Microcolumn and coelution hydration of oil seal blood spot for efficient screening of newborn α-thalassemia via chip isoelectric focusing

The global prevalence of α-thalassemia necessitates effective newborn screening strategies due to its severe clinical consequences. Traditional methods such as liquid chromatography (LC), capillary electrophoresis (CE), and isoelectric focusing (IEF) face limitations, including low separation efficiency, poor sensitivity for detecting Hb Bart's, and time-intensive operations, particularly with dried blood spots (DBS). These limitations hinder timely and accurate screening. This study addresses the need for a more efficient, sensitive, and rapid method for detecting Hb Bart's in newborns. We enhanced IEF separation and sensitivity by designing a microfluidic IEF (mIEF) system with shortened columns and employing a coelution sample loading technique using oil-sealed blood spots for rapid sample pretreatment. Our experiments demonstrated significant improvements: the total analysis time was reduced from 1110 min (IPG IEF) and 46 min (LC) per batch to 36 min per batch. For individual samples, the focusing time decreased from 6 min (previous mIEF) to 3 min, with the microcolumn length shortened by 50 %, from 30 mm to 15 mm. The developed method showed excellent consistency with clinical Bart's detection and PCR diagnosis, achieving 100 % sensitivity and 98 % specificity for α-thalassemia screening. Our novel mIEF method provides an efficient, sensitive, and rapid tool for screening newborns for α-thalassemia. This advancement addresses the limitations of traditional techniques, improving early diagnosis and intervention strategies and ultimately enhancing health outcomes for at-risk newborns. [Display omitted] •Theory and design of shortened mIEF columns enhance Hb Bart's focusing efficiency and sensitivity.•Co-elution sample loading with oil-sealed blood spots enables rapid Hb Bart's analysis with minimal oxidation.•mIEF screening for neonatal α-thalassemia shows 100% sensitivity and 98% specificity, consistent with clinical PCR results.