A prospective method for non-invasive diagnosis of iron deficiency anemia using microfluidic paper-based analytical devices and a natural reagent from red rose (Rosa damascena) flower extract

A non-invasive, low-cost, non-toxic, and user-friendly microfluidic paper-based analytical device (μPAD) was developed for Fe(II) determination in artificial urine samples. The determination method is based on the color change of anthocyanin from Rosa damascena flower extract (RDFE) through the formation of an Fe(II)-anthocyanin complex. The image of the complex was captured using a smartphone camera and analyzed for color intensity using ImageJ. The “mickey mouse” hydrophobic pattern on the μPAD was created by printing a pattern consisting of one circular sample zone, two circular detection zones of the same size (7.5 mm i.d.), and two channel zones with dimensions of 3.3 × 2.5 mm. The optimal conditions for the μPAD were 1.2 μL of extract (8% v/v), 28 μL of the sample, and a reaction time of 25 minutes. The proposed μPAD method offers a linear dynamic range of 0–4 mg L⁻¹, with LOD and LOQ values of 0.075 and 0.25 mg L⁻¹, respectively. The validation of the method using recovery tests yielded results ranging from 93% to 106%, which are considered accurate. This method successfully determined Fe(II) ions in artificial urine samples, with Fe(II) levels mimicking those of patients with iron deficiency anemia, and it is highly applicable to other sample types.