Paper-based miniaturized immunosensor for naked eye ALP detection based on digital image colorimetry integrated with smartphone.

Alkaline phosphatase (ALP) is a metalloprotein found naturally in raw milk samples and is considered as an important biomarker in quality control of milk. Its easy, personalized, as well as instrument-less detection is important to ensure the pasteurization and its differentiation form raw milk. In view of such importance of APL, we have developed an office punching machine crafted paper biosensor for naked eye detection of ALP in milk samples. The quantitative estimation is done by digital image colorimetry (DIC) based technique integrated with smartphone. The sensor-probe was developed by the covalent immobilization of ALP antibody (anti-ALP) onto the functionalized paper surface. The fabrication of the biosensing probe was characterized using DIC, Fourier transform infrared spectroscopy (FTIR), and atomic force microscopy (AFM). The detection was based on immunocomplexation between the sensor-probe and ALP, which generates blue-green precipitate as an analytical signal by exploiting the catalytic activity of ALP towards 5-bromo-4-chloro 3-indolyl phosphate (BCIP). The dose dependent appearance of the blue-green complex was captured using smartphone camera and DIC was employed using Red, Green, and Blue (RGB) profiling system, where the maximum sensitivity was obtained for the red color channel. Based on the DIC analysis, a wide dynamic range for the ALP detection is obtained from 10 to 1000 U/mL with the detection limit of 0.87 (± 0.07) U/mL. The designed paper-based biosensor is successfully applied to detect ALP in commercial and raw milk samples. Interferences due to components present in the milk samples was evaluated and the long-term stability of the designed biosensor was examined. Based on the detection principle, a miniaturized kit [20.0 mm (L) × 20.0 mm (W) × 2.15 mm (H)] was developed and applied for the ALP detection to demonstrate the instrument-free direct in-kitchen applicability.