Automated passive serial dilution microfluidic chip for calcium quantification based on the Arsenazo III method

Abstract

In this study, a valveless, resistance-based method was used to design and develop a passive microfluidic chip to implement an automated calcium assay in urine samples using the Arsenazo III method with a 3D printed mold. The resistive design of the channels provides a precise mixing in the assay at a ratio of 250 : 25 : 2 for water, reagents, and samples, respectively, with an added advantage of reduced process cost due to the 3D printed mold. The reaction between Arsenazo III and calcium in the urine results in a purple-colored complex that is quantified using a visible light source spectrophotometer, a portable fiber-optic spectrophotometer with a UV (ultraviolet) source and a smartphone camera capable of capturing RGB values. A linear range from 1.62–9.59 mg dL⁻¹ (limit of detection: 0.205 mg dL⁻¹) for calcium concentration in urine was obtained using a visible light spectrophotometer with R² = 0.99. Similarly, the linear range obtained for the portable spectrometer and smartphone camera was 1.62–6.50 mg dL⁻¹ with R² = 0.93 and R² = 0.97, respectively. The chip fabrication method and assay were found to be highly reproducible (σ < 0.001). The approach becomes highly portable with the use of a smartphone camera for quantification. The proposed microfluidic system can successfully quantify calcium in a relevant range in urine samples and is sensitive enough for an automated urinalysis system inside a smart toilet. Additionally, it should also be applicable to other assays by changing channel resistance according to the desired dilution or mixing ratio.