8-Hydroxypyrene-1,3,6-trisulfonate

The first intravascular sensor for simultaneous and continuous monitoring of the pH, pC>2, and pCC>2 was developed by CDI-3M Health Care (Tustin CA)14 based on a system designed and tested by Gehrich et al.15. Three optical fibres (core diameter = 125 pm) are encapsulated in a polymer enclosure, along with a thermocouple embedded for temperature monitoring (Figure 3). pH measurement is carried out by means of a fluorophore, hydroxypyrene trisulfonic acid (HTPS), covalently bonded to a matrix of cellulose, attached to the fibre tip. Both the acidic ( eXc=410 nm) and alkaline ( exc=460 nm) excitation bands of the fluorophore are used, since their emission bands are centred on the same wavelength (/-cm 520 nm). The ratio of the fluorescence intensity for the two excitations is measured, to render the sensor relatively insensitive to fluctuations of optical intensity. 

Membrane-covered optochemical sensors (optodes) with O2 sensitive or pH sensitive fluorescence indicators (e.g. pyrene butyric acid or hydroxypyrene trisulfonic acid) have been coupled with different enzyme reactions, such as the conversion of glucose, lactate, ethanol, or xanthine, and with antigen-antibody couples (Opitz and Lubbers, 1987). 

The effect of salt on the rate of proton dissociation from excited hydroxypyrene trisulfonate is demonstrated in Figure 6. The effect on the steady-state fluorescence is similar to that shown in Figure 3. The emission of the neutral form is intensified while that of the anion decreases. 

Figure 7. The variation of the rate of proton dissociation from excited hydroxypyrene trisulfonate on the molar concentration of the salt (O, ) time-resolved fluorescence measurements ( , ) steady-state fluorescence measurements (A) proton diffusion coefficient, normalized for pure water (data from Glietenberg et al. 1968). Open symbols, MgCl2 closed symbols, LiC104.

Figure 10. The reduction of water activity by high concentration of NaCl. Water activity was measured by the rate of proton dissociation from excited hydroxypyrene trisulfonate (A), excited 2-naphthol-6-suIfonate ( ), or using the published vapor pressure ( ) (Grollman, 1928 Kracek, 1928).

Figure 11. Correlation between water activity coefficient of MgCl2 and NaC104 solutions as estimated from the rate of proton dissociation from two proton emitters, 2-naphthol-6-sulfonate (ordinate) and hydroxypyrene trisulfonate (abcissa). ( ) MgCl2 ( ) NaC104.

Figure 12 (line A) depicts the emission spectrum of hydroxy pyrene trisulfonate dissolved in diluted buffer (pH 5.0). At this pH, the ground state is fully protonated (pK0 = 7.7), but not so the first excited singlet state (pK = 0.5). The excited molecules dissociate and 95% of the emission is at the wavelength of the excited anion (515 nm). The dissociation can be prevented if the compound is dissolved in acid solution, pH < pK., such as 2MHC1 (line B). Under such conditions, we observe the emission of the neutral form with maximum at 445 nm. Upon ligation to apomyoglobin, the fluorescence of hydroxypyrene trisulfonate consists of two... 

Figure 12. Steady-state fluorescence emission of hydroxypyrene trisulfonate. Fluorescence of 20pAf hydroxypyrene trisulfonate. (A) at pH 5.0, (B) in 2M HC1, and (C) in 30pAf apomyoglobin pH 5.0. Excitation at 400 nm. Fluorescence measured in arbitrary units at identical instrumental set up.

Figure 13. Fluorescence emission spectra of free and protein-bound hydroxypyrene trisulfonate. 1.16(iM hydroxypyrene trisulfonate in water (A) or 1 % bovine serum albumin (B) adjusted to pH 6.0. The samples were excited at 400 nm and the emission spectra were measured under identical instrumental set up.

Figure 14. Fluorescence decay time of hydroxypyrene trisulfonate bound to apomyoglobin (A) 60pAf apomyoglobin, 50pAf hydroxypyrene trisulfonate in lOmAf Mes buffer pH 5.0. The emission was measured (in arbitrary units) at a streak speed of 15 mm/nsec at the wavelengths 400—450 nm using BG-3 (3 mm) and GG 400 Schott glass filters. (B) The excitation laser pulse measured under identical conditions as seen by reflection from the front and back surfaces of an empty 0.5-cm cuvette.

