Three-dimensional microfluidic paper-based analytical device fabricated by cutting technique for highly sensitive and specific colorimetric detection of triglycerides in whole blood utilizing a mixed indicator system

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In this study, we developed a three-dimensional microfluidic paper-based analytical device (3D-µPAD) for quantifying triglycerides (TGs) in whole blood samples. This method provides an alternative to conventional TGs detection and offers several advantages. The 3D-µPAD was fabricated using an in-house computer-controlled X-Y knife plotter, eliminating the need for hydrophobic reagents. This approach results in a more rapid, simple, highly reproducible, and cost-effective mass production process. To separate plasma from a drop of whole blood, the LF1 membrane was incorporated as a sample zone on the 3D-µPAD. The hydrolysis of TGs was catalyzed by lipase, producing H+ ions detected by a phenol red indicator read against cyan ink to reduce background interference. A spectrophotometer (i1 Pro 3 mini) measured the green color intensity in the 3D-µPAD’s detection zone, which was proportional to the TGs concentration and quantified within 10 min. Results were consistent with clinical methods, with a detection limit of 0.182 mmol L−1 in the 0.1–6 mmol L 0207B1 TGs concentration range. Using mixed indicators successfully reduced background interference and enhanced sensitivity for TGs determination. The 3D-µPAD demonstrated a storage stability of approximately 30 days. Our 3D-µPAD prototype offers a rapid, cost-effective, and user-friendly approach for pre-clinical TGs diagnostics, expanding the potential for point of care whole blood sample analysis.