Tuning the p Ka of a pH Responsive Fluorophore and the Consequences for Calibration of Optical Sensors Based on a Single Fluorophore but Multiple Receptors.

Since Sørensen and Bjerrum defined the pH scale, we have relied on two methods for determining pH, the colorimetric or the electrochemical. For pH electrodes, calibration is easy as a linear response is observed in the interesting pH range from 1 to ∼12. For colorimetric sensors, the response follows the sigmoidal Bjerrum diagram of an acid-base equilibrium. Thus, calibration of colorimetric sensors is more complex. Here, seven pH responsive fluorescent dyes based on the same diazaoxatriangulenium (DAOTA) fluorophore linked to varying receptor groups were prepared. Photoinduced electron transfer (PeT) quenching from appended aniline or phenol receptors generated the pH response of the DAOTA dyes, and the position of the p Ka value of the dye was tuned using the Hammett relationship as a guideline. The fluorescence intensity of the dyes in a sol-gel matrix environment was measured as a function of pH in universal buffer, and it was found that the dyes behave as perfect pH responsive probes under these conditions. The response of optical pH sensors is nonlinear and was found to be limited to 2-3 pH units for a precision of 0.01 pH unit. As sensors with a broader sensitivity range can be achieved by mixing multiple dyes with different p Ka values, mixtures of dyes in solution were investigated, and a broad range pH sensor with a precision of 0.006 pH units over a range of 3.6 pH units was demonstrated. Further, approximating the sensor response as linear was considered, and a limiting precision for this approach was determined. As the responses of the pH responsive DAOTA dyes were found to be ideally sigmoidal and as the six dyes were shown to have p Ka values scattered over a range from ∼2 to ∼9, this allows for design of a broad range optical pH sensor in the pH range from 1 to 10. This hypothesis was tested using quaternary mixtures of the different DAOTA dyes, and these were found to behave as a direct sum of the individual components. Thus, while linear calibration is limited to a precision of 0.02 in a range of 2-3 pH units, calibration using ideal sigmoidal functions is possible in the range of 1-10 with a precision better than 0.01, and as good as 0.002 pH units.

Optical Chemical Sensor Using Intensity Ratiometric Fluorescence Signals for Fast and Reliable pH Determination.

Optical pH sensors enable noninvasive monitoring of pH, yet in pure sensing terms, the potentiometric method of measuring pH is still vastly superior. Here, we report a full spectrometer-based optical pH sensor system consisting of sensor chemistry, hardware, and software that for the first time is capable of challenging the performance of an electrode-based pH meter in specific applications such as biopharmaceutical process monitoring and in single-use bioproduction. A highly photostable triangulenium fluorophore emitting at 590 nm was immobilized in an organically modified silicon matrix that allows for fast time-response by rapid diffusion of water in and out of the resulting composite polymer deposited on a polycarbonate substrate. Fluctuations from the fiber optical sensor hardware have been reduced by including a highly photostable terrylene-based reference dye emitting at 660 nm, thus enabling intensity-based ratiometric readouts. The dyes were excited by 505 nm light from a light emitting diode. The sensor was operational within a pH range of 4.6-7.6, and was characterized and demonstrated to have properties that are comparable to those of commercial pH electrodes considering time-response ( t90 < 90 s), precision (0.03 pH-units), and drift.

Biocompatible Microporous Organically Modified Silicate Material with Rapid Internal Diffusion of Protons.

A new four-component organically modified silicate (ORMOSIL) material was developed with optical pH sensors in mind. Through a sol-gel process, the porosity of an ORMOSIL framework was optimized to allow rapid diffusion of protons, ideal for fast response to pH in an optical sensor. The optically transparent material was produced by catalyzing the dual polymerization of 3-(glycidoxy)propyltrimethoxysilane (GPTMS) and propyltriethoxysilane (PrTES) with boron trifluoride diethyl etherate. The performance of the resulting material in fluorescence based optical pH sensors was evaluated by incorporation of active dye components in the inorganic polymer framework. It is demonstrated that the material has a short response time ( t90 < 30 s) and high stability in medium and during storage, and resulting sensor spots are biocompatible. It is concluded that this ORMOSIL material has excellent properties for optical pH sensors.