Collapse and recovery of green fluorescent protein chromophore emission through topological effects.

Housed within the 11-stranded ß-barrel of the green fluorescent protein (GFP) is the arylideneimidazolidinone (AMI) chromophore, the component responsible for fluorescence. This class of small-molecule chromophore has drawn significant attention for its remarkable photophysical and photochemical properties, both within the intact protein and after its denaturation. All of the proteins so far isolated that have visible light fluorescence have been found to contain an AMI chromophore. These proteins comprise an extensive rainbow, ranging from GFP, which contains the simplest chromophore, p-hydroxybenzylideneimidazolidinone (p-HOBDI), to proteins having molecules with longer conjugation lengths and a variety of intraprotein interactions. The fluorescence invariably almost vanishes upon removal of the protective ß-barrel. The role of the barrel in hindering internal conversion has been the subject of numerous studies, especially in our laboratories and those of our collaborators. A better understanding of these chromophores has been facilitated by the development of numerous synthetic protocols. These syntheses, which commonly use the Erlenmeyer azlactone method, have evolved in recent years with the development of a [2 + 3] cycloaddition exploited in our laboratory. The synthetic AMI chromophores have allowed delineation of the complex photophysics of GFP and its derivatives. Upon denaturation, AMI chromophores are marked by 4 orders of magnitude of diminution in emission quantum yield (EQY). This result is attributed to internal conversion resulting from conformational freedom in the released chromophore, which is not allowed within the restrictive ß-barrel. To date, the photophysical properties of the AMI chromophore remain elusive and have been attributed to a variety of mechanisms, including cis-trans isomerization, triplet formation, hula twisting, and proton transfer. Advanced studies involving gas-phase behavior, solvent effects, and protonation states have significantly increased our understanding of the chromophore photophysics, but a comprehensive picture is only slowly emerging. Most importantly, mechanisms in structurally defined chromophores may provide clues as to the origin of the "blinking" behavior of the fluorescent proteins themselves. One approach to examining the effect of conformational freedom on rapid internal conversion of the chromophores is to restrict the molecules, both through structural modifications and through adjustments of the supramolecular systems. We thus include here a discussion of studies involving the crystalline state, inclusion within natural protein-binding pockets, complexation with metal ions, and sequestration within synthetic cavities; all of this research affirms the role of restricting conformational freedom in partially restoring the EQY. Additionally, new photochemistry is observed within these restricted systems. Many of the studies carried out in our laboratories show promise for these molecules to be adapted as molecular probes, wherein inclusion turns on the fluorescence and provides a signaling mechanism. In this Account, we present an overview of the AMI chromophores, including synthesis, overall photophysics, and supramolecular behavior. A significant amount of work remains for researchers to fully understand the properties of these chromophores, but important progress achieved thus far in photophysics and photochemistry is underscored here.

Inhibition of twisting of a green fluorescent protein-like chromophore by metal complexation.

Substitution of a pyridyl for the hydroxyphenyl moiety in the Green Fluorescent Protein analog p-hydroxybenzylidene-dimethylimidiazolinone produces a chromophore which "turns on" fluorescence in the presence of Zn(2+) or Cd(2+) ions. Such a phenomenon provides "proof of principle" for using GFP chromophores in a variety of sensing applications.

Topochemistry and photomechanical effects in crystals of green fluorescent protein-like chromophores: effects of hydrogen bonding and crystal packing.

