In this work we present optical pH sensors especially designed for pH measurements in marine environment. Embedded in an uncharged, highly proton-permeable hydrogel matrix, the two novel lipophilic carboxyfluorescein derivatives 2',7'-dihexyl-5(6)-N-octadecyl-carboxamidofluorescein (DHFA) and 2',7'-dihexyl-5(6)-N-octadecyl-carboxamidofluorescein ethyl ester (DHFAE) have apparent dissociation constants of ca. 8.4. The pH transition range of the sensors perfectly matches the pH range occurring in seawater and marine sediment (ca. pH 7.2-9.2). The cross-sensitivity towards ionic strength (IS) was found to be low for DHFA-containing membranes and was even negligible when using DHFAE as indicator. The quantum yield (QY) of DHFA (0.94(basic)) is similar to that of fluorescein (0.97(basic)). QYs of 0.62(basic) and 0.22(acidic) were found for DHFAE. The optical properties of the indicators enable referenced measuring schemes. Lactonisation of the DHFAE chromophore is prevented by esterification of the carboxyl group in 2 position. Thus, internally referenced dual wavelength measurements are possible since the emission maxima of the basic and acidic form of DHFAE differ by 30 nm. Dual lifetime referenced (DLR) measurements were made with pH sensors incorporating ruthenium-(II)-tris-4,7-diphenyl-1,10-phenanthroline (Ru(dpp)(3))-containing reference particles in addition to the indicator. This type of sensor can be applied for pH imaging or in phase-modulation measurements of pH.

A laboratory-based optical pH sensor for 2-dimensional pH imaging at benthic interfaces is presented. The sensor consists of a single-layer hydrogel matrix embedding the fluorescent pH indicator 2′, 7′-dihexyl-5(6)-Noctadecyl-carboxamidofluorescein ethyl ester (DHFAE) and the phosphorescent ruthenium(II)-Tris-4,7-diphenyl-1,10-phenanthroline incorporated in nanoparticles, serving as an inert reference standard. The measuring principle is based on time domain dual-lifetime referencing (t-DLR). The fluorophore/phosphor couple is simultaneously excited by a green LED (λ max 530 nm) pulsed in the microsecond range, and the sensor emission is recorded by a fast-gateable CCD camera. For each pH image, intensity images in 2 time windows (1 during and 1 after the excitation phase) are taken. The ratio of these 2 images is proportional to the pH of the sample and not affected by the overall signal intensity. The sensor has a dynamic range suitable for marine conditions (pH 7.3 to 9.3) with an apparent pK a of 8.3. The sensor was long-term stable (months) when kept in darkness and had a response time of < 200 s when going from pH 8.3 to 7.6. Light proved to have a negative effect on the sensor performance due to photobleaching of the pH indicator, resulting in a negative drift in the signal ratio at higher pH after prolonged light exposure. The spatial resolution (83 by 83 μm/pixel) of the sensor was capable of resolving smallscale spatial variability in the pH at a heterogeneous sediment-water interface, and time series of calibrated pH images also expressed a marked temporal variability in the pH distribution across this interface. Hotspots with intensified microbial activity were observed, and pH minima along burrow walls of polychaetes indicated elevated diagenetic activity in these zones. Individually extracted profiles from the pH images agreed well with independently measured pH microelectrode profiles, confirming the robustness of the approach.

A method for simultaneous and referenced 2D mapping of pH and pO2 is described. The experimental setup combines a fast gateable CCD camera as detector, a LED as excitation light source and a single-layer sensor membrane as optical transducer. The planar optode comprises a lipophilic fluorescein derivative (lifetime approximately 5 ns) and platinum(II) mesotetrakis(pentafluorophenyl)porphyrin (approximately 70 micros in the absence of a quencher) immobilized in a hydrogel matrix. Depending on the fluorescent pH indicator, a pH transition in the physiological range (pH 6-pH 8) or in the near-basic region (pH 7-pH 9) can be achieved. The measuring scheme involves the time-resolved acquisition of images in three windows during a series of square-shaped excitation pulses. A method allowing the calculation of both parameters from these three images is presented. The pH/pO2 hybrid sensor incorporating the pH indicator 2',7'-dihexyl-5(6)-N-octadecyl-carboxamidofluorescein was characterized in detail. The pH and pO2 were determined with a maximum deviation of 0.03 pH unit and 6.5 hPa pO2, respectively, within the range of pH 7.6-pH 8.7 and 0-200 hPa pO2 in test measurements. The ionic strength (IS) cross-sensitivity was found to be relatively small (pH/IS < 3.5 x 10(-4) mM(-1) and pO2/IS <-0.053 hPa mM(-1) at a transition from 0.5 to 0.1 M IS). Whereas a strong temperature effect on the sensor signal was observed (DeltapH/DeltaT = 0.011-0.034 K-1 and DeltapO2/DeltaT = 1.85-7.17 hPa K-1 in the range from 277 to 308 K). Examples of pH/pO2 images obtained in natural marine sediment are presented.

No abstract