We present dye-doped polymer nanoparticles that are able to detect mercury in aqueous solution at parts per billion levels via fluorescence resonance energy transfer (FRET). The nanoparticles are ...prepared by reprecipitation of highly fluorescent conjugated polymers in water and are stable in aqueous suspension. They are doped with rhodamine spirolactam dyes that are nonfluorescent until they encounter mercury ions, which promote an irreversible reaction that converts the dyes to fluorescent rhodamines. The rhodamine dyes act as FRET acceptors for the fluorescent nanoparticles, and the ratio of nanoparticle-to-rhodamine fluorescence intensities functions as a ratiometric fluorescence chemodosimeter for mercury. The light harvesting capability of the conjugated polymer nanoparticles enhances the fluorescence intensity of the rhodamine dyes by a factor of 10, enabling sensitive detection of mercury ions in water at levels as low as 0.7 parts per billion.
Rhodamine spirolactams (RSLs) have recently emerged as popular fluorescent pH probes due to their fluorescence turn‐on capability and ease of functionalization at the spirolactam nitrogen. Design of ...RSLs is often driven by biological targeting or compatibility concerns, rather than the pH sensitivity of the probe, and the relationship between RSL structure and pKa is not well understood. To elucidate the relationship between pKa values and the properties of substituents attached to the spirolactam nitrogen, a series of 19 aniline‐derived RSLs is presented. RSLs derived from di‐ortho‐substituted anilines exhibit pKa tunability across the moderately acidic region (ca. pH 4–6). Evaluation of pKa data using the Fujita–Nishioka model for ortho substituent effects reveals that both steric and electronic substituent properties influence RSL pH responsiveness, with pKa values increasing as substituent size and electron withdrawing character increase. These trends are attributed to changes in the RSL structure induced by large substituents, and to electronic influences on the protonated spirocyclic reaction intermediate. To demonstrate the practical applicability of these probes in completely aqueous environments, RSL‐doped conjugated polymer nanoparticles that exhibit a ratiometric fluorescence response to changing pH levels are presented.
Deformed dye: The quest for a structure‐pKa relationship for fluorescent rhodamine pH probes yields a tale of steric and electronic substituent effects, linear free energy relationships, a crystal structure, and ratiometric fluorescent pH sensing in water with doped conjugated polymer nanoparticles. The pKa values of aniline‐derived rhodamine spirolactams can be increased by 2 pH units by employing large substituents to deform the xanthene ring system and electron withdrawing ones to destabilize a key intermediate.
Conjugated polymer nanoparticles doped with a reverse photochromic dye exhibit highly quenched fluorescence that can be reversibly activated by controlling the form of the photochrome with visible ...light.
Nanoparticles prepared from conjugated polymers (CPNs or Pdots) are powerful fluorophores for sensing and imaging applications, including those that require fluorescence photoswitching. We and others ...have developed CPNs doped with photochromic dyes that act as acceptors in only one of their two forms via fluorescence resonance energy transfer (pcFRET). We recently developed visible-light-responsive CPNs doped with a reverse photochromic spirooxazine dye that has a thermally stable merocyanine form. Off-to-on fluorescence switching occurs when the merocyanine is switched to its nonquenching spirooxazine form. The on-state fluorescence intensity is more than 100 times greater when photoswitching occurs via FRET from the CPNs than when the dyes are excited directly. Temporal control of fluorescence intensity is achieved using a single-color input of variable intensity for fluorescence excitation (read-only mode) and photoswitching (read–write mode). Here, we present photokinetic measurements and simulations that establish the origin of the observed fluorescence behavior. We find that highly efficient CPN-to-merocyanine energy transfer sensitizes the photochromic reaction, which has a quantum yield of 0.03 in the absence of FRET. Simulated fluorescence trajectories reveal that the low photochromic quantum yield is the necessary condition for single-color intensity control in this system. From the simulations, we determine that the product of the photochromic quantum yield and the energy transfer efficiency (Φ × E) must be no more than 0.05 to produce analogous fluorescence behavior. These findings suggest that the CPNs could be used as an amplifier to drive other low-quantum-yield photoreactions to completion with intensity control.
