostics in the Developing World. Nature. 2006;444:73–79. doi: 10.1038/nature05448. [DOI] [PubMed] [Google Scholar]
  3. Kim S., Sikes H. D.. Radical Polymerization Reactions for Amplified Biodetection Signals. Polym. Chem. 2020;11(8):1424–1444. doi: 10.1039/C9PY01801H. [DOI] [Google Scholar]
  4. Sikes H. D., Hansen R. R., Johnson L. M., Jenison R., Birks J. W., Rowlen K. L., Bowman C. N.. Using Polymeric Materials to Generate an Amplified Response to Molecular Recognition Events. Nat. Mater. 2008;7(1):52–56. doi: 10.1038/nmat2042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kaastrup K., Sikes H. D.. Using Photo-Initiated Polymerization Reactions to Detect Molecular Recognition. Chem. Soc. Rev. 2016;45:532–545. doi: 10.1039/C5CS00205B. [DOI] [PubMed] [Google Scholar]
  6. Wu Y., Liu S., He L.. Electrochemical Biosensing Using Amplification-by-Polymerization. Anal. Chem. 2009;81(16):7015–7021. doi: 10.1021/ac9011254. [DOI] [PubMed] [Google Scholar]
  7. Zhao L., Yang H., Zheng X., Li J., Jian L., Feng W., Kong J.. Dual Signal Amplification by Polysaccharide and eATRP for Ultrasensitive Detection of CYFRA 21–1 DNA. Biosens. Bioelectron. 2020;150:111895. doi: 10.1016/j.bios.2019.111895. [DOI] [PubMed] [Google Scholar]
  8. Zheng X., Liu Q., Li M., Feng W., Yang H., Kong J.. Dual Atom Transfer Radical Polymerization for Ultrasensitive Electrochemical DNA Detection. Bioelectrochemistry. 2020;133:107462. doi: 10.1016/j.bioelechem.2020.107462. [DOI] [PubMed] [Google Scholar]
  9. Pollard J., Rifaie-Graham O., Raccio S., Davey A., Balog S., Bruns N.. Biocatalytically Initiated Precipitation Atom Transfer Radical Polymerization (ATRP) as a Quantitative Method for Hemoglobin Detection in Biological Fluids. Anal. Chem. 2020;92(1):1162–1170. doi: 10.1021/acs.analchem.9b04290. [DOI] [PubMed] [Google Scholar]
  10. Ma N., Zhang J., Kong J., Zhang X.. Fluorescence Sensing of Eclampsia Biomarkers via the Immunosorbent Atom Transfer Radical Polymerization Assay. Anal. Chem. 2024;96(21):8450–8457. doi: 10.1021/acs.analchem.4c00110. [DOI] [PubMed] [Google Scholar]
  11. Allen Z. T., Sackey-Addo J. R., Hopps M. P., Tahseen D., Anderson J. T., Graf T. A., Cooley C. B.. Fluorogenic Atom Transfer Radical Polymerization in Aqueous Media as a Strategy for Detection. Chem. Sci. 2019;10(4):1017–1022. doi: 10.1039/C8SC03938K. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Tahseen D., Sackey-Addo J. R., Allen Z. T., Anderson J. T., McMurry J. B., Cooley C. B.. Fluorogenic Monomer Activation for Protein-Initiated Atom Transfer Radical Polymerization. Org. Biomol. Chem. 2022;20(31):6257–6262. doi: 10.1039/D2OB00175F. [DOI] [PubMed] [Google Scholar]
  13. Simakova A., Averick S. E., Konkolewicz D., Matyjaszewski K.. Aqueous ARGET ATRP. Macromolecules. 2012;45(16):6371–6379. doi: 10.1021/ma301303b. [DOI] [Google Scholar]
  14. Feng H., Dan Y., Zhao Y.. A Sensitive Fluorescence Method for Monitoring the Kinetics of Microemulsion Polymerization. Can. J. Chem. 2010;88(3):185–191. doi: 10.1139/v09-161. [DOI] [Google Scholar]
  15. Matyjaszewski K., Jakubowski W., Min K., Tang W., Huang J., Braunecker W. A., Tsarevsky N. V.. Diminishing Catalyst Concentration in Atom Transfer Radical Polymerization with Reducing Agents. Proc. Natl. Acad. Sci. U.S.A. 2006;103(42):15309–15314. doi: 10.1073/pnas.0602675103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Averick S., Simakova A., Park S., Konkolewicz D., Magenau A. J. D., Mehl R. A., Matyjaszewski K.. ATRP under Biologically Relevant Conditions: Grafting from a Protein. ACS Macro Lett. 2012;1(1):6–10. doi: 10.1021/mz200020c. [DOI] [PubMed] [Google Scholar]
