A novel planar optical sensor for simultaneous monitoring of oxygen, carbon dioxide, pH and temperature

The first quadruple luminescent sensor is presented which enables simultaneous detection of three chemical parameters and temperature. A multi-layer material is realized and combines two spectrally independent dually sensing systems. The first layer employs ethylcellulose containing the carbon dioxide sensing chemistry (fluorescent pH indicator 8-hydroxy-pyrene-1,3,6-trisulfonate (HPTS) and a lipophilic tetraalkylammonium base). The cross-linked polymeric beads stained with a phosphorescent iridium(III) complex are also dispersed in ethylcellulose and serve both for oxygen sensing and as a reference for HPTS. The second (pH/temperature) dually sensing system relies on the use of a pH-sensitive lipophilic seminaphthorhodafluor derivative and luminescent chromium(III)-activated yttrium aluminum borate particles (simultaneously acting as a temperature probe and as a reference for the pH indicator) which are embedded in polyurethane hydrogel layer. A silicone layer is used to spatially separate both dually sensing systems and to insure permeation selectivity for the CO2/O2 layer. The CO2/O2 and the pH/temperature layers are excitable with a blue and a red LED, respectively, and the emissions are isolated with help of optical filters. The measurements are performed at two modulation frequencies for each sensing system and the modified Dual Lifetime Referencing method is used to access the analytical information. The feasibility of the simultaneous four-parameter sensing is demonstrated. However, the practical applicability of the material may be compromised by its high complexity and by the performance of individual indicators

New fluorescent perylene bisimide indicators—a platform for broadband pH optodes

Asymmetric perylene bisimide (PBI) dyes are prepared and are shown to be suitable for the preparation of fluorescence chemosensors for pH. They carry one amino-functional substituent which introduces pH sensitivity via photoinduced electron transfer (PET) while the other one increases solubility. The luminescence quantum yields for the new indicators exceed 75% in the protonated form. The new indicators are non-covalently entrapped in polyurethane hydrogel D4 and poly(hydroxyalkylmethacrylates). Several PET functions including aliphatic and aromatic amino groups were successfully used to tune the dynamic range of the sensor. Because of their virtually identical spectral properties, various PBIs with selected PET functions can easily be integrated into a single sensor with enlarged dynamic range (over 4 pH units). PBIs with two different substitution patterns in the bay position are investigated and possess variable spectral properties. Compared with their tetrachloro analogues, tetra-tert-butyl-substituted PBIs yield more long-wave excitable sensors which feature excellent photostability. Cross-sensitivity to ionic strength was found to be negligible. The practical applicability of the sensors may be compromised by the long response times (especially in case of tetra-tert-butyl-substituted PBIs)

