This study examines the binding of wild-type streptavidin and streptavidin mutants to biotinterminated self-assembled monolayers (SAMs) as a model of biomolecular recognition at solid-liquid interfaces. The types of streptavidin proteins employed in this work were wild-type, Tyr43Ala (Y43A), and Trp120Ala (W120A), which have biotin-binding affinities that span several orders of magnitude (K a varies from ∼10 13 M -1 for wild-type to 10 7 M -1 for W120A). Two types of biotin-terminated monolayers were examined: those formed by chemisorption of 11-mercaptoundecanoic-(8-biotinoylamido-3,6-dioxaoctyl) amide (1) and those formed from mixtures of 12-mercaptododecanoic-(8-biotinoylamido-3,6-dioxaoctyl) amide ( 2) and 11-mercapto undecanol (3). Our findings support two previously published studies that found that 1 forms monolayers on gold that are disordered, while 2 and mixtures of 2 and 3 form closely packed, well-organized SAMs. The kinetics of binding and desorption of wild-type streptavidin and the mutants to and from these monolayers were measured using surface plasmon resonance spectroscopy. Adsorption of the proteins was found to occur at a diffusion-limited rate and to saturate at different surface coverages depending on their biotin-binding affinity. On disordered monolayers formed from 1, only a fraction of the bound mutants could be dissociated by exposure to free biotin. The fraction of undissociated mutants correlated with the biotin-binding affinity, suggesting that the formation of nonspecific interactions depends on the residence time of the protein on the surface. On mixed SAMs formed from 2 and 3, complete dissociation of the proteins occurred upon exposure to free biotin in solution. The kinetics of desorption of streptavidin from mixed SAMs was analyzed using a model that included the possibility of bivalent protein binding to the SAM at high surface concentrations of biotin. It was found that rate constants of dissociation were larger for the dissociation of a streptavidin-biotin bond on the surface than in solution. On biotinylated SAMs, the kinetic constants of dissociation were dependent on the surface concentration of biotin. Slow dissociation rates at higher surface coverage result from attractive protein-protein interactions. The results demonstrate the importance of the preparation and the structure of the solid surface and the complexity of biomolecular recognition at solid-liquid interfaces. Molecular recognition is affected by interactions between the adsorbed proteins and the surface and also by interactions among adsorbed proteins. These conclusions have important implications for the development of reversible biosensors.

Intramolecular electron transfer quenching following metal-to-ligand charge transfer (MLCT) excitation of complexes of Run containing the ligand N-((4'-methy1-2,2'-bipyridy1)-4-methy1)-9,10-anthraquinone-2-carboxamide (bpy-AQ) have been investigated by transient emission and absorption measurements. For [Run(bpy)2(bpy-AQ)I2+ (bpy is 2,2'-bipyridine) in acetonitrile at room temperature, partial quenching of the initial (Rulnbpy'-) excited state occurs as evidenced by decreased emission quantum yields (0.062 vs 0.011) and lifetimes (980 vs 158 ns) relative to [Ru1"(bpy*-)(bpy)(dmb)l2+* (dmb is 4,4'-dimethyl-2,2'-bipyridine). In [Ru"(tmb)z(bpy-AQ)I2+ (tmb is 4,4',5,5'-tetramethyl-2,2'-bipyridine), electron transfer quenching is nearly complete (@em = 0.002, t = 6 ns). In neither case is there evidence for AQ'-(Amax = 590 nm) by transient absorption measurements, and it is concluded that the expected redox-separated states, e.g., [Rum(bpy)2(bpy-AQ'-)I2+, undergo rapid back electron transfer to return to the ground states. MLCT excitation of the donor-chromophore-acceptor complex [Ru"(bpy-AQ)2(bpy-PTZ)12+ [bpy-FTZ is { 10-[(4'-methyl-2,2'-bipyridin-4-yl)methyl]phenothiazine)] leads to formation of the redox-separated state [Run(bpy-AQ)(bpy-AQ'-)(bpy-PTZ'+)I2+ (n,,(-F"+) = 5 10 nm) in approximately 40% yield. It undergoes back-electron-transfer to give the ground state with k(25 "C, CH3CN) = 6.7 x lo6 s-l.

Competitive energy and electron transfer occur following excitation of the chromophore-biquencher [Re I (MebpyCH 2 OCH 2 An)(CO) 3 (MQ + )](PF 6 ) 2 (MebpyCH 2 OCH 2 An is 4-[(9-anthrylmethoxy)methyl]-4′-methyl-2,2′-bipyridine; MQ + is N-methyl-4,4′,-bipyridinium ion). Following excitation with a 392-nm, 4-ns laser pulse in 1,2-dichloroethane (DCE), the initial excited state energy is distributed in a ∼2:1 ratio between 3 An* and the lower lying Re II (MQ ‚ ) metal-to-ligand charge transfer (MLCT) excited state. The appearance of the two states was monitored by transient absorption spectroscopy, which detected the triplet-triplet band for 3 An* at λ max ) 420 nm (τ ) 35 ( 2 µs, k ) (2.9 ( 0.2) × 10 4 s -1 ) and a π f π* band of MQ • at 610 nm (τ ) 37 ( 2 ns, k ) (2.7 ( 0.2) × 10 7 s -1 ). These states result from a competitive partitioning in the initial excited state or states. They do not interconvert to any significant degree on the time scale of the shorter lived, higher energy Re II (MQ*) state.

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