Ratiometric Singlet Oxygen Detection in Water Using Acene-Doped

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Ratiometric Singlet Oxygen Detection in Water using Acene Doped Conjugated Polymer Nanoparticles Fanny Frausto, and Samuel W. Thomas ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b02034 • Publication Date (Web): 14 Apr 2017 Downloaded from http://pubs.acs.org on April 22, 2017

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ACS Applied Materials & Interfaces

Ratiometric Singlet Oxygen Detection in Water using Acene Doped Conjugated Polymer Nanoparticles Fanny Frausto and Samuel W. Thomas III*

Department of Chemistry, Tufts University, 62 Talbot Avenue Medford MA 02155

Keyword: Singlet Oxygen, Fluorescent Probe, Conjugated Polymer Nanoparticles, Ratiometric, Sensitizer

Abstract: While fluorescent probes for the detection of singlet oxygen (1O2) have been an active area of research, most such probes rely upon change in intensity of a single band. Herein we report a FRET-based, 1

O2-sensitive aqueous suspension of conjugated polymer nanoparticles (CPNs) comprising the energy

donating host polymer poly[{9,9-dioctyl-2,7-divinylene-fluorenylene}-alt-co-{2-methoxy-5-(2-ethylhexyloxy)1,4-phenylene}] (PFPV) doped with an energy acceptor, the 1O2-sensitive thienoacene 5,12-bis(4methoxyphenyl)-2-butyltetraceno[2,3-b]thiophene (DA-TMT). Using a phthalocyanine-based sensitizer, IRDye 700DX, our probe shows a rapid, ratiometric response to photosensitized 1O2 in water in both cuvettes and 96-well plates that compares favorably to the commercial 1O2-sensitive dye Singlet Oxygen Sensor Green (SOSG). The response to irradiation of even nanomolar concentrations of photosensitizer demonstrates the sensitivity of our ratiometric probe.

INTRODUCTION

This paper describes fluorescent ratiometric responses of acene-doped conjugated polymer nanoparticles (CPNs) to externally generated singlet oxygen in water. Singlet oxygen (1O2) is an electronically excited state of molecular oxygen and is highly reactive compared to its ground state. It can be generated through

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photosensitization using appropriate dyes such as methylene blue and rose bengal. Its cytotoxicity makes it an important therapeutic agent in photodynamic therapy,1-3 and it is a key reactive oxygen species involved in the degradation of materials.4-6 It has also been implicated in the mechanisms for damage to plants upon overexposure to light.7,8 Due to the short lifetime of 1O2 in aqueous environments, however, effective detection of singlet oxygen remains a challenging problem. The phosphorescence of 1O2 itself at 1270 nm is a direct and specific method of detection, but the low quantum yield of phosphorescence inhibits broad applicability and has led to the active research of indirect, luminescent probes for 1O2. A current benchmark in singlet oxygen detection in aqueous media is the commercially available fluorophore Singlet Oxygen Sensor Green (SOSG).9 Quenching by photoinduced electron transfer (PET) from a covalently bound anthracene moiety deactivates fluorescence from a xanthene fluorophore. 1O2 and anthracene react through cycloaddition, however, to form the 9,10endoperoxide, which does not quench xanthene fluorescence. Thus, Φf of SOSG increases after exposure to 1

O2. Mechanistically similar probes from the groups of Ogibly10 and Majima11 use other fluorophores

covalently bound to 9,10-disubstituted anthracenes that quench fluorescence through PET. In contrast to the types of turn-on fluorescence probes described above, ratiometric luminescent responses mitigate environmentally and experimentally dependent effects that can interfere with those fluorophores that only show uniform changes in intensity. Examples include a series of ratiometric probes for 1O2 that comprise fluorophores coupled to 1,3-diarylisobenzofurans through π-conjugated linkages.12 1O2induced oxidation of the isobenzofurans to dibenzyoylbenzenes shortens the π-conjugation of the chromophores, yielding blue-shifted emission spectra. Our lab has published several studies regarding the response of 1O2 using conjugated polymers (CPs) incorporating acene units both in organic solvent, and as thin films in aqueous environments.13-15 The CP backbones transfer energy efficiently to pendant or doped acenes. Upon exposure to 1O2, however, the resulting acene endoperoxides no longer accept energy from the CP backbones. Instead, fluorescence from the CP backbone occurs, leading to a ratiometric response to 1O2. Our lab has also demonstrated water


