Development of a paper strip test for colorimetric detection of urea in raw materials for animal feed

Aiming at developing an easily implementable method for on-site analysis to detect urea adulteration in feed ingredients, a simple and inexpensive paper strip for urea detection via colorimetric assay is herein presented. The paper strip can be simply fabricated by immobilizing urease with bromothymol blue (BTB) as a pH indicator on cellulose fiber. Upon dipping the paper strip into the target sample, the release of ammonia during the reaction between urea in the sample and urease on the paper strip causes a pH change that results in the development of a blue color, thus indicating the presence of urea. A semiquantitative detection method was developed on the basis of the color change on the paper strip, which can be detected by naked eyes and compared with a color chart made by spiking urea at concentrations varying from 0.10% to 1.0% (w/w) in animal protein and fishmeal samples. Moreover, quantitative data were obtained by taking a picture with a smartphone camera and measuring the color intensity using ImageJ software. A comparison between BTB and phenol red as pH indicators revealed that the former afforded better results in terms of resolution. Under optimal conditions, good linear responses of blue intensity were obtained in a concentration range of 0.10%-1.0% (w/w). The recovery was determined to range between 98.1% and 118.3% with a relative standard deviation of <5%. The developed paper strip assay was applied to determine urea in animal protein and fishmeal, finding good agreement with the official AOAC method (No. 967.07). The present paper strip is rapid and requires neither sophisticated devices nor skilled personnel, allowing its use by quality controllers for the routine on-site detection of the urea adulteration of raw materials.