Instant and low-cost detection of urinary microalbumin using smartphone technology and a paper-based platform with a colorimetric ratio analysis

Abstract

Early detection of microalbuminuria is essential for preventing the progression of nephropathy and improving patient management. The present research proposes a simple, low-cost dye-binding method based on a standard addition principle for determining urinary albumin at clinically significant levels. The assay principle of the proposed paper-based analytical device is based on utilizing a specific and sensitive dye, bis(3',3″-diiodo-4',4″-dihydroxy-5',5″-dinitrophenyl) -3,4,5,6-tetrabromosulfonphthalein (DIDNTB), which binds to urinary albumin under extreme low pH conditions. The strategy was based on testing with three different albumin concentrations in the urine samples to obtain different color intensity ratios denoted as R1, R2, and R3, and further used to calculate a semi-quantitative amount of albumin. Under the optimized conditions, the linear range from 10 to 200 mg/L albumin and the detection limit 8.2 mg/L (R² = 0.99) was obtained. Interference studies revealed that glucose, ascorbic acid, uric acid, L-histidine, and globulin were in the acceptable recovery range (89-109%). Reproducibility assessments showed that the coefficients of variation (CV) for this method ranged between 3.7% and 7.7%. Additionally, receiver operating characteristic (ROC) plots were generated to establish the cut-off value for instant semi-quantitative interpretation of the results. Results from real sample analysis revealed that the proposed method is comparable to a widely used quantitative immunoturbidity method (r = 0.94, n = 50).

Keywords: Colorimetry; Microalbuminuria; Paper-based analytical device; RGB detector; Smartphone.

Fig. 1

Fabrication process of the paper platform (A) and the ready-to-use platform of paper-based colorimetric ratio analysis. Created in BioRender. Laiwattanapaisal, W. (2025) https://BioRender.com/wcv3zrd.

Fig. 2

Schematic illustration of the urine microalbumin screening procedure (A); the interface of the RGB Color Detector App on the Android mobile operating system, and instructions for use of the functions involved in the screening of urine microalbumin (B); the interpretation of test results ratios (R1, R2, and R3) along with the in-house spin wheel MAU reader (C); and the image of the in-house spin wheel MAU reader (D). Created in BioRender. Laiwattanapaisal, W. (2025) https://BioRender.com/nasw24z.

Fig. 3

Albumin concentration-dependence with the mean brightness intensity, R/(R + G + B), on Whatman No. 3 paper substrate (A) and albumin concentration-dependence with the mean brightness intensity of corrected R/(R + G + B) on Whatman No. 3 paper substrate (B).

Fig. 4

The calibration curve of ratio 1, ratio 2, and ratio 3 for the determination of microalbumin with paper-based colorimetric ratio analysis in the range of 10–100 mg/L. The Y-axis represents the signal ratio of [sample − blank] to [spiked-sample − blank] wherein the sample is an albumin standard solution mimicking several concentrations of albumin in urine.

Fig. 5

Passing–Bablok regression analysis for the comparison of results between paper-based colorimetric ratio analysis and immunoturbidity in determining concentrations of urine microalbumin.

Fig. 6

A Bland–Altman bias plot comparing the results obtained using paper-based colorimetric ratio analysis and immunoturbidity for the determination of urine microalbumin concentrations.