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soluble acrylic polymers with pendant acenes that showed selective ratiometric responses to singlet oxygen in water.16 Conjugated polymer nanoparticles (CPNs) enable use of highly hydrophobic materials for function in aqueous media. The tight packing of polymer chains in these nanoparticles allow the exciton mobility and signal amplification found in CP thin films to occur in CPNs when they are in aqueous environments. CPNs are reported to be bright, non-cytotoxic, non-blinking, and photostabile.17-19 CPNs can also encapsulate chromophores yielding red-shifted fluorescence through energy transfer, making chromophore-doped CPNs promising for biological labeling and sensing.19,20 Recent efforts to extend CPNs as analytical tools have yielded ratiometric and turn-on fluorescent probes, as well as CPNs that generate 1O2 21-25 We report herein acene-doped conjugated polymer nanoparticles that show highly sensitive ratiometric fluorescent responses to photosensitized singlet oxygen in water. EXPERIMENTAL SECTION

Materials: 5,12-Bis(4-methoxyphenyl)-2-butyltetraceno[2,3-b]thiophene (DA-TMT) was prepared as previously reported.16 The copolymer poly[{9,9-dioctyl-2,7-divinylene-fluorenylene}-alt-co-{2-methoxy-5-(2ethylhexyloxy)-1,4-phenylene}] (PFPV, MW = 107 kDa , PDI = 4.3) was purchased from ADS Dyes, Inc. Neutravidin was purchased from ThermoFisher and IRDye 700DX (Si-Pc) was purchased from Licor. HPLCgrade THF was purchased from VWR. Singlet Oxygen Sensor Green (SOSG) was purchased from ThermoFisher Scientific. All commercially available chemicals were used without further purification. Neutravidin-Phthalocyanine (NA-Pc) Formation: Neutravidin was conjugated with Si-Pc in the following manner. The pH of 1 mL of a 1 mg/mL solution of protein in PBS was raised to 8.5 with 1 M potassium phosphate dibasic. 18.6 µL of a 7 µg/mL an aqueous solution of the NHS ester of IRDye 700DX was then added to the protein solution and allowed to react at room temperature in the dark for 1 hour. The dyeconjugated protein was isolated using a size exclusion column or dialysis and stored at 4 °C. NA-Pc had an average of 2.5 dyes per protein as determined by UV/Vis.


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Nanoparticle Formation: A stock solution of CPs and DA-TMT in THF were prepared. The stock solutions were filtered through a 0.2 µm PTFE filter prior to dilution. A working solution of 20 ppm (18 ppm PFPV and 2 ppm TMT) was made from the stock solutions, and 2 mL of that solution was injected rapidly into 8 mL of water under ultrasonication. The THF was removed by partial vacuum evaporation on a rotary evaporator and the resulting mixture was passed through a 0.2 µm PTFE syringe filter. DLS measurements showed nanoparticles to have hydrodynamic diameters of ~60 nm with PDI = 0.2. These CP colloids were stable for up to 4 months. The maximum absorbance values of the resulting nanoparticle suspensions in the visible region of the spectrum were 0.2 – 0.9. Instrumentation: Electronic absorbance spectra were acquired with a Varian Cary-100 spectrophotometer in double beam mode using a solvent-containing cuvette for background subtraction spectra. Fluorescence emission spectra were obtained with a PTI Quantum Master 4 equipped with a 75 W Xe lamp. All fluorescence spectra were corrected for the output of the lamp and the dependence of detector response to the wavelength of emitted light. Dynamic light scattering (DLS) measurements were made with a Malvern Zetasizer Nano-ZS. Fluorescent quantum yields were determined relative to Coumarin 6 in ethanol. Timeresolved fluorescence data was collected using a time-correlated single-photon counting instrument with a pulsed LED operating at 403 nm. Irradiation for production of singlet oxygen was performed with a 200 W Hg/Xe lamp (Newport-Oriel) equipped with a water filter, manual shutter, a focusing lens, and both 630 and 665 nm long-pass filters, giving an average power density of 64 mW/cm2. For irradiation experiments using microwell plates, the experiments were performed as described above with the following modifications: the focusing lens was removed and a mirror at 90° was placed in the light path to irradiate a 4 well by 4 well area of a clear uncoated polystyrene flat-bottom 96 well plate previously blocked by 2% BSA in 1X PBS. The total power density over this area was 15 mW/cm2.The fluorescence measurements were taken with a Tecan well plate reader using λex = 450 nm λem = 511 nm and 571 nm for DA-TMT/PFPV nanoparticles and λex = 490 λem = 535 for SOSG.


