Monitoring cellulose oxidation for protein immobilization in paper-based low-cost biosensors

The oxidation of paper by periodate was investigated and systematically characterized by Fourier-transform infrared (FTIR) spectroscopy, scanning electron microscopy, X-ray diffraction, goniometry, and dynamic mechanical analysis. For the first time, in situ FTIR microscopy analysis was performed, yielding chemical images of carbonyl groups on the cellulose fibers. The enhancement of protein immobilization on oxidized paper was quantified by a colorimetric assay with Ponceau dye, demonstrating that 0.5-h oxidation suffices to functionalize the paper-based devices. The oxidized paper was applied as a sensor for protein quantification in urine, a test able to detect levels of proteinuria and even microalbuminuria. The quantification was based on the capture of proteins through covalent bonds formed with the carbonyl groups on the oxidized paper followed by the staining of the region with Ponceau dye. There is a linear dependency between human serum albumin (HSA) concentration and the length of the stained blot from 0.1 to 3 mg mL^-1. This method correlated linearly with a reference method showing a higher sensitivity (0.866 cm mL mg^-1) than the latter. The limit of quantification was 0.1 mg mL^-1, three times lower than that of the commercial strip. Graphical abstract Paper oxidation with periodate and extensive characterization, including microspectroscopy. The conversion of cellulose hydroxyl groups to aldehyde enhances covalent immobilization of protein on paper for application as analytical device. The oxidized paper determined protein in urine, suitable for proteinuria diagnosis.