New fluorescent optical pH sensors with minimal effects of ionic strength

The thesis describes the development, characterization and application of fluorescence-based, optical pH sensors. Special attention is given to the dependence of the sensor signal and changes of ionic strength in the analyte solution. Based on three different methods for minimization of this dependence, various sensor membranes are presented in detail. Further, a new concept to reference fluorescence intensity signals is introduced and applied to an optical pH sensor. Chapter 1 emphasizes the necessity of precise pH control and measurements by means of examples. An overview of possible fields of pH sensors is given in general. In particular, three different formats of optical pH sensors are presented. Furthermore, the cross-sensitivity of the calibration curve of optical sensors towards ionic strength is mentioned. At the beginning of chapter 2, a short, review on the development of the term �pH� is given, followed by the explanation of the principle of optical pH sensors. The effect of ionic strength on the signal of optical pH sensor is explained by means of the law of Debye and Hückel and the definition of activity coefficients. A paragraph about the state of the art in optical pH sensor technology is followed by the second half of chapter 2, concerning the phenomena luminescence. Beside from basics, also methods for referencing and measurement techniques are explained. Chapter 3 describes the application and spectral properties of commercially available, pH-sensitive fluorescent dyes. HPTS, carboxyfluorescein and fluorescein were checked on their cross-sensitivity towards IS in the range from 25 to 500 mM. According to the theory of Debye and Hückel, the two-fold negative charged indicator fluorescein is less affected by IS than HPTS which carries four negative charges. A novel, partially positive charged indicator shows a contrary change of the dissociation constant. In an equimolar mixture with carboxyfluorescein, the effect of IS was distinctly reduced. In chapter 4, two methods are presented based on the principle described previously for the compensation of the effect of IS. The pH-indicator carboxyfluorescein was immobilized on partially amino-modified carboxycellulose. For the first method, the remaining carboxy groups were converted to positively charged groups. Again, mixing positively and negatively charged celluloses made an improvement of the cross-sensitivity towards IS in the range from 25 mM to 500 mM possible. For the second method, the negatively charged cellulose strand was partially loaded with negative charges. Six differently charged sensors were checked on their cross-sensitivity towards IS. One sensor shows a minimal cross-sensitivity towards IS and it shows the smallest zeta-potential, meaning a low charge density and a successful compensation of negative and positive charges. Chapter 5 deals with the third method for minimization the cross-sensitivity towards IS. Novel, fluorescein-based, lipophilic pH-indicators were embedded in an ion-permeable, charge-free polymer. The indicators were made lipophilic by esterification of the carboxy group with a C18 alkyl chain. This ester-modification reduces the number of charges to one and zero for basic and acid form, respectively. As a result of the charge reduction, the effect of IS is reduced to minimum and becomes negligible in the range from 25 to 500 mM. The fluorescent dyes differ in their substituents at 2�- and 7�-position of the xanthene structure. This variation of substituents results in dissociation constants between 5.5 and 8.5. Sensor properties like photo stability, temperature dependence and fluorescent lifetime were analyzed and discussed in detail. Finally, two applications using these sensor membranes were demonstrated. The chapter 6 deals with new amino-modified polymers. The polymers were embedded in hydrogel together with pH-inert reference particles. The fluorescence intensity of the sensors is converted into a phase shift by means of a novel referencing method (Dual Lifetime Referencing) using luminophores with different fluorescent decay times

pH Fluorosensors for use in marine systems

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-octadecylcarboxamidofluorescein (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

Fluorescent pH sensors with negligible sensitivity to ionic strength

Optical pH determination has the fundamental disadvantage of measuring a signal that depends on the ionic strength of the sample. The problem originates from the complex relationship between the proton activity and the concentration of the pH-sensitive dye. The effect of ionic strength on the signal depends on the charge of the indicator and its environment, e.g. the immobilisation matrix. We present novel lipophilic fluorescein esters carrying one negative charge. They are embedded in an uncharged, highly proton-permeable hydrogel to give optical pH sensors that show a negligible cross-sensitivity towards ionic strength. The fluorescent dyes differ in their substituents. This variation of substituents results in dissociation constants between 5.5 and 8.5. The indicators were made lipophilic by esterification of the carboxy group with a C18 alkyl chain. Since their spectral properties are quite similar, two indicators may be used in one sensor. This results in an optical pH sensor with a dynamic range that extends from pH 4.5 to 8

Set of fluorochromophores in the wavelength range from 450 to 700 nm and suitable for labeling proteins and amino-modified DNA

We describe the synthesis, purification, and spectral properties of new dyes and reactive labels. They absorb in the visible range between 450 and 700 nm and display analytically useful fluorescence. They were made amino-reactive by esterification with N-hydroxysuccinimide (NHS). The resulting oxysuccinimide (OSI) esters were covalently linked to the amino groups of human serum albumin (HSA) or certain DNA oligomers. Except for dyes 9 and 13, they contain one reactive group only in order to avoid cross linking of biomolecules. Labeling of amino-modified biomolecules was performed by standard protocols, and the labeled proteins and oligonucleotides were separated from the unreacted dye by gel chromatography using Sephadex G25 as the stationary phase in the case of proteins, and reversed-phase HPLC in the case of DNA oligomers. The dyes also have been used as donor–acceptor pairs in fluorescence energy transfer systems and in energy transfer cascades