Three-Minute-Long Chemiluminescent Immunoassay Using Dually Accelerated Immunoreaction by Infrared Heating and Passive Mixing

Abstract

A rapid chemiluminescent (CL) immunoassay method was developed by integrating a newly designed infrared-radiation technique with a pressure-driven fluidic system. The fluidic system combined a three-dimensional helical glass tube for rapid mixing of immunoreagents with two spiral glass tubes for magnetic separation and CL detection, respectively. The mixture passively formed in the helical glass tube could be quickly heated and kept at about 37 degrees C by the infrared radiation. The immunoreaction could be finished within 90 s due to the dual acceleration by the improved mass transport and enhanced reaction kinetics. The horseradish peroxidase-labeled sandwich immunocomplex formed on paramagnetic particles was then separated by a permanent magnet and mixed with CL substrate in a long spiral tube, and the detection mixture was immediately injected through another spiral tube in front of the photomultiplier for collecting the CL signal. Using alpha-fetoprotein as a proof-of-principle analyte, the immunoassay could be completed within 3 min with a linear calibration range of 0.2-90 microg/L. This programmable method showed acceptable detection and fabrication reproducibility and good accuracy, indicating a promise of automated high-throughput clinical application.