This paper describes a prototype of an integrated fluorescence detection system that uses a microavalanche photodiode (microAPD) as the photodetector for microfluidic devices fabricated in poly(dimethylsiloxane) (PDMS). The prototype device consisted of a reusable detection system and a disposable microfluidic system that was fabricated using rapid prototyping. The first step of the procedure was the fabrication of microfluidic channels in PDMS and the encapsulation of a multimode optical fiber (100-microm core diameter) in the PDMS; the tip of the fiber was placed next to the side wall of one of the channels. The optical fiber was used to couple light into the microchannel for the excitation of fluorescent analytes. The photodetector, a prototype solid-state microAPD array, was embedded in a thick slab (1 cm) of PDMS. A thin (80 microm) colored polycarbonate filter was placed on the top of the embedded microAPD to absorb scattered excitation light before it reached the detector. The microAPD was placed below the microchannel and orthogonal to the axis of the optical fiber. The close proximity (approximately 200 microm) of the microAPD to the microchannel made it unnecessary to incorporate transfer optics; the pixel size of the microAPD (30 microm) matched the dimensions of the channels (50 microm). A blue light-emitting diode was used for fluorescence excitation. The microAPD was operated in Geiger mode to detect the fluorescence. The detection limit of the prototype (approximately 25 nM) was determined by finding the minimum detectable concentration of a solution of fluorescein. The device was used to detect the separation of a mixture of proteins and small molecules by capillary electrophoresis; the separation illustrated the suitability of this integrated fluorescence detection system for bioanalytical applications.

Flash photolysis investigations of horse heart metmyoglobin bound with NO (Mb(3+)NO) reveal the kinetics of water entry and binding to the heme iron. Photodissociation of NO leaves the sample in the dehydrated Mb(3+) (5-coordinate) state. After NO photolysis and escape, a water molecule enters the heme pocket and binds to the heme iron, forming the 6-coordinate aquometMb state (Mb(3+)H2O). At longer times, NO displaces the H2O ligand to reestablish equilibrium. At 293 K, we determine a value k(w) approximately 5.7 x 10(6) s(-1) for the rate of H2O binding and estimate the H2O dissociation constant as 60 mM. The Arrhenius barrier height H(w) = 42 +/- 3 kJ/mol determined for H2O binding is identical to the barrier for CO escape after photolysis of Mb(2+)CO, within experimental uncertainty, consistent with a common mechanism for entry and exit of small molecules from the heme pocket. We propose that both processes are gated by displacement of His-64 from the heme pocket. We also observe that the bimolecular NO rebinding rate is enhanced by 3 orders of magnitude both for the H64L mutant, which does not bind water, and for the H64G mutant, where the bound water is no longer stabilized by hydrogen bonding with His-64. These results emphasize the importance of the hydrogen bond in stabilizing H2O binding and thus preventing NO scavenging by ferric heme proteins at physiological NO concentrations.

We have performed resonance Raman and optical absorption studies on ferric cytochrome P450cam complexed with oxidized putidaredoxin. Optical absorption and resonance Raman measurements demonstrate that this complexation shifts the spin-state equilibrium of P450cam to the low-spin form. In the resonance Raman spectra, the ν3 heme skeletal mode characteristic of low-spin P450cam intensifies upon complexation with putidaredoxin. Its frequency is indistinguishable to that of the usual low-spin species, indicating that this low-spin form originates from a water-bound, hexacoordinate heme. This observation suggests that putidaredoxin binding affects the distal side of P450cam and allows water entry into the heme pocket. We also examined the effects of putidaredoxin binding on the heme axial ligand (Fe−S) stretching mode. The binding of putidaredoxin upshifts the mode by ∼3 cm-1. Investigations of the Fe−S mode for the Thr252 → Ala distal pocket mutant and P450cam bound to various camphor analogues demonstrate that the frequency of the νFe - S is sensitive to distal pocket water. The water lowers the Fe−S stretching frequency by ∼1 cm-1, demonstrating that the putidaredoxin-induced ∼3-cm-1 increase in the Fe−S stretching frequency is not simply caused by the increased hydration of the heme pocket. We also found that the Fe−S frequency is increased by ∼0.5 cm-1 in the presence of high salt concentrations. This result suggests that the shielding of positive charges at the proximal face of P450cam leads to an increased νFe - S frequency. The salt effect is consistent with the observation that the binding of negatively charged putidaredoxin at the proximal side of the enzyme also increases the νFe - S frequency. Taken together, these results strongly suggest that electrostatic shielding of charged groups on the proximal face of P450cam takes place when putidaredoxin binds and contributes to the observed upshift of the Fe−S mode.