To obtain insight into the effects of the environment on the photophysics and photochemistry of the green fluorescence protein (GFP), eight crystal structures of six synthetic aryl-substituted analogues (2-fluoro, 2-methyl, 3-hydroxy, 3-methoxy, 2,4-dimethyl and 2,5-dimethyl) of the GFP chromophore (4-hydroxy-benzylidenedimethylimidazolinone) were determined and correlated with their two-dimensional steady-state and time-resolved solid-state excitation-emission spectra. The stacking between the molecules greatly affected the emission energy and the lifetime of the emission of the chromophore, implying that pi-pi interactions could be critical for the photophysics of GFP. The reaction pathways were dependent on the excitation energy, resulting either in [2 + 2] photodimerization at the bridging double bond (UV excitation) or flipping of the imidazolone ring (visible excitation). The meta-hydroxy chromophore (3-HOBDI) was the only GFP-chromophore analogue that was obtained as more than one stable polymorph in the pure state thus far. Due to the asymmetric substitution with hydrogen bond donors and acceptors, 3-HOBDI is tetramorphic, the forms showing distinctly different structure and behavior: (1) while one of the polymorphs (3-HOBDI-A), having multilayer structure with alternating stereochemistry of linear hydrogen-bonded motifs, undergoes photodimerization under UV light, (2) another (3-HOBDI-C), which has dimeric head-to-tail structure, shows Z-to-E isomerization via tau-one-bond flip of the imidazolone ring by excitation in the visible region. X-ray diffraction analysis of a partially reacted single crystal of 3-HOBDI-C provided the first direct evidence of tau-one-bond flip occurring in a GFP-like compound. Moreover, the cooperative action of the photodimerization of 3-HOBDI-A appears as a photomechanical effect of unprecedented magnitude for a single crystalline specimen, where photoexcited single crystals bend to more than 90 degrees without breaking.

Combining scanning electrochemical microscopy with infrared attenuated total reflection spectroscopy for in situ studies of electrochemically induced processes.

The combination of scanning electrochemical microscopy (SECM) with single-bounce attenuated total reflection Fourier-transformed infrared spectroscopy (FT-IR-ATR) has been developed for in situ studies on electrochemically induced processes at IR waveguide surfaces via evanescent field absorption spectroscopy. The feasibility of the combined microelectrochemical FT-IR setup was demonstrated by spectroscopically monitoring microstructured polymer depositions induced via feedback mode SECM using a 25 mum Pt disk ultramicroelectrode (UME). The surface of a ZnSe ATR crystal was initially coated with 2,5-di-(2-thienyl)-pyrrole (SNS) layer, which was then locally polymerized during Ru(bpy)(3)(2+) mediated feedback mode SECM experiments. The polymerization reaction was simultaneously monitored by recording absorption intensity changes of SNS specific IR bands, thereby providing information on the polymerization mechanism and on the percentage of surface modification.

Isomerization in fluorescent protein chromophores involves addition/elimination.

The green fluorescent protein (GFP) chromophore undergoes both photochemical and thermal isomerizations. Typically, the Z form is more stable and undergoes photochemical conversion to the E form followed by thermal reversion over a period of seconds or minutes. Although the mechanism of the thermal reversion has been the subject of some investigations, the surprisingly low activation energy for this process has not sparked any controversy. We now show that the chromophore is surprisingly stable in both E and Z forms and that the facile thermal reversion is the result of a novel nucleophilic addition/elimination mechanism. This observation may have implications for the intervention of such processes, as well as blinking and kindling, in fluorescent proteins.

Label-free DNA detection of hepatitis C virus based on modified conducting polypyrrole films at microelectrodes and atomic force microscopy tip-integrated electrodes.

We present a new strategy for the label-free electrochemical detection of DNA hybridization for detecting hepatitis C virus based on electrostatic modulation of the ion-exchange kinetics of a polypyrrole film deposited at microelectrodes. Synthetic single-stranded 18-mer HCV genotype-1-specific probe DNA has been immobilized at a 2,5-bis(2-thienyl)-N-(3-phosphoryl-n-alkyl)pyrrole film established by electropolymerization at the previously formed polypyrrole layer. HCV DNA sequences (244-mer) resulting from the reverse transcriptase-linked polymerase chain reaction amplification of the original viral RNA were monitored by affecting the ion-exchange properties of the polypyrrole film. The performance of this miniaturized DNA sensor system was studied in respect to selectivity, sensitivity, and reproducibility. The limit of detection was determined at 1.82x10(-21) mol L(-1). Control experiments were performed with cDNA from HCV genotypes 2a/c, 2b, and 3 and did not show any unspecific binding. Additionally, the influence of the spacer length of 2,5-bis(2-thienyl)-N-(3-phosphoryl-n-alkyl)pyrrole on the behavior of the DNA sensor was investigated. This biosensing scheme was finally extended to the electrochemical detection of DNA at submicrometer-sized DNA biosensors integrated into bifunctional atomic force scanning electrochemical microscopy probes. The 18-mer DNA target was again monitored by following the ion-exchange properties of the polypyrrole film. Control experiments were performed with 12-base pair mismatched sequences.

