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

Coupling polymerization initiators to molecular recognition events provide the ability to amplify these events and detect them using the formation of a cross-linked polymer as an inexpensive readout that is visible to the unaided eye. The eosin-tertiary amine co-initiation system, activated by visible light, has proven utility in this context when an average of three eosin molecules are coupled to a protein detection reagent. The present work addresses the question of how detection sensitivity is impacted when the number of eosin molecules per binding event increases in the range of two to fifteen. Unlike in other initiation systems, a non-monotonic relationship is observed between the number of initiators per binding event and the observed detection sensitivity.


Introduction
Rapid detection tests (RDTs) that enable molecular diagnosis and monitoring of disease have had significant impact on human health, and current research efforts focus on increasing the number and types of disease states that may be routinely and reliably monitored in this way. [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. 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. [17] To date, several polymerization initiators have been investigated for use in PBA. [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. [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.

Results and Discussion
Scheme 1 shows the syntheses of amine-terminated eosin 1 and macroinitiators 6-9, and a conceptual representation of PBA for colorimetric detection. The aminated eosin was synthesized from eosin 5-isothiocyante (EITC) through thiourea bond formation. Briefly, tertbutoxycarbonyl (Boc)-protected amine was employed here to prevent the formation of undesired products, such as dimer eosin. After the formation of a thiourea bond, cleavage of the Boc group using TFA [20] afforded aminated eosin 1, which was tethered to poly(acrylamide-co-acrylic acid) by an amide coupling reaction (see Supporting Information).
Eosin-functionalized copolymers 2-5 were purified by dialysis, precipitation, and size exclusion chromatography and subsequently conjugated with neutravidin; as a result, macroinitiators 6-9 were obtained (see Supporting Information). As depicted in Scheme 1B, macroinitiators were used to connect molecular recognition with a polymeric readout.
Neutravidin, incorporated in the macroinitiator, recognized biotin-labeled DNA coupled to a glass surface. Upon exposure to visible light in an air environment, the eosin macroinitiators reacted with tertiary amine co-initiators to initiate a polymerization of acrylate monomers.
The resulting hydrogel was stained with a dye for colorimetric read-out as previously reported. 19 Eosin-functionalized copolymers 2-5 were characterized by 1 H NMR and UV-visible spectroscopy. In the proton NMR spectra with D 2 O as a solvent (Figure 1), the x-axis values were chosen to highlight the range of chemical shifts displayed by the aromatic protons of eosin, and the peaks were assigned by 1 H NMR measurement of free EITC in deuterated DMSO. We monitored a noticeable peak-broadening trend as a function of reaction stoichiometry (Figure 1a-d). In the other words, as the molar ratio of eosin to the copolymer increased in the reaction mixture, the spectral features arising from the eosin moiety broadened. For example, while the spectra of 2 and 3 had several characteristic peaks in the range of 7.2 to 8.0 ppm (Figure 1a-b), these features broaden markedly and shift slightly to lower ppm for compounds 4 and 5 (Figure 1c-d). Although the broadness hindered a determination of the number of eosin molecules per copolymer for all cases, the integration results support the increasing trend for yield as a function of stoichiometry, where the protons of the copolymer backbone were used as a reference peak at 1.2-2.2 ppm. As a complementary characterization strategy that we have successfully used in past work [19] , UVvisible spectroscopy was employed to determine the average number of eosin per copolymer.
This analysis yielded the following average numbers of eosin per copolymer: 2 for 2, 6 for 3, 10 for 4, and 15 for 5.
After neutravidin was coupled to the eosin/copolymers 2-5 via an amide bond, macroinitiators 6-9 were obtained with yields > 70% as measured by the HABA (4'hydroxyazobenen-2-carboxylic acid) assay. [19] The macroinitiators were tested for specific binding capabilities using glass surfaces covalently modified with replicate spots of biotinlabeled DNA at 5 different surface densities (Figure 2a). Detailed information on the test surface, including density of biotin and spot size, was obtained by fluorescence techniques as previously described. [9,19] Figure 2b-d shows fluorescence responses resulting from the incubation of 6-9 with the test surface. Interestingly, macroinitiator 8 generates higher 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 the case of macroinitiator 9, a slight increase in background intensity (i.e. nonspecific binding) was accompanied by a dramatic decrease in the average spot intensity (i.e. specific binding) compared with the results using 8 (Figure 2e).
The finding that an average of 15 initiators per binding event leads to a lower surfacelocalized signal than an average of 10 initiators per binding event is contrary to results obtained in past work with other initiation systems, namely fluorescein/tertiary amine and Irgacure 2959. [6,19] In these previous systems, increasing the number of initiators per binding event led to higher surface-localized initiator density as a function of specific binding interactions. While it was possible to synthesize macroinitiators that coupled >100 fluorescein molecules to each binding event, 15 was the observed limit for eosin, a tetrabromo derivative of fluorescein. The aqueous solubility of eosin macroinitiators exhibited a strong inverse proportionality to the number of hydrophobic bromo groups per copolymer. We hypothesize that poor solubility not only interferes with the specific binding of the eosin macroinitiator to the biotin-DNA functionalized surface, but it also increases nonspecific adsorption to the surface in regions that have not been modified with biotinylated DNA. Efforts to optimize the response from molecular recognition using pH did not yield substantial improvements.
Because the eosin moiety is known to exist in its dianionic form above pH 7 [21] and the acrylic acid residues of poly(acrylic acid-co-acrylamide) are also ionizable, macroinitiator 8 was treated under alkaline conditions in order to increase solubility. Resulting signal and background intensities at pH 11 were both 2-5 times lower than at pH 7.4 (Figure 3a and b). Figure 4 shows the polymerization response resulting from molecular recognition between 8 and the biotin-functionalized surface at pH 7.4. Interfacial polymerizations coupled to molecular recognition were not observed using macroinitiators 6, 7 or 9 with the monomer solution and illumination conditions as previously described. [19] In Figure 4, macroinitiator 8 yielded colorimetric responses from the first 3 rows of features with as few as 50 possible molecular recognition events per square micrometer. This finding represents a three-fold higher detection limit than that attainable with an average of three eosin molecules coupled directly to streptavidin. 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] Figure S2 for a detailed mechanism). The fact that interfacial polymerization coupled to binding events was possible using 8 but not 9 supports the idea that reduced binding of 9, rather than a quenching mechanism, causes the reduced fluorescence signal. Our observations of reduced solubility with increasing eosin substitution of the macroinitiators suggest the possibility that solution phase aggregates may form at the highest degree of eosin substitution in this study. Unfortunately, dynamic light scattering and x-ray techniques that are commonly used to characterize solution phase structure require concentrations in the range of 2-10 mg/mL, and these concentrations are much higher than the µg/mL concentrations relevant for this work.

