Development of Integrated Turbidity SpectrophotometricBiosensor System with Osmotic Fragility/DichlorophenolIndophenol Precipitation Assays for Thalassemia Red Blood CellScreening

Introduction: Thalassemia remains a major public health burden, particularly in low-resource regions where access to advanced laboratory diagnostics is limited. Although high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) provide reliable hemoglobin characterization, their cost, infrastructure requirements, and turnaround time restrict large-scale screening. From a practical screening perspective, there is a clear need for a rapid, portable, and objective point-of-care (POC) approach that supports early identification and appropriate referral. Methods: We developed an integrated turbidity spectrophotometric biosensor that quantitatively combines the osmotic fragility (OF) test and the dichlorophenolindophenol precipitation (DCIP) clear assay. The system employs a portable LEDbased spectrophotometric reader controlled by an ESP32 microcontroller, integrated with real-time web-based visualization and automated interpretation. Ninety-nine EDTA blood specimens were analyzed. Red blood cell fragility and hemoglobin stability were assessed using time-resolved absorbance slope measurements at 540 and 600 nm for OF and DCIP, respectively. Analytical reproducibility, diagnostic performance, and agreement with reference methods were evaluated Results: The biosensor demonstrated high analytical reproducibility, with mean coefficients of variation of 2.8% for OF slopes and 3.5% for DCIP slopes. Diagnostic performance was robust, yielding AUC values of 0.94 for OF and 0.90 for DCIP. An integrated decision algorithm achieved an overall concordance of 90.2% with goldstandard methods and provided preliminary interpretation within 3 minutes through a secure web-based dashboard. Discussion: Quantitative integration of OF and DCIP assays within a single platform improves screening objectivity while maintaining rapid turnaround, supporting decentralized and point-of-care testing. Conclusion: This integrated turbidity biosensor offers a rapid and scalable POC solution for preliminary thalassemia screening in underserved settings. Future development will focus on mobile integration, secure linkage with public health systems, and incorporation of additional hematologic or molecular parameters to further enhance diagnostic confidence