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Irradiation: For irradiation with the 200 W Hg/Xe lamp, the cuvette was placed at the focal point of the focusing lens and irradiated with 630 nm and 663 nm long pass filters for the stated irradiation interval. Both absorbance and fluorescence spectra of the sample were acquired after each irradiation interval. For irradiation experiments using microwell plates, the fluorescence measurements of samples in the well plates were taken after 15-minutes of irradiation. Formation of DA-TMT Endoperoxide: For NMR experiment: To an NMR tube was added a solution of DATMT and tetraphenylporphyrin in CDCl3. The sample was irradiated at the focal point of a 200 W Hg/Xe lamp with a 630 nm long pass filter. Air was bubbled through the sample. The degradation of DA-TMT was monitored by 1H and 13C NMR. After 1 hour and 50 minutes, no DA-TMT was detected, and characteristic peaks of endoperoxides appeared in the NMR spectra. For UV/Vis and Emission Spectroscopy: In a quartz cuvette was added stock solutions of methylene blue and DA-TMT in CDCl3 to form a dilute solution of both compounds. This was then irradiated as with the NMR experiment, and the reaction was monitored through UV/Vis and emission spectroscopy. Figure 1: Response of DA-TMT of photochemically generated 1O2 by selective irradiation of singlet oxygen


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sensitizers under ambient conditions. a) chemical structures of DA-TMT and the endoperoxide oxidation products, as well as the two sensitizers used to collect data in this Figure, methylene blue (MB) and tetraphenylporphyrin (TPP) b) 1H NMR spectra of DA-TMT and TPP solution before (blue) and after (green) irradiation with a spectrum of TPP (black) solution in CDCl3. c) absorbance spectrum of dilute solution of DATMT in CHCl3 before and after selective irradiation of MB. The absorbance of MB has been subtracted from these spectra to highlight the hypsochromic shift of absorbance of DA-TMT upon oxidation with 1O2. RESULTS AND DISCUSSION

Incorporating previously discussed methods of CP-acene 1O2 detection, our design strategy combines the brightness and versatility of CPNs in aqueous environments with the reactivity of diarylacenes. We envisioned that energy transfer from the majority component of the CPNs—the conjugated polymers—to acceptor acene guests would yield an overall red-shifted emission spectrum for the CPNs, as the major contribution to emission would be from the red-emitting acene guest. Upon exposure to 1O2, we expected that the acene-endoperoxide products (Figure 1), which have different absorbance and excited state energies than the reactant acenes due to interruption of conjugation, would limit energy transfer sufficiently to yield emission from the polymer host. The effect would be a blue shift of the overall emission spectrum in the CPNs that results in ratiometric response of fluorescence, with emission intensity of the low energy band decreasing concomitantly with the emission intensity of the higher energy band increasing. We based our design for the formulation of CP nanoparticles (Figure 2) on two important factors: i) the reactivity of the dopant acene with 1O2 and ii) the photophysical properties of the host conjugated polymer

Figure 2: Design of ratiometric 1O2 detection in water using CPNs comprising the conjugated polymer PFPV doped with tetracenothiophene DA-TMT. ACS Paragon Plus Environment 6

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and acene. We chose the highly reactive DA-TMT molecule based on our previous studies26, which indicated rapid reactivity with 1O2 (k ~ 108 M-1 s-1) and a high quantum yield of fluorescence (Φf = 0.55). Upon exposure to 1O2 in CDCl3, DA-TMT forms two different endoperoxides—either across the aryl-substituted ring of the tetraceneothiophene core or across one of the unsubstituted rings—based on 13C NMR resonances at ~78 ppm and ~84 ppm (Figure S1). Although 1O2 oxidizes 6,13-diarylpentacenes approximately twice as fast as it does DA-TMT, such diarylpentacenes have significantly lower efficiencies of fluorescence (Φf ~ 0.1). The commercially available polymer PFPV was chosen as the host since the absorbance spectrum of DA-TMT and the fluorescence spectrum of PFPV overlap between 475 nm and 575 nm (Figure 3a), which is important for efficient Förster energy transfer, although the Dexter electron-exchange energy transfer mechanism may also be operational in these nanoparticles.27 Förster energy transfer is dependent on the spectral overlap of the donor and acceptor as well as their spatial separation. With the spectral data, the overlap can be quantified by the spectral overlap integral J(λ) :