Rhodamine spirolactams (RSLs) have recently emerged as popular fluorescent pH probes due to their fluorescence turn-on capability and ease of functionalization at the spirolactam nitrogen. Design of ...RSLs is often driven by biological targeting or compatibility concerns, rather than the pH sensitivity of the probe, and the relationship between RSL structure and pK
is not well understood. To elucidate the relationship between pK
values and the properties of substituents attached to the spirolactam nitrogen, a series of 19 aniline-derived RSLs is presented. RSLs derived from di-ortho-substituted anilines exhibit pK
tunability across the moderately acidic region (ca. pH 4-6). Evaluation of pK
data using the Fujita-Nishioka model for ortho substituent effects reveals that both steric and electronic substituent properties influence RSL pH responsiveness, with pK
values increasing as substituent size and electron withdrawing character increase. These trends are attributed to changes in the RSL structure induced by large substituents, and to electronic influences on the protonated spirocyclic reaction intermediate. To demonstrate the practical applicability of these probes in completely aqueous environments, RSL-doped conjugated polymer nanoparticles that exhibit a ratiometric fluorescence response to changing pH levels are presented.
We investigate amplified energy transfer in conjugated polymer nanoparticles (CPNs or Pdots) by studying both fluorescence quenching of CPN donors and the sensitization of reactive dye acceptors. By ...delivering excitation energy to dye dopants via a combination of Förster energy transfer and exciton diffusion, CPNs act as powerful light-harvesting antennae. This phenomenonamplified energy transferis used to sensitize dye dopants, producing a higher concentration of the dye’s excited state than would be observed upon direct excitation. Here, we study CPN sensitization of a low-efficiency photochemical reaction to determine the CPN size and dye loading that yield optimized outputs in the form of energy transfer efficiency, the antenna effect (AE), and reaction duration. Our model system is the cycloreversion reaction of a diarylethene (DAE) photochrome as the dye dopant and CPNs of the conjugated polymer poly(9,9-dioctylfluorenyl-2,7-diyl)-co-1,4-benzo-{2,1′-3}-thiadiazole as the sensitizer. In their visible-absorbing form, DAE dyes are localized on the particle surface and are effective fluorescence quenchers of 15, 20, and 26 nm diameter CPNs. Quenching is most efficient for the smallest particles, and high dye loadings are necessary to offset reduced efficiency as CPN size increases. Our photokinetic studies of DAE acceptors demonstrate the crucial importance of dye loading: both energy transfer efficiency and the AE show abrupt declines when the dye concentration is increased beyond a critical threshold. We find that CPNs with a 15 nm diameter exhibit the most efficient energy transfer (99–100%) and the largest AE (32) of the CPNs studied. For CPNs of all sizes and dye loadings, a photoselection phenomenon reveals that the energy-transfer-accepting ability of the DAE dyes varies tremendously within the dye ensemble. These findings are used to develop design recommendations for CPN sensitizers.
Fluorescent turn-on probes based on a rhodamine spirolactam (RSL) structure have recently become a popular means of detecting pH, metal ions, and other analytes of interest. RSLs are colorless and ...non-fluorescent until the target analyte induces opening of the spirocyclic ring system, revealing the fully conjugated and highly fluorescent rhodamine dye. Among RSLs opened by acid, we have observed wide variation in the kinetics of the fluorescence turn-on process such that some probes would not be usable in situations where a rapid reading is desired or the pH fluctuates temporally. Herein we present a systematic investigation of the fluorescence turn-on kinetics of RSLs to probe the hypothesis that the reaction rates are influenced by the electronic properties of the spirolactam ring system. A series of 8 aniline-derived RSLs with para substituents ranging from electron-donating to electron-withdrawing was prepared from rhodamine B. The fluorescence turn-on rates are observed to increase by a factor of four as the substituent is tuned from methoxy to nitro. This effect is explained in terms of the destabilization of the reaction intermediate by the substituent. As the reaction rates increase across the series, a concomitant increase in fluorescence intensity is also observed. This result is attributed to an increase in the concentration of the fluorescent form of the dye and is consistent with the expected equilibrium properties of this system. These findings are applied to the design of a faster-reacting and more intensely fluorescent RSL pH probe.