Ultrafast photoinduced charge separation dynamics in polythiophene/SnO2 nanocomposites.

We present a study of photoinduced interfacial electron transfer (ET) dynamics of SnO2 nanocrystalline thin films sensitized by polythiophene derivatives (regioregular poly(3-hexylthiophene) (P3HT) and regiorandom poly(3-undecyl-2,2'-bithiophene) (P3UBT)). ET dynamics were measured by following the dynamics of injected electrons in SnO2 and polarons in the conjugated polymer using ultrafast mid-IR transient absorption spectroscopy. The rate of electron transfer from P3HT and P3UBT to SnO2 films was determined to occur on sub-picosecond time scale (120 +/- 20 fs). In P3HT/SnO2 composite, interchain charge transfer was found to compete with and reduce the quantum efficiency of interfacial electron transfer at high polymer loading. This interchain charge separation processes can be reduced in non-regioregular polymer or at low polymer loading levels.

Label-free DNA detection based on modified conducting polypyrrole films at microelectrodes.

A label-free electrochemical detection method for DNA hybridization based on electrostatic modulation of the ion-exchange kinetics of a polypyrrole film deposited at microelectrodes is reported. Synthetic single-stranded 27-mer oligonucleotides (probe) have been immobilized at 2,5-bis(2-thienyl)-N-(3-phosphorylpropyl)pyrrole film formed by electropolymerization on the previously formed polypyrrole layer. The 27- or 18-mer target oligonucleotides were monitored via the electrochemically driven anion exchange of the inner polypyrrole film. The performance of the miniaturized DNA biosensor system was studied in respect to selectivity, sensitivity, reproducibility, and regeneration of the sensor. Control experiments were performed with a noncomplementary target of 27-mer DNA and 12 base-pair mismatched 18-mer sequences, respectively, and did not show any unspecific binding. Under optimized experimental conditions, the label-free electrochemical biosensor enabled the detection limits of 0.16 and 3.5 fmol for the 18- and 27-mer DNA strand, respectively. Furthermore, we demonstrate reusability of the electrochemical DNA biosensor after successful recovery of up to 100% of the original signal by regenerating the DNA "label-free" electrode with 50 mM HCl at room temperature.

Pentacene disproportionation during sublimation for field-effect transistors.

At moderate temperatures in flowing gas, pentacene undergoes a disproportionation reaction to produce 6,13-dihydropentacene (DHP) and a series of polycondensed aromatic hydrocarbons, including the previously unknown peripentacene (PP). The process requires activation by heating to 320 degrees C and is possibly catalyzed by impurities such as DHP, 6,13-pentacenequinone (PQ), Al, or Fe found in the starting materials. These impurities also result in a decrease in the intrinsic field-effect mobility (FEM) of pentacene crystals. Subsequent purifications remove such impurities, thus inhibiting the formation of the disproportionation products and increasing the FEM of pentacene (2.2 cm(2)/Vs). These results clarify the importance of purification of semiconductive materials for measurements of intrinsic mobility and optimal device performance.

Label-free DNA hybridization probe based on a conducting polymer.

A new approach to the realization of the electrochemical DNA hybridization probe is described. It is based on the exchange of chloride ion between the polypyrrole layer and the buffer. The shape of the cyclic voltammogram is modulated by the negative charge density at this interface, resulting from the immobilized target DNA. The negative charge density increases when the complementary DNA hybridizes with the probe DNA. The hybridization event can be clearly seen in the cyclic voltammogram before and after the addition of the probe DNA. The immobilization is accomplished via the Mg2+ bridging complex between phosphonic acid groups of the poly[2,5-dithienyl-(N-3-phosphorylpropyl)pyrrole] grafted at the polypyrrole surface and the phosphate groups of the target DNA.