A homogeneous chemiluminescent immunoassay method.

A new homogeneous chemiluminescent immunoassay method featuring the use of specific binding members separately labeled with an acridan-based chemiluminescent compound and a peroxidase is reported. Formation of an immunocomplex brings the chemiluminescent compound and the peroxidase into close proximity. Without any separation steps, a chemiluminescent signal is generated upon addition of a trigger solution, and the intensity is directly correlated to the quantity of the analyte.

Vibrational coherence in polar solutions of Zn(II) tetrakis(N-methylpyridyl)porphyrin with Soret-band excitation: rapidly damped intermolecular modes with clustered solvent molecules and slowly damped intramolecular modes from the porphyrin macrocycle.

Ground-state coherent wavepacket motions arising from intermolecular modes with clustered, first-shell solvent molecules were observed using the femtosecond dynamic absorption technique in polar solutions of Zn(II) meso-tetrakis(N-methylpyridyl)porphyrin (ZnTMPyP) with excitation in the Soret absorption band. As was observed previously in bacteriochlorophyll a solution, the pump-probe transients in ZnTMPyP solutions are weakly modulated by slowly damped (effective damping time gamma > 1 ps) features that are assigned to intramolecular modes, the skeletal normal modes of vibration of the porphyrin. The 40 cm(-1) and 215 cm(-1) modes from the metal-doming and metal-solvent-ligand modes, respectively, are members of this set of modulation components. A slowly damped 2-4 cm(-1) component is assigned to the internal rotation of the N-methylpyridyl rings with respect to the porphyrin macrocycle; this mode obtains strong resonance Raman intensity enhancement from an extensive delocalization of pi-electron density from the porphyrin in the ground state onto the rings in the pi* excited states. The dominant features observed in the pump-probe transients are a pair of rapidly damped (gamma < 250 fs) modulation components arising from intermolecular modes with solvent molecules. This structural assignment is supported by an isotope-dependent shift of the average mode frequencies in methanol and perdeuterated methanol. The solvent dependence of the mean intermolecular mode frequency is consistent with a van der Waals intermolecular potential that has significant contributions only from the London dispersion and induction interactions; ion-dipole or ion-induced-dipole terms do not make large contributions because the pi-electron density is not extensively delocalized onto the N-methylpyridyl rings. The modulation depth associated with the intermolecular modes exhibits a marked dependence on the electronic structure of the solvent that is probably related to the degree of covalency; the strongest modulations are observed in acetonitrile and dimethylsulfoxide. The results strongly support a structural assignment of the low-frequency modes that are coupled to the primary and secondary electron-transfer reactions in photosynthetic reaction centers to intermolecular modes between the redox-active chromophores and first-solvation shell groups from the surrounding protein, and an important additional function of the intermolecular modes in the stabilization of charged intermediates is suggested.

Intermolecular vibrational coherence in the bacteriochlorophyll proteins B777 and B820 from Rhodospirillum rubrum.

The low-frequency vibrational coherence in the bacteriochlorophyll (BChl)-containing subunit proteins B777 and B820 from the LH1 light-harvesting complex isolated from Rhodospirillum rubrum G9 exhibits rapidly damped modulation components arising from intermolecular, formally nonbonding interactions between the BChl macrocycle and polar groups in the surrounding detergent or protein. The vibrational coherence observed in the monomeric B777 system resembles that observed previously with BChl in acetone because it contains a pair of broad overlapping line shapes with a mean frequency of 191 cm(-1), but the 10:1 intensity ratio of the librational and translational components is distinctive of the motions of the polar head groups in the nonionic detergent micelle that solvates the BChl macrocycle. In contrast, the vibrational coherence observed with the dimeric B820 complex is almost 20 times weaker in intensity and exhibits narrower line shapes and lower average frequencies than observed in B777. The structure of the B820 complex sterically protects the pair of BChl macrocycles from the surrounding solvent, so modulation components assigned to intrinsic interactions between the BChl and the protein and between the pair of BChl's are revealed. A relatively well-ordered interaction between the BChl macrocycle and a tryptophan residue in each alpha-helical polypeptide accounts for a 28 cm(-1) component with a narrow line shape, but most of the intensity arises from a broader 46 cm(-1) component that is assigned to the interaction between the paired BChl macrocycles. The breadth of the line shape for this component is a measure of the disorder in the ensemble of B820 subunits. The results support the hypothesis that the excited-state vibrational dynamics and the optical and/or Marcus charge-transfer reorganization energies of BChl in photosynthetic light-harvesting proteins and reaction centers are strongly controlled by van der Waals modes with neighboring molecules, with dominant contributions to the intermolecular potential arising from the London dispersion and dipole-dipole interactions.

Intermolecular vibrational coherence in bacteriochlorophyll a with clustered polar solvent molecules.

We show that resonant impulsive excitation of the Qy absorption band of bacteriochlorophyll a (BChl) launches a rapidly damped (gamma < 200 fs) ground-state coherent wave-packet motion that arises from intermolecular modes with clustered solvent molecules. Femtosecond pump-probe, dynamic-absorption signals were obtained at room temperature with BChl solutions in pyridine, acetone, and 1-propanol. The vibrational coherence observed in the 0-800-fs regime is modeled in the time domain by two (or three, in the case of 1-propanol) modulation components with asymmetric, inhomogeneously broadened line shapes and frequencies in the 100-200-cm(-1) range. The mean frequency of the vibrational coherence exhibits at least a quadratic dependence on the dipole moment of the solvent molecules and a y-intercept in the 100-cm(-1) regime. This trend is modeled by an expression for the natural frequency of a "6-12" potential composed of attractive terms from van der Waals forces and a repulsive term from the exchange (Pauli exclusion) force. The model suggests that comparable contributions to the potential are provided by the dipole-dipole and London dispersion interactions. These results support the hypothesis that the low-frequency vibrational modes in the 100-cm(-1) regime that are coupled to the light-driven charge-separation reactions in the reaction center from purple bacteria are derived from intermolecular vibrational modes between the chromophores and the surrounding protein medium.

Vibrational coherence from the dipyridine complex of bacteriochlorophyll a: intramolecular modes in the 10-220-cm(-1) regime, intermolecular solvent modes, and relevance to photosynthesis.

We present the first observations of vibrational coherence in the 10-220-cm-1 region from bacteriochlorophyll a (BChl) in solution. A distinction can be made for the first time between BChl's intramolecular normal modes and intermolecular modes between BChl and solvent. The results show that the low-frequency vibrations that accompany the initial electron-transfer reaction from the paired BChl primary electron donor, P, in photosynthetic reaction centers arise predominantly from intramolecular modes of histidine-ligated BChl macrocycles. The results also suggest that polar-solvent interactions can significantly perturb the electronic properties of BChl in a manner that might have important functional consequences.