A high-precision ratiometric fluorosensor for pH: implementing time-dependent non-linear calibration protocols for drift compensation

We present a versatile time-dependent non-linear calibration protocol for optical sensors, implemented on the pH sensitive ratiometric fluorophore 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) immobilized in ethyl-cellulose. The calibration protocol individually compensated for the progressive drift of calibration parameters, whereby sensor precision and accuracy, as well as applicable lifetime were improved. A severely reduced photoacidity was observed for the immobilized fluorophore, for which excited state dynamics was characterized and benefited from during measurements. Due to the significantly reduced photoacidity of HPTS immobilized in the ethyl-cellulose sensing membrane, a dual excitation/dual emission (F₁, ex/em: 405/440 nm and F₂, ex/em: 465/510 nm) ratiometric (RF₁,F₂ = F₁/F₂) sensing scheme could be used to amplify sensor response. The signal to noise (S/N) ratio was enhanced by ∼400% utilizing the dual excitation/dual emission ratiometric sensing scheme, rather than the more commonly used protocol of dual excitation/single emission for HPTS fluorescence. Apparent pK_a of the fluorophore ranged from 6.74 to 8.50, mainly determined by the immobilization procedure. The repeatability (IUPAC, pooled standard deviation) over three pH values (6.986, 7.702 and 7.828) was 0.0044 pH units for the optical sensor, compared to 0.0046 for the electrode used for standardization. Sensor analytical characteristics were thereby in principle limited by the performance of the standardization procedure.