=

Where εA is the extinction coefficient of the acceptor at wavelength λ and FD(λ) is the fluorescence intensity of the donor without acceptor with a normalizing constant. This integral can also be used to calculate the Förster radius Ro, defined as the distance that energy transfer efficiency is diminished by 50% of maximum value and given by the following equation: /

Φ = 0.211

where κ is the orientation factor, set to 2/3 in a randomly oriented donor-acceptor pair, D is the quantum yield of the donor in the absence of acceptor28, and n is the refractive index of water. Using this information, the overlap integral is 2.7×1014 M-1cm-1nm4 and Ro is 25 Å. The overlap between the PFPV emission and DATMT absorption (Figure 3a) and the spatial confinement of the acenes in the nanometer scale of the CPNs enables efficient energy transfer between the polymer host and the dopant DA-TMT in the CPNs in the nonsolvent water. As shown in Figure S2, diluting CPNs 1:2 THF/water solution prevents this spatial


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confinement, no efficient energy transfer is possible, and, accordingly, emission from the donor PFPV predominates. To fabricate DA-TMT/PFPV nanoparticles, we followed the straightforward nanoprecipitation procedure of McNeil and coworkers,29 which afforded nanoparticles with monomodal distribution of size, with average diameter of ~ 60 nm and PDI of 0.2 (Figure 3) as determined by dynamic light scattering (DLS). Consistent with previous reports of other acceptor-doped CP nanoparticles,19,21,30 the hydrophobic polymer matrix encapsulates the acene within these nanoparticles, providing a platform for efficient energy transfer from polymer donor to acene acceptor. Absorbance spectra of these filtered samples of nanoparticles with 10% DA-TMT doping shows small peaks at both 310 nm and 550 nm, indicating the presence of DA-TMT (Figure 3b). Moreover, the fluorescence properties of these nanoparticles also reflect the encapsulation of and emission from DA-TMT (Figure 3b). Steady-state fluorescence spectroscopy of the DA-TMT (10%)/PFPV nanoparticles upon excitation of PFPV at 480 nm causes almost complete emission from the acene, with the resulting peak in the emission spectrum from DA-TMT at 568 nm having twenty-fold larger peak intensity than the peak from PFPV at 513 nm; absorbance by DA-TMT at 480 nm accounts for less than 2% of the total absorbance. Furthermore, while monitoring emission intensity from DA-TMT at 570 nm, the excitation spectrum is nearly identical to that of undoped PFPV nanoparticles (Figure S3). The fluorescence lifetime of DA-TMT/PFPV nanoparticles is close to the fluorescence lifetime of dilute DA-TMT in dichloromethane,26 providing additional evidence of DA-TMT encapsulation (Figure S4). Combining the ~300 ps lifetime of undoped PFPV nanoparticles with an estimate of 90% efficiency energy transfer from PFPV to DA-TMT at a doping level of 10% (w/w), we can approximate a lower limit of rate of energy transfer from PFPV to DATMT of 3x1010 s-1.


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Figure 3: a) Spectral overlap of the emission of conjugated polymer PFPV and diaryltetracenothiophene DATMT. b) UV/vis absorbance, fluorescence excitation (λem = 571 nm), and fluorescence emission (λex =480 nm) of DA-TMT doped PFPV nanoparticles. c) Dynamic light scattering results for DA-TMT doped PFPV nanoparticles. The doping level of DA-TMT in PFPV nanoparticles influences the initial efficiency of energy transfer (Figure 4). At a doping feed ratio for DA-TMT of 0.1% relative to PFPV (w/w %) nanoparticles displayed no energy transfer due to the low probability of PFPV-based excitons diffusing to the small concentration of energy accepting DA-TMT molecules trapped in the PFPV matrix. The extent of energy transfer increases up to a doping level of 7%, when the ratio of acceptor to donor emission is greater than 90%. We chose 10% doping (w/w%) to mitigate the effect of unintended oxidation on fluorescence spectra. Based on the density and degree of polymerization of PFPV, as well as the volume of the nanoparticles, we estimate that such nanoparticles have an average of 2600 polymers per particle. In addition, based on the


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extinction coefficient of DA-TMT and PFPV, we estimate that the actual level of DA-TMT incorporation in these particular nanoparticles is approximately 14%.

