Accuracy improvement via novel ratiometry design in distance-based microfluidic paper based analytical device: instrument-free point of care testing

Developing accurate, precise, instrument-free, easily prepared, and point-of-need microfluidic paper-based devices is highly significant in clinical diagnosis and biomedical analysis. In the present work, a ratiometric distance-based microfluidic paper-based analytical device (R-DB-µPAD), along with a three-dimensional (3D) multifunctional connector (spacer), was designed to improve the accuracy and detection resolution analyses. Specifically, the novel R-DB-µPAD was used for the accurate and precise detection of the ascorbic acid (AA) as a model analyte. In this design, two channels were fabricated as detection zones, with a 3D spacer located between the sampling and detection zones to improve the detection resolution by preventing the reagents mixing from overspreading between these zones. Two probes for AA were used: Fe3+ and 1,10-phenanthroline were deposited in the first channel, and oxidized 3,3ʹ,5,5ʹ-tetramethylbenzidine (oxTMB) was added to the second channel. Accuracy improvement of this ratiometry-based design was achieved by enhancing the linearity range and reduce the volume dependency of the output signal. Moreover, the 3D connector improved the detection resolution by eliminating systematic errors. Under optimal conditions, the ratio of the distances of the color bands in the two channels was used to construct an analytical calibration curve in the range from 0.05 to 1.2 mM, with a limit of detection of 16 µM. The proposed R-DB-µPAD combined with the connector was successfully used for the detection of AA in orange juice and vitamin C tablets with satisfactory accuracy and precision. This work opens the gates for multiplex analysis of various analytes in different matrices.