Conjugated Polyelectrolyte-Based Real-Time Fluorescence Assay for Alkaline Phosphatase with Pyrophosphate as Substrate

The fluorescence of the anionic, carboxylate-substituted poly(phenylene ethynylene) polymer PPECO2 is quenched very efficiently via the addition of 1 equiv of Cu2+. Addition of pyrophosphate (PPi) into the weakly fluorescent solution of PPECO2 and Cu2+ induces recovery of the polymer’s fluorescence; the recovery occurs because PPi complexes with Cu2+, effectively sequestering the ion so it cannot bind to the carboxylate groups of the polymer. A calibration curve was developed that relates the extent of fluorescence recovery to [PPi], making the PPECO2−Cu2+ system a sensitive and selective turn-on sensor for PPi. Using the PPECO2−Cu2+ system as the signal transducer, a real-time fluorescence turn-off assay for the enzyme alkaline phosphatase (ALP) using PPi as the substrate is developed. The assay operates with [PPi] in the micromolar range, and it offers a straightforward and rapid detection of ALP activity with the enzyme present in the nanomolar concentration range, operating either in an end point or real-time format. Kinetic and product inhibition parameters are derived by converting time-dependent fluorescence intensity into PPi (substrate) concentration, thus allowing calculation of the initial reaction rates (vo). Weak, nonspecific fluorescence responses are observed concomitant to addition of other proteins to the assay solution; however, the signal response to ALP is demonstrated to arise from the ALP catalyzed hydrolysis of PPi to phosphate (Pi)

Meta-Linked Poly(phenylene ethynylene) Conjugated Polyelectrolyte Featuring a Chiral Side Group: Helical Folding and Guest Binding

A water soluble, meta-linked poly(phenylene ethynylene) featuring chiral and optically active side groups based on l-alanine (mPPE-Ala) has been studied by using absorption, fluorescence, and circular dichroism spectroscopy. Studies of mPPE-Ala in methanol/water solvent mixtures show that the polymer folds into a helical conformation, and the extent of helical folding increases with the volume % water in the solvent. The presence of the helical conformation is signaled by the appearance of a broad, excimer-like visible fluorescence band, combined with a strong bisignate circular dichroism signal in the region of the π,π* absorption of the polymer backbone. The circular dichroism signal exhibits negative chirality, suggesting that the left-handed (M-form) of the helix is in enantiomeric excess. Binding of the metallointercalator [Ru(bpy)2(dppz)]2+ (where bpy = 2,2-bipyridine and dppz = dipyrido[3,2-a:2‘,3‘−c]phenazine) with the helical polymer is accompanied by the appearance of the orange-red photoluminescence from the metal complex. This effect is directly analogous to that observed when [Ru(bpy)2(dppz)]2+ binds to DNA via intercalation, suggesting that the metal complex binds to mPPE-Ala by intercalating between the π-stacked phenylene ethynylene residues. Cationic cyanine dyes also bind to the periphery of the helical polymer in a manner that is interpreted as “groove binding”. A circular dichroism signal is observed that is believed to arise from exciton coupling within the chiral cyanine dye chromophore aggregate that is formed as the dye molecules are oriented by the helical mPPE-Ala “template”

Radical Cation Probes for Photoinduced Intramolecular Electron Transfer in Metal−Organic Complexes

Two transition metal complexes of the type fac-(bpy)ReI(CO)3(DA)+ (where bpy = 2,2‘-bipyridine and DA is a pyridine ligand that is substituted with a 1,2-diamine electron donor) have been prepared. The 1,2-diamine serves as a “reactive donor ligand” owing to its propensity to undergo rapid C−C bond fragmentation when activated by single electron transfer oxidation. Photoexcitation of the diamine complexes affords a ligand-to-ligand charge transfer (LLCT) state via intramolecular electron transfer quenching of a metal-to-ligand charge transfer (MLCT) state, [(bpy)ReI(CO)3(DA)]+ + hν → [(bpy•-)ReII(CO)3(DA)]+*(MLCT) → [(bpy•-)ReI(CO)3(DA•+)]+*(LLCT). Photochemical product and quantum efficiency studies indicate that the diamine reactive donor ligand undergoes photoinduced C−C bond fragmentation with high efficiency, presumably via the radical cation (DA•+) which is present in the LLCT excited state. Laser flash photolysis allows direct detection of the metal complex based radicals that are formed by C−C bond fragmentation. Quantitative kinetic information gathered through luminescence, laser flash photolysis, and quantum yield studies allows estimation of the rates for formation of the LLCT state by forward electron transfer (kFET), decay of the LLCT state by back electron transfer (kBET), and the rate of diamine radical cation bond fragmentation in the LLCT state (kBF). The relationship between these kinetic parameters and the driving force for electron transfer and bond fragmentation as well as the structure of the reactive donor ligands is discussed