Figure 16. The lifetime of the excited anion of hydroxypyrene trisulfonate bound to apomyoglobin. The sample was excited by a nitrogen laser (337 nm, 1 nsec full width at half maximum) and the emission of the anionic form was monitored (in arbitrary units) through a KV 550 filter by a photomultiplier attached to a Tektronix 7912 AD transient digitizer equipped with a 7A19 vertical amplifier.

Figure 18. Kinetic analysis of the fluorescence decay of neutral hydroxypyrene trisulfonate bound to apomyoglobin (A) or bovine serum albumin (B). The results are taken from experiments carried out as detailed in Figures 15 and 17, using various streak speeds. The values of y-i, y2 and AR are listed in Table II.

These results can be subjected to more rigorous analysis. The rate constant of protonation of the excited anion of hydroxypyrene trisulfonate is k = 5 x 10loM-1 sec-1 (Weller, 1958). Thus, the effective concentration of H+ in the reaction sphere (R) is... 

Despite this convenience, such approximation is seldom permitted by the initial conditions of the experiment. Suppose that a dilute neutral solution (lOOpM, pH = 7) of hydroxypyrene trisulfonate (pK = 7.7) is pulsed... 

Figure 26. The effect of initial pH on the macroscopic parameters characterizing the Bromo Cresol Green-hydroxypyrene trisulfonate system. The macroscopic parameters were calculated for the experimental conditions described in Figure 23 and the rate constant listed in Table IV. X0 = 4.25(jlM. The experimental values are drawn with their error bars. (A) y, vs. pH (B) y2 vs. pH (C) Tmax vs. pH.

Figure 46. The effect of buffer on the rate of 0 relaxation after a laser pulse. lOOpAf hydroxypyrene trisulfonate, 0.1- lOmAf of imidazol. The pH of the experiment was varied between 6.5 to 8.5. Intercept = 1.8 x 109 M-1 sec-1 slope = 7.7 x 10s M-1 sec-1. The reaction was monitored at 445 nm using CW HeCd laser as a probing light. 

Figure 47. The effect of pH on the rate of < >0- reprotonation in an emitter—buffer system. lOOpJVf hydroxypyrene trisulfonate, 2mM imidazol buffer. The reaction was followed at 440 nm using CW dye laser. The continuous line is computed for the emitter-buffer system using the rate constants ki = 1.6 x 10u M sec-1 A5 = 3.5 x 1010 Af-1 sec-1 io = 2.1 x 109 AT-1 sec-1. Measurements in the absence of buffer ( ) or in the presence of 2mM imidazol ( ).

The pH-sensitive chemistry consists of a cellulosic material to which hydroxypyrene trisulfonate (HPTS) is covalently bonded. The C02-sensitive material is a fine emulsion of a hydrogen-carbonate buffer (plus HPTS) in a two-component silicone. The oxygen-sensitive chemistry is simply a solution of chemically modified decacyclene (which is strongly quenched by oxygen) in a one-component silicone. To make it insensitive toward halothane (an inhalation narcotic), it is covered with a thin layer of black PTFE, which also serves as an optical isolation. The fluorescence intensities of the three sensing spots can be related to PO2, pH, and PCO2 via modified Stem-Volmer or Henderson-Hasselbalch algorithms. 

Following the initial feasibility studies of Lubbers and Opitz, Cardiovascular Devices (GDI, USA) developed a GasStat extracorporeal system suitable for continuous online monitoring of blood gases ex vivo during cardiopulmonary bypass operations. The system consists of a disposable plastic sensor connected inline with a blood loop through a fiber optic cable. Permeable membranes separate the flowing blood from the system chemistry. The C02-sensitive indicator consists of a fine emulsion of a bicarbonate buffer in a two-component silicone. The pH-sensitive indicator is a cellulose material to which hydroxypyrene trisulfonate (HPTS) is bonded covalently. The 02-sensitive chemistry is... 

The first studies of the photoprotolytic dissociation of 1-naphthol, 2-naphthol and 1-aminopyrene cation in micellar solutions were made by Klein and Hauser [116] and also by Weller and SeUnger [117,118] who have shown that the photoprotolytic dissociation slows down in anionic micelles. Fendler [119] observed the photoprotolytic dissociation of 1-hydroxypyrene-trisulfonate in CTAB micelles. The inhibition of dissociation was explained in terms of the relatively low polarity and high microviscosity of the micellar phase [116-119] and also the effect of the negative micellar potential. 

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