Conclusions
In summary, a limit has been determined in the eosin/tertiary amine initiation system where increasing the number of initiators per macroinitiator beyond ten begins to impede the function of the macroinitiator in the context of PBA. This result contrasts with observations using other initiation systems. [19,[22][23][24] In our view, even with this limitation, the eosin/tertiary amine initiation system provides superior performance to other alternatives as detailed in Table 1 of [9] . For example, other photoinitiation systems such as Irgacure 2959 [6] and fluorescein/tertiary amine [19] are more sensitive to oxygen inhibition than the eosin/tertiary amine system. Initiators of AGET ATRP have been used to detect molecular recognition in air environments, but a tradeoff between reaction time and sensitivity exists with hours required for maximal sensitivity. In addition, AGET ATRP produces thinner films [24] that require silicon or gold substrates for visualization and expensive instrumentation for quantitation of analytes. Because of the capabilities of the eosin/tertiary amine initiation system in comparison to alternatives, increasing the number of eosin initiators localized per binding event remains an attractive strategy for improving the detection limit if the problem of aqueous solubility can be addressed in future efforts.

Supporting Information
Supporting Information is available from the Wiley Online Library.     was purified by dialysis, size exclusion column chromatography, and precipitation to remove completely any remaining free aminated eosin after reaction. We initially dialyzed the crude product with a 13 kDa molecular weight cutoff membrane tube against a mixture of deionized water and methanol at room temperature. After the dialysis, the resulting solution was concentrated with ethanol by rotary evaporation and then precipitated with a mixture of acetone (6 mL) and ethyl acetate (12 mL) in a vial. Precipitated red solid was dried under vacuum for 1 day.
Synthesis of macroinitiator 6-9. EDC (100 equiv) and NHSS (150 equiv) were added into the solution of compound 2-5 (approximately 0.3 mg, 1 equiv) dissolved in pH 8.0 buffer solution (100-150 µL) at room temperature for 10 min. Neutravidin (1 equiv) dissolved in pH 8.0 buffer solution (50 µL) was added into the reaction mixture at room temperature, and the resulting mixture was incubated at 4 °C for overnight. The crude product was filed using a centrifugal filter device (100 kDa molecular weight cutoff) by three times with pH 8.0 buffer solution. The filtered solution was collected, and then used for photopolymerization.
Photopolymerization. The solution containing the co-initiator, triethanolamine, and monomers, 1-vinyl-2-pyrrolidinone, and poly(ethylene glycol) diacrylate, was purchased from InDevR, and used without the addition of the supplied enhancer solution.
Photopolymerization was carried out with an ampliPHOX TM Reader (InDevR) at room temperature without inert gas purging. For the preparation of sample, we mixed macroinitiator   Figure S2. A current proposal for the mechanism by which eosin and tertiary amines initiate radical polymerization of acrylate monomers. (Incorporates findings from D. C. Neckers et al.