Protein Induced Aggregation of Conjugated Polyelectrolytes Probed with Fluorescence Correlation Spectroscopy: Application to Protein Identification

The interaction of a series of water-soluble conjugated polyelectrolytes with varying backbone structure, charge type (cationic and anionic), and charge density with a set of seven different proteins is explored by using fluorescence correlation spectroscopy (FCS). The FCS method affords the diffusion time for a particular CPE/protein pair, and this diffusion time is a reflection of the aggregation state of the polymer/protein in the solution. The diffusion time is larger for oppositely charged CPE/protein combinations, reflecting the tendency toward the formation of CPE/protein aggregates in these systems. However, by careful analysis of the data, other factors emerge, including possible effects of hydrophobic interaction in specific CPE/protein systems. The final diffusion time for each CPE/protein mixture varies and the diffusion time response pattern created by the six-CPE array for a typical protein is unique, and this effect was leveraged to develop a sensor array for protein identification by using linear-discriminant analysis (LDA) methods. By application of multimode linear discrimination analysis, the unknown protein samples have been successfully identified with a total accuracy of 93%

Conjugated Polyelectrolyte Based Real-Time Fluorescence Assay for Adenylate Kinase

Addition of adenosine 5′-triphosphate (ATP) to a solution of the anionic conjugated polyelectrolyte PPECO2 and copper(II) ion (Cu2+) recovers the Cu2+-quenched fluorescence of PPECO2 to a significantly greater extent compared with the addition of adenosine 5′-diphosphate (ADP) or adenosine 5′-monophosphate (AMP) at the same concentration levels. Taking advantage of the differential response of the PPECO2−Cu2+ system to ATP, ADP and AMP, we have developed fluorescence turn-off and turn-on assays that monitor the catalytic activity of adenylate kinase (ADK) in the equilibrium transphosphorylation reaction (ATP + AMP ⇔ 2ADP). The fluorescence turn-on and turn-off assays monitor the forward and reverse transphosphorylation reactions, respectively. The forward assay operates with ATP substrate present at the submillimolar concentration range and offers a straightforward and rapid detection of ADK catalytic activity with the enzyme present in the nanomolar range, in either end-point or real-time formats. The real-time fluorescence intensity from PPECO2 can be converted to substrate (ATP) concentration in the forward reaction assay by using an ex-situ calibration curve, allowing ADK catalyzed reaction rates and kinetic parameters to be determined. ADK activation by Mg2+ and inhibition by Ag+ and product are analyzed using the optimized assay system. Non-specific interactions are observed between the assay complex and other proteins, but the signal response to the ADK assay is demonstrated to mainly arise from the specific enzyme catalyzed transphosphorylation reaction

Elucidating the Effects of Solvating Side Chains on the Rigidity and Aggregation Tendencies of Conjugated Polymers with Molecular Dynamics Simulations Using DFT Tight Binding

Poly­(phenylene ethynylene) (PPE) and a series of PPE derivatives were studied using density functional theory tight binding to generate molecular dynamics simulations in the gas phase. Dihedral angles between adjacent phenylene units were measured over time to generate a histogram of conjugation lengths, where the conjugation length was defined by planarity. The average effective conjugation lengths for these polymers were extracted from this data. Notably, it was found that PPE with alkoxy substituents on the phenylene ring of each repeat unit is attributed to causing an increased average conjugation length relative to unsubstituted PPE from 4.7 to 6.4 repeat units. Comparatively, alkyl substituents caused a decrease in the conjugation length to 4.5 repeat units. The methods developed here were extended to a wider series of PPE derivatives, where a direct link was found between polymer planarity and the electron-donating/-withdrawing ability of substituents. These results indicate that the solvating side chains frequently employed in conjugated polymers have an innate effect on the rigidity of the polymer backbone