Fluorescent systems that can undergo intensity photomodulation in aqueous environments are finding increasing applications, particularly in high-resolution imaging of biological samples. We seek to ...develop conjugated polymer nanoparticles (CPNs) with bright fluorescence that can be modulated with a light signal. Here, we present CPNs, doped with a photochromic diarylethene dye, that exhibit efficient fluorescence photomodulation that is thermally irreversible. In their UV-absorbing open form, the diarylethenes have no effect on the fluorescence properties of the bright CPNs. A brief period of UV irradiation converts the dyes to their visible-absorbing closed form, which is an efficient fluorescence quencher for the CPNs, likely via a fluorescence resonance energy transfer mechanism. Aqueous suspensions of dye-doped CPNs prepared from the homopolymer poly2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene (MEH-PPV) or a polyfluorene-phenylenevinylene copolymer (PFPV) exhibit thermally stable bright and dark levels. The dye-doped MEH-PPV CPNs also exhibit photomodulation in single-nanoparticle imaging experiments, which reveal that nearly all CPNs retain a small amount of residual emission in the dark state. Their PFPV counterparts undergo irreversible fluorescence photobleaching rather than photomodulation in single-nanoparticle studies. The photostability of the CPNs under the UV irradiation conditions required for photochromic conversion is investigated on the single-particle level, and PFPV CPNs are found to be particularly susceptible to photobleaching upon 254 nm irradiation. These results will guide the selection of polymers and photochromes for CPNs intended for single-particle photomodulation.
We present studies of fluorescence photomodulation and solvatochromism in nanoparticles of the conjugated polymer poly2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene (MEH-PPV) doped with a ...photochromic spirooxazine dye. The fluorescence properties of doped nanoparticles with dyes in the spirooxazine form are identical to those in undoped control nanoparticles. UV irradiation converts some of the dyes to their visible-absorbing merocyanine form, which is an efficient quencher of MEH-PPV fluorescence. The fluorescence intensity of the nanoparticles drops to less than 10% of its initial value and recovers when the merocyanines undergo thermal reversion to spirooxazines. The fluorescence modulation can be cycled many times without fatigue or photodegradation, and the degree of quenching is linear with merocyanine concentration. The photochromic conversion can also be used as a probe of the environment within the nanoparticles as both the kinetics of the thermal merocyanine-to-spirooxazine conversion and the merocyanine absorption spectrum are sensitive to the dye environment. The kinetics of the thermal dye reversion in the nanoparticles are first order and nearly as fast as those in THF, while those in a MEH-PPV film are biexponential and substantially slower. The position of the merocyanine absorption within the nanoparticles is likewise distinct from that in a MEH-PPV film and implies a liquid-like environment that is more polar than THF. We hypothesize that those dyes that undergo spirooxazine-to-merocyanine conversion are adhered to solution-exposed MEH-PPV segments within the nanoparticles or to the particle surface and thus have ample free volume for the photochromic conversion. These findings will be useful in designing future stimulus-responsive nanoparticle systems.
The ability to modulate protein function through minimal perturbations to amino acid structure represents an ideal mechanism to engineer optimized proteins. Due to the novel spectroscopic properties ...of green fluorescent protein, it has found widespread application as a reporter protein throughout the fields of biology and chemistry. Using site-specific amino acid mutagenesis, we have incorporated various fluorotyrosine residues directly into the fluorophore of the protein, altering the fluorescence and shifting the pKa of the phenolic proton associated with the fluorophore. Relative to wild type GFP, the fluorescence spectrum of the protein is altered with each additional fluorine atom, and the mutant GFPs have the potential to be employed as pH sensors due to the altered electronic properties of the fluorine atoms.
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