Paper Based Assay of Copper (II) Ion Using Egg White as a Natural Chromogenic Reagent

A green analytical paper based assay of Cu (II) has been developed using egg white protein as a natural reagent. Either crayon and pencil graphite, or laser engraver may be used to effectively create hydrophobic barriers and hydrophilic reaction zones on the filter paper substrate. The reaction is observed via the Biuret test's principle of Cu (II) ions reacting with nitrogen in the peptide bonds of the egg white under alkaline conditions to form a purple complex. Graphics editing software was used to determine the RGB values or grayscales of the reaction for the construction of a calibration graph. The volumes of reagents and samples were as low as 3 to 5 µL each. The limits of detection and limit of quantitation were determined to be 0.0002 M (0.04 µg Cu) and 0.0007 M (0.13 µg Cu), respectively. The main benefits of the proposed paper based assay compared to the use of a spectrophotometer are its ability to work with turbid reagents and samples and its portability. In addition, the color of egg white-Cu (II) complex is stable for a long period (at least two weeks) which allows flexible time for data analysis. This method has been shown to tolerate the presence of other ions, i.e., Al (III), Ca (II), Mg (II), and Zn (II) at concentrations at least 50 times of those of the analyte, and can tolerate the presence of Fe (II) up to the same concentration of Cu (II). The proposed paper based assay was demonstrated to successfully determine various Cu (II) salts in supplement samples.