Novel microfluidic device for factor VIII quantification by chemiluminescence in hemophilia A patients.

Point-of-care (PoC) testing can revolutionize diagnostics, enabling early detection and disease monitoring for chronic disorders. However, current PoC systems often lack the adaptability to detect multiple analytes. We present a capillary-based microfluidic detection platform with integrated optical sensors, that allows for an array of 16 simultaneous measurements using a chemiluminescent readout. The device consists of a disposable part containing microfluidics and a reusable part with integrated optical sensors. To demonstrate proof-of-principle, the current study specifically focuses on quantifying clotting Factor VIII (FVIII), a critical parameter in Hemophilia A management. Therefore, a chemiluminescent-based FVIII assay (FVIIIlum) was designed to quantify the activity levels in citrated plasma samples. All reagents necessary for the assay are contained in the disposable part of the device. To detect FIXa/FVIIIa tenase activity, a caged chemiluminescent substrate specific for activated Factor X (FXa) was synthesized for ultrasensitive real-time measurement of FVIII-mediated FXa generation. Experiments with the FVIIIlum, performed with the microfluidic cartridge with dried-in reagents, showed high level of precision. In addition, the FVIIIlum was capable to distinguish FVIII deficient plasma from plasma with only 0.017 IU/mL activity, demonstrating its applicability for Hemophilia A management. Finally, proof-of-principle was obtained by pharmacokinetic monitoring of FVIII activity in patient samples, demonstrating good agreement with the reference method. This study successfully presents a new miniaturized chemiluminescent platform, incorporating microfluidics and photon sensors, designed to detect multiple parameters in parallel per cartridge.