An integrated real-time sensing system that uses a portable Raman spectrometer and a micropillar array chip has been developed for field analysis. The problem of poor detection sensitivity, caused by miniaturization in the portable Raman spectrometer, was overcome by using the surface-enhanced Raman scattering (SERS) technique. The problem of poor reproducibility in the SERS detection, caused by different particle sizes and inhomogeneous degrees of aggregation, was also overcome by using continuous flow and homogeneous mixing between the analytes and nanocolloidal silver in a micropillar array microfluidic chip. Two hazardous materials, dipicolinic acid and malachite green, were quantitatively analysed using our integrated portable Raman sensor system. The observed limit of detection was estimated to be 200 ppb and 500 ppb, respectively. Our proposed analytical method, using a micropillar array PDMS chip and a portable SERS system, offers a rapid and reproducible trace detection capability for hazardous materials in the field.
In order to determine the molecular origin of the differential photocurrent from bacteriorhodopsin (bR), the photoelectric response of bR film deposited on an indium tin oxide (ITO) conductive glass electrode under CW excitation is compared with that under pulsed laser excitation at different pH and with opposite membrane orientation with respect to the ITO electrode surface. The characteristics (sign and magnitude) of the dominant component of the differential photocurrent (appearing on the millisecond time scale) are found to correlate with the process of proton release into, or uptake from, the aqueous solution during the photocycle under different experimental conditions. This suggests that the differential current results mainly from the change in the H + concentration at the bR-ITO electrode interface.
When excited with a pulsed laser, an electric field-oriented bacteriorhodopsin (bR) film on an indium−tin oxide (ITO) conductive electrode generates a photocurrent composed of at least three different components: B1 (<100 ps), B2 (∼60 μs), and B3 (∼ms). When excited with an electronic shutter modulated CW light pulse (>200 ms in duration), a differential photocurrent (components D1 and D2 with decay times in milliseconds) is observed from the bR film. D1 is observed when the CW light is turned on, and D2 is observed when the CW light is turned off. In this paper, we compare the amplitudes and lifetimes of B2, B3, and D1 at various values of pH and ionic strength of the electrolyte solution in which the photocurrent is measured. It is found that changing the film orientation changes the polarity (sign) of B1 and B2, while it does not affect the polarity of B3 and D1. It is also found that B3 and D1 change their polarity upon changing the pH of electrolyte solution, whereas B1 and B2 do not. These results suggest that the origin of B3 and D1 is different from that of B1 and B2. Our results suggest that B3 and D1 are due to the formation of a transient proton capacitor between the two ITO electrodes resulting from the proton pumping in bR. The magnitude and sign of B3 and D1 are determined by the transient proton concentration change (accumulation or disappearance) occurring near the bR-modified ITO electrode interface on the millisecond time scale. The change of sign in B3 and D1 as a function of pH is due to the sequence of proton release/uptake in the bR photocycle: It first releases protons into the aqueous solution at high pH, while it first takes up protons from the aqueous solution at low pH. The effects of buffer and ionic strength on B3 and D1 are discussed in terms of the kinetics of proton release/uptake and of the transportation of positive and negative ions in the electrolyte solution.
The first section of this paper is a detailed summary of studies made by us and others on metal cations binding to deionized bacteriorhodopsin (dIbR) and its variants. Our studies include the luminescence experiments of Eu 3 + binding to dIbR and potentiometric studies of Ca" binding to dIbR, to deionized bR mutants, to bacterioopsin, and to dIbR with its C-terminus removed. The results suggest the presence of two classes of binding sites, one class has two high-affinity constants, and one has one low-affinity constant. For Ca" binding, there is one metal cation in each of the two high-affinity sites which are coupled to the charged aspartates 85 and 212 (known to be in the retinal cavity) but not coupled to each other. The lowaffinity class can accommodate 0-6 Ca" ions and most of them are bound to the surface. Mg" has a slightly smaller value for its binding constant to the highestaffinity site. Thus, one expects more Ca" than Mg" bound to the two high-affinity sites. In the second section, we summarize our recent study on the effect of metal cation charge density (Ca'", Mg", Eu 3 +, Tb 3 +, H03+, Dy3+) on the kinetics of both Schiff base deprotonation and proton transport to the extracellular surface. For all metal cations, the apparent rate constant of the slow components of the deprotonation process is the same as that for the transport process at 22°C. The temperature studies, however, show this apparent equality to be fortuitous and to result from cancellation of the contribution of the energy and entropy of activation. Thus, while the entropy of activation is positive for the deprotonation process, it is negative for the proton transport process. These kinetic parameters depend weakly on the charge density, but in an opposite sense for the two processes. These results suggest that the deprotonation is not the rate-limiting step for the proton transport process. A possible mechanism is proposed in which a hydrated metal cation is used to induce the deprotonation of the protonated Schiff base and to dissociate one of its HzO molecules to donate the proton in the L~M process.b~-:>(bR)*~I460~J625~~IO~L550~M4l2~N~b- H+ +H+In the L sso~M412 step, a proton is transferred from
Bacteriorhodopsin contains Ca2+ and Mg2+ ions whose removal inhibits its proton pump function. The binding constants of Ca2+ to the high-affinity sites were determined by the use of a calcium ion specific electrode. The unavailability of magnesium ion specific electrode prevented a similar determination for Mg2+. The binding constant of Mg2+ to the binding site of highest affinity is determined by using a calcium ion selective electrode to measure the concentration of free Ca2+ in competition with Mg2+ for the binding. The binding constant of Mg2+ to the second high affinity site is determined spectrally. The two high-affinity binding constants for Mg2+ are compared with those obtained for Ca2+ in the absence and the presence of Mg2+. The fact that the presence of low concentration of one metal ion does not affect the binding constant of the other metal ion to the other binding site supports the assumption of the independence of the two high-affinity sites of one another. The difference in the observed values of the binding constants of the two high-affinity sites for Ca2+ and Mg2+ is qualitatively discussed in terms of the enthalpy and entropy changes in the binding equilibrium.
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