Figure 4: a) Ratio of PFPV emission to total emission as a function of DA-TMT doping percentage from feed ratio used to prepare nanoparticles b) fluorescence emission (λex =480 nm) of DA-TMT doped PFPV nanoparticles with varying doping levels of DA-TMT. To test the fluorescence response of the DA-TMT/PFPV nanoparticles, we exposed them to 1O2 generated by irradiating a photosensitizer selectively in an air-equilibrated aqueous suspension. Phthalocyanines are established 1O2 sensitizers that absorb red light, multiple derivatives of which have been incorporated into polymeric platforms to generate 1O2 in vitro and in vivo. 2,31-33 We chose a commercially available hydrophilic silicon-phthalocyanine IRDye 700DX (Si-Pc, λmax = 689 nm) with absorbance spectrum red-shifted from both


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PFPV and DA-TMT (Figure S5), which allowed us to irradiate the sensitizer selectively and generate 1O2. In particular, the Si-Pc derivative used in this study has proven useful in both electron microscopy imaging33 and as a potent cytotoxic agent for photoimmunotherapy when bound to target-specific antibodies, primarily through a Type II (1O2-mediated) mechanism.1,32 A challenge in the development of 1O2-responsive materials is the rapid rates of physical quenching of 1O2 with electron donors, including the electron rich side chains of amino acids such as histidine and lysine.34-36. We therefore conjugated the commercially available NHS ester derivative of Si-Pc to the protein Neutravidin; this labeled protein (NA-Pc) was used as the sensitizer in subsequent experiments. The fluorescence emission spectrum of DA-TMT/PFPV nanoparticles showed a ratiometric response upon exposure to 1O2 generated through selective irradiation of NA-Pc (absorbance of 0.1 at 689 nm). As shown in Figure 5, emission from DA-TMT (571 nm) decreases in intensity, while emission from PFPV (511 nm) increases, as the nanoparticles are exposed to 1O2 through selective irradiation of the sensitizer. Emission by the polymer host PFPV replaced emission from DA-TMT within ten minutes of exposure to 1O2. This behavior is consistent with our design: 1O2 oxidizes DA-TMT to endoperoxides with larger excited state energies, precluding its ability to accept energy from PFPV. Negative control experiments in which irradiation, NA-Pc, or both were withheld (See Figure S6) exhibited no change in emission. The potential quenching of 1O2 by the Neutravidin protein did not prevent the ratiometric response of the nanoparticles. Exposure to other reactive oxygen species showed reactivity to NaOCl, mostly likely due to the alkene groups in PFPV (Figure S7).


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Figure 5. Fluorescent response (λex = 480 nm) of DA-TMT/PFPV nanoparticles after exposure to photochemically generated 1O2 via selective irradiation of Si-Pc-labeled Neutravidin under ambient conditions in a) H2O and b) D2O. c) Ratio of emission intensity of PFPV (λ = 511 nm) to the sum of PFPV and DA-TMT (λ = 571 nm) emission intensity in H2O and D2O as a function of irradiation time. d) Schematic of the experimental procedure and the effects of photochemically generated 1O2 on the CPNs. One noteworthy observation is that the recovery of the emission from the donor PFPV does not reach the same intensity as the initial emission from the acceptor. To understand the origin for this behavior, we measured the quantum yields of fluorescence of three different samples of CP NPs in the presence of NA-Pc (absorbance of 0.1 at 689 nm): i) undoped PFPV nanoparticles (ΦF = 0.1), ii) unoxidized DA-TMT doped (10% w/w) PFPV nanoparticles (ΦF = 0.2), and iii) DA-TMT doped (10% w/w) PFPV nanoparticles after complete


