Development of Alpha-thalassemia 1 Southeast Asia deletion detection by multiplex polymerase chain reaction coupled with electrochemical impedance spectroscopy biosensor

Alpha(α)-thalassemia 1 Southeast Asia (SEA) deletion is a common inherited blood disorders commonly found in Southeast Asian. Many countries define the α-thalassemia 1 diagnosis in prenatal program to control severe thalassemia. Polymerase Chain Reaction (PCR) based method is used as a definitive diagnosis. However, the post PCR process required gel electrophoresis which time-consuming, laborious, and hazardous DNA staining. Electrochemical Impedance Spectroscopy (EIS) biosensor is highly sensitive, label-free, and real-time analysis for DNA detection. Therefore, the α-thalassemia 1 EIS biosensor was developed to detect α-thalassemia 1 (SEA deletion) based on multiplex PCR (mPCR) and EIS biosensor coupled with DNA binding dye (Hoechst-33258). The optimal condition and diagnostic characteristics of α-thalassemia 1 EIS biosensor were determined. Under the optimal condition, a criterion cut-off value of the negative imaginary impedance (-Z”) at 26.84 kΩ and charge transfer resistance (Rct) at 48.83 kΩ was established for distinguishing between normal α-globin individuals and α-thalassemia 1 (SEA deletion) carriers. The reliability analysis demonstrated a strong correlation with Pearson’s correlation coefficient of 0.88. Moreover, Bland-Altman plot analysis showed mean difference with 55.8 kΩ. The diagnostic characteristics of the developed α-thalassemia 1 EIS biosensor using 81 clinical blood samples represented 100% sensitivity, 82.4% specificity, 76.9% positive predictive value (PPV), 100% negative predictive value (NPV), and 88.9% accuracy for -Z" measurement. For Rct measurement, the results represented 100% sensitivity, 88.2% specificity, 83.3% PPV, 100% NPV, and 92.6% accuracy. The developed α-thalassemia 1 EIS biosensor showed significant potential as a valuable tool for detection of α-thalassemia 1 (SEA deletion) carrier in risk couples, which can aid in preventing Hb Bart's hydrops fetalis, the most severe form of thalassemia.