Enhancing performance in triglyceride detection: fabrication of microfluidic paper-based analytical devices (μPADs) using cutting methods and a mixed indicator for improved colorimetric analysis of whole blood samples

This thesis presents the enhancement of triglyceride detection using mixed indicators for improved colorimetric analysis in whole blood samples on a three-dimensional microfluidic paper-based analytical device (3D-μPAD). Triglycerides(TG) are the primary transporters of dietary fats throughout the bloodstream. However, the traditional detection methods are time-consuming, require expertise, and involve complicated steps requiring many enzymes and high-cost instruments. This research successfully developed a 3D-μPAD for quantitative colorimetric analysis of TG in whole blood samples based on the pH change resulting from the hydrolysis of TG catalyzed by only lipase enzyme to generate an H+ product. Phenol red, as an indicator combined with cyan ink, was used to adjust the background color and reduce background interference. However, the main limitations are the low solvent resistivity and the use of hydrophobic reagents of the traditional μPAD. Therefore, an in-house computer-controlled X-Y knife plotter was used to fabricate the non-hydrophobic reagent 3D-μPAD, resulting in rapidity, high reproducibility, simplicity, and low cost.
For TG detection, we performed colorimetric reactions on the 3D-μPAD by applying a single drop of whole blood on the LF1 membrane attached to the sample support without sample preparation. The LF1 membrane separates plasma from whole blood and yields plasma for further enzymatic reaction. After the enzymatic reaction, the green color product corresponding to the TG concentrations in the sample was easily observed and detected using an il Pro mini spectrophotometer directly on the 3D-μPAD in approximately 10 minutes. The results agreed with the hospital method, with a detection limit (LOD) of 0.182 mM (16.12 mg dL-1). The 3D-μPAD was developed as a prototype that enables rapid, low-cost, simple fabrication and short analysis time and is easy to apply for pre-clinical and point-of-care diagnostic testing. It could contribute to expanding the applicability of well-known markers in diagnosis that deal with a whole blood sample and can be used in areas with limited resources and lacking specialized personnel and tools.