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Nanoliter-Fabricated Paper-Based Colorimetric Lateral Flow Strip for Urea Detection.

Supatinee Kongkaew^1,2,3, Suparat Cotchim^1,2,3, Warakorn Limbut^1,2,3,4

¹Division of Health and Applied Sciences, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand.

|October 28, 2025

Summary

A novel colorimetric lateral flow strip (Urea-CLFS) was developed for sensitive urea detection using nanoliter reagent volumes. Vertical enzyme alignment enhances performance, enabling accurate human diagnostics.

Keywords:

biosensor colorimetric lateral flow strip miniaturization urea

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Area of Science:

Biomedical Engineering
Analytical Chemistry
Biosensors

Background:

Urea detection is crucial for diagnosing various health conditions.
Existing methods may require complex instrumentation or large sample volumes.
Development of rapid, sensitive, and portable urea detection tools is needed.

Purpose of the Study:

To develop a nanoliter-scale colorimetric lateral flow strip for urea detection (Urea-CLFS).
To optimize the Urea-CLFS design and fabrication for enhanced sensitivity and accuracy.
To evaluate the performance of Urea-CLFS for potential human health diagnostics.

Main Methods:

Fabrication of a Urea-CLFS using nitrocellulose membrane (NC-Mb) for urease immobilization and chromatography paper (CH-PP) with phenol red indicator.
Investigation of urease enzyme alignment (horizontal vs. vertical) on NC-Mb.
Optimization of parameters including phenol red layers, concentrations, reaction time, and sample volume.
Colorimetric analysis of urea concentration based on enzyme-catalyzed color change.

Main Results:

Vertical alignment of urease on NC-Mb resulted in prolonged reaction time and higher product yield.
Optimized Urea-CLFS demonstrated a linear detection range of 0.25-8.0 mmol L^-1.
A limit of detection (LOD) of 0.34 mmol L^-1 was achieved, suitable for diagnostics.
Accurate urea quantification in human urine samples with recovery rates of 95-103%.

Conclusions:

The nanoliter-scale Urea-CLFS offers a sensitive and accurate method for urea detection.
The device's design, particularly vertical enzyme alignment, significantly improves performance.
Urea-CLFS shows promise as a portable and cost-effective tool for point-of-care diagnostics.