  17. Dhara D., Shah D. O.. Stability of Sodium Dodecyl Sulfate Micelles in the Presence of a Range of Water-Soluble Polymers: A Pressure-Jump Study. J. Phys. Chem. B. 2001;105(29):7133–7138. doi: 10.1021/jp003072l. [DOI] [Google Scholar]
  18. Dhara D., Shah D. O.. Effect of Poly­(Ethylene Glycol)­s on Micellar Stability of Sodium Dodecyl Sulfate. Langmuir. 2001;17(23):7233–7236. doi: 10.1021/la001030i. [DOI] [Google Scholar]
  19. Fatma N., Panda M., Ansari W. H., Kabir-ud-Din. Solubility Enhancement of Anthracene and Pyrene in the Mixtures of a Cleavable Cationic Gemini Surfactant with Conventional Surfactants of Different Polarities. Colloids Surf., A. 2015;467:9–17. doi: 10.1016/j.colsurfa.2014.11.010. [DOI] [Google Scholar]
  20. Odian, G. Principles of Polymerization; John Wiley & Sons, Inc.: Hoboken, New Jersey, USA, 2004. [Google Scholar]
  21. Pandey S.. Cetylpyridinium Chloride Micelles as a Selective Fluorescence Quenching Solvent Media for Discriminating between Alternant versus Nonalternant Polycyclic Aromatic Hydrocarbons. Talanta. 1997;45(1):39–45. doi: 10.1016/S0039-9140(97)00108-2. [DOI] [PubMed] [Google Scholar]
  22. Fantin M., Chmielarz P., Wang Y., Lorandi F., Isse A. A., Gennaro A., Matyjaszewski K.. Harnessing the Interaction between Surfactant and Hydrophilic Catalyst To Control e ATRP in Miniemulsion. Macromolecules. 2017;50(9):3726–3732. doi: 10.1021/acs.macromol.7b00530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gaynor S. G., Qiu J., Matyjaszewski K.. Controlled/“Living” Radical Polymerization Applied to Water-Borne Systems. Macromolecules. 1998;31(17):5951–5954. doi: 10.1021/ma980724j. [DOI] [Google Scholar]
  24. Teo V. L., Davis B. J., Tsarevsky N. V., Zetterlund P. B.. Successful Miniemulsion ATRP Using an Anionic Surfactant: Minimization of Deactivator Loss by Addition of a Halide Salt. Macromolecules. 2014;47(18):6230–6237. doi: 10.1021/ma501379q. [DOI] [Google Scholar]
  25. Tang W., Kwak Y., Braunecker W., Tsarevsky N. V., Coote M. L., Matyjaszewski K.. Understanding Atom Transfer Radical Polymerization: Effect of Ligand and Initiator Structures on the Equilibrium Constants. J. Am. Chem. Soc. 2008;130(32):10702–10713. doi: 10.1021/ja802290a. [DOI] [PubMed] [Google Scholar]
  26. Averick S. E., Bazewicz C. G., Woodman B. F., Simakova A., Mehl R. A., Matyjaszewski K.. Protein–Polymer Hybrids: Conducting ARGET ATRP from a Genetically Encoded Cleavable ATRP Initiator. Eur. Polym. J. 2013;49(10):2919–2924. doi: 10.1016/j.eurpolymj.2013.04.015. [DOI] [Google Scholar]
  27. Fantin M., Isse A. A., Gennaro A., Matyjaszewski K.. Understanding the Fundamentals of Aqueous ATRP and Defining Conditions for Better Control. Macromolecules. 2015;48(19):6862–6875. doi: 10.1021/acs.macromol.5b01454. [DOI] [Google Scholar]
  28. Porter, M. R. Handbook of Surfactants, 2nd ed.; Blackie Academic & Professional: London, UK, 1994. [Google Scholar]
  29. Yeow J., Chapman R., Gormley A. J., Boyer C.. Up in the Air: Oxygen Tolerance in Controlled/Living Radical Polymerisation. Chem. Soc. Rev. 2018;47(12):4357–4387. doi: 10.1039/C7CS00587C. [DOI] [PMC free article] [PubMed] [Google Scholar]

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