Elucidating the Effects of Solvating Side Chains on the Rigidity and Aggregation Tendencies of Conjugated Polymers with Molecular Dynamics Simulations Using DFT Tight Binding

Poly­(phenylene ethynylene) (PPE) and a series of PPE derivatives were studied using density functional theory tight binding to generate molecular dynamics simulations in the gas phase. Dihedral angles between adjacent phenylene units were measured over time to generate a histogram of conjugation lengths, where the conjugation length was defined by planarity. The average effective conjugation lengths for these polymers were extracted from this data. Notably, it was found that PPE with alkoxy substituents on the phenylene ring of each repeat unit is attributed to causing an increased average conjugation length relative to unsubstituted PPE from 4.7 to 6.4 repeat units. Comparatively, alkyl substituents caused a decrease in the conjugation length to 4.5 repeat units. The methods developed here were extended to a wider series of PPE derivatives, where a direct link was found between polymer planarity and the electron-donating/-withdrawing ability of substituents. These results indicate that the solvating side chains frequently employed in conjugated polymers have an innate effect on the rigidity of the polymer backbone

Free Energy Dependence of Photoinduced Electron Transfer in Octathiophene-Diimide Dyads

Donor–acceptor dyads consisting of octathiophene (T8) paired with three (di)­imide acceptors (naphthalene diimide (NDI), benzene diimide (BDI), and naphthalimide (NI)) were synthesized and probed for their photoinduced forward electron transfer (ET) and charge recombination kinetics by using ultrafast transient absorption (TA) spectroscopy. The three acceptors have different electron affinities, leading to variation in the energy of the charge-separated state and the driving force (ΔG) for forward ET and charge recombination. Analysis of the TA spectra and kinetics allows assignment of rates for forward ET and charge recombination for each of the oligomers. Electrochemistry and photoluminescence spectroscopy are used to determine the ΔG values for the ET processes. For two of the oligomers (T8NDI and T8BDI), the rates for forward ET and charge recombination are very rapid (k > 3 × 1010 s–1). By contrast, for the third oligomer (T8NI), the rates for both processes are considerably slower (k < 5 × 109 s–1). Analysis of the rate/free energy correlation for the series of oligomers reveals generally good agreement with the Marcus semiclassical theory. In all of the oligomers, the ET reactions are nonadiabatic, in part, due to weak coupling caused by out-of-plane twisting of the phenylene spacer that lies between the T8 segment and the (di)­imide acceptors. The rapid ET dynamics for T8NDI and T8BDI are explained as arising due to the processes occurring near the barrierless region (−ΔG ≈ λ) or slightly into the Marcus inverted region (−ΔG > λ). The slower dynamics for T8NI are explained as arising because the forward ET is weakly exothermic, whereas charge recombination is deep into the inverted region. This study is the first to produce experimental results that match a full Marcus bell-shaped curve with ET rates in the normal, barrrierless, and inverted regions in dyads based on a π-conjugated oligomer donor

Synthesis of Monodisperse Platinum Acetylide Oligomers End-Capped with Naphthalene Diimide Units

We report the synthesis and structural characterization of a series of monodisperse platinum acetylide oligomers with the general structure NDI-[Ph−CC−Pt(PBu3)2−CC−]n−Ph−NDI, where n = 2, 3, 6, or 10, Ph = 1,4-phenylene, NDI is a substituted 1,4,5,8-naphthalene diimide, and the geometry at the Pt centers is trans. The oligomers were synthesized via an iterative-convergent approach utilizing organometallic synthons that feature orthogonally protected terminal acetylene units. The 31P NMR spectra of the oligomers are especially revealing as to their structure, due to a difference in chemical shift for the internal and terminal Pt(PBu3)2 units. The oligomers were also characterized by electrochemistry, UV−visible absorption, and photoluminescence spectroscopy. The emission spectroscopy reveals that the triplet exciton is efficiently quenched in the NDI end-capped oligomers, and the quenching is thought to arise due to photoinduced charge separation