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thienoacene oxidation with 1O2. (ΦF = 0.1). Furthermore, exposure to undoped PFPV nanoparticles to 1O2 by irradiation of the sensitizer at 0.1 OD under identical irradiation conditions showed no detectable decrease in nanoparticle quantum yield. We therefore conclude that the doped nanoparticles are inherently more fluorescent than the undoped nanoparticles, and that this difference explains the smaller intensity of the PFPV emission upon complete oxidation when compared to the DA-TMT emission before oxidation. Faster oxidation in perdeuterated solvents can provide additional evidence for the involvement of 1O2 due to the longer lifetimes of 1O2 in solvents that lack hydrogen atoms.37 We therefore exposed nanoparticles to irradiated sensitizer in D2O under otherwise identical conditions. These samples showed a clear decrease in irradiation time required to show complete polymer donor emission (~3 min), in line with the increased lifetime of 1O2 in D2O. We then compared the fluorescence response of our DA-TMT/PFPV nanoparticles to the commercially available 1O2-responsive fluorophore, Singlet Oxygen Sensor Green (SOSG) by determining the limit of detection (LoD) of photosensitizer (Figure 6). In these experiments, we varied the concentration of sensitizer in separate wells of a 96 well plate while irradiating all samples for 15 minutes. As expected, the magnitudes of ratiometric responses of the acene-doped nanoparticles were lower than those seen with higher concentrations of NA-Pc and higher irradiation power density in earlier experiments performed in cuvettes. Under these conditions of lower power density and sensitizer concentration, the ratiometric changes of fluorescence of the probes were approximately linear, and neither SOSG nor the CP nanoparticles approached plateau values of response. Calculated as concentration of sensitizer corresponding to three times the standard deviation of the blank, we determined LoD values of 8 ng/mL of NA-Pc for DA-TMT/PFPV nanoparticles and 1.2 µg/mL of NA-Pc for SOSG. We attribute the high precision of signal for the blank of the nanoparticles to the self-referencing nature of their ratiometric output. We experimentally demonstrated observable response of DA-TMT/PFPV nanoparticles to concentrations of NA-Pc as low as 12.6 ng/mL, with a ratiometric response of 0.14 after 3 hours of irradiation (Figure 6c). Conclusion


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we have demonstrated robust fluorescence response of heteroacene-doped conjugated polymer nanoparticles to photogenerated 1O2 in aqueous environments. Our design has a number of distinct advantages: i) stable colloidal suspensions of nanoparticles that are easily fabricated from readily available hydrophobic components, ii) a ratiometric response of fluorescence that is resistant to the error inherent in simple fluorescence intensity measurements, iii) rapid oxidation of DA-TMT by 1O2, iv) the close packing of polymer chains in the nanoparticles, together with encapsulation of the reactive heteroacene, enables efficient energy harvesting and the use of small concentrations of acceptor. This readily prepared probe responds rapidly compared to previous studies done in our lab with thin films, solvated small molecules, polymers, and shows promising sensitivity compared to an intensity-only probe.13,16,26 Practical challenges yet to be addressed with these materials include sensitization of 1O2 by the probe itself, aggregation of these nanoparticles with molecules found in biologically relevant samples, and the rapid quenching of 1O2 by electron donors such as amines.


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Figure 6: Fluorescent response of a) SOSG and b) DA-TMT/PFPV nanoparticles with varying concentrations of NA-Pc with a 15-minute irradiation time. c) NP response to 12.6 ng/mL NA-Pc after irradiation for 15 minutes and 3 hours. Error bars show standard deviations of n = 3. λex = 480 nm for CP NPs; λex = 490 nm for SOSG.


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ASSOCIATED CONTENT

Supporting Information. The Supporting Information is available free of charge on the ACS website. 13

C NMR spectra of DA-TMT and its endoperoxides, fluorescence emission of DA-TMT/PFPV with and

without an aqueous THF dilution, comparison of absorbance and excitation spectra of doped and undoped PFPV with DA-TMT, fluorescent decay traces of PFPV and DA-TMT / PFPV nanoparticles, absorbance and structure of Si-Pc, and control experiments for the irradiation experiment of NA-Pc in the presence of DATMT/PFPV nanoparticles, DA-TMT/PFPV nanoparticles in the presence of various reactive oxygen species.

AUTHOR INFORMATION Corresponding Author * Email: [email protected]

ACKNOWLEDGMENT The authors thank the National Science Foundation (CHE-1305832) for generous support of this work. We are also grateful for assistance from Ms. Syrena Fernandes and Prof. Charles Mace (Tufts Chemistry) regarding protein labeling and purification, as well as the laboratory of Prof. Rebecca Scheck (Tufts Chemistry) for use of their microplate reader.

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Liu, Y.; Pauloehrl, T.; Presolski, S. I.; Albertazzi, L.; Palmans, A. R. A.; Meijer, E. W. Modular Synthetic

Platform for the Construction of Functional Single-Chain Polymeric Nanoparticles: From Aqueous Catalysis to Photosensitization. J. Am. Chem. Soc. 2015, 137, 13096–13105.


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Ratiometric Singlet Oxygen Detection in Water Using Acene-Doped

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