CARBONIC ANHYDRASE-BASED PAPER-BASED COLORIMETRIC DEVICE FOR ACETAZOLAMIDE DETERMINATION IN BIOLOGICAL SAMPLES

Acetazolamide (ACZ) is a drug commonly used to treat edema from heart failure, epilepsy, and glaucoma; moreover, ACZ, due to its diuretic activity, is also employed as a masking agent for doping. Several methods for analyzing ACZ have been reported in the literature, but there remains a lack of smart analytical devices capable of evaluating ACZ in biological samples with intra-sulphonamide selectivity. In this presentation, a carbonic anhydrase-based, paper-based colorimetric device (cPAD) is presented for the direct determination of ACZ in urine and serum. The cPAD, which operates via ACZ-driven inhibition of carbonic anhydrase, was fully designed and fabricated in-house using benchtop instruments. The cPAD resulted in an origami setup composed of the ‘sampling zone’ devoted to sample pretreatment via paper filtration and the ‘colorimetric zone’ housing the enzyme and the corresponding colorimetric reaction. The colorimetric signal relies on the carbonic anhydrase-catalyzed conversion of 4-nitrophenyl acetate (4-NA) to p-nitrophenol, resulting in a yellow color on paper. The sensing principle is based on ACZ inhibition of enzyme activity in a concentration-dependent manner, resulting in a proportional color ‘switch-off’. The paper-device operating strategy consists of: (i) sample loading onto the dedicated zone, (ii) overlaying with the ‘colorimetric zone’, and (iii) the 4-NA loading followed by a 10-min incubation. Eventually, the colorimetric signal was collected using a smartphone. The cPAD paper components for sample collection and enzymatic-assay hosting were carefully optimized with respect to paper type and operating geometries. The enzymatic reaction was carefully optimized to maximize assay sensitivity and the selectivity toward other sulphonamides. The proposed cPAD allowed the determination of ACZ in both urine and serum using matrix-matched calibration in the range of 0.05-0.5 ppm for urine (R2=0.994; LOD = 0.011 ppm) and 0.1-0.8 ppm for serum (R2=0.993; LOD = 0.024 ppm), proving for both matrices good reproducibility (RSD ≤ 13%, n=3). Eventually, the cPAD exploitability was successfully demonstrated for ACZ determination in urine and serum samples, yielding satisfactory recoveries (86-121%; RSD ≤ 14%, n=3). Summing up, the proposed cPAD enables smartphone-based ACZ determination at clinically relevant concentrations within 20 minutes, requiring only 10 μL of sample, representing a step toward the sustainable expansion of point-of-care devices.