Multiplexed chemiluminescence determination of three acute myocardial infarction biomarkers based on microfluidic paper-based immunodevice dual amplified by multifunctionalized gold nanoparticles

Acute myocardial infarction (AMI) causes significant mortality and morbidity. The determination of multiple AMI biomarkers is very important for the timely diagnosis of AMI. In this work, simultaneous determination of three AMI biomarkers were achieved by virtue of a three-dimensional (3D) microfluidic paper-analytical device (mu PAD) with temporally resolved chemiluminescence (CL) emissions for the first time. A dual-signal amplification strategy was introduced including by employing primary antibody functionalized gold nanoparticles (Ab(1)-GNPs) immobilized on the detection zone as amplified capture probes, and Co(II) catalyst, secondary antibody, luminol multifunctionalized gold nanoparticles (Co(II)-Ab(2)-luminol-GNPs) with excellent CL activity as amplified signal probes. CL immunoreactions were performed at three detection zone of the fabricated 3D PAD by assembling Ab(1)-GNPs, antigen, and Co(8)-Ab(2)-luminol-GNPs to form sandwich-type immunocomplexes. Auto separated CL signals with temporal resolution were obtained by time delayed transport of H2O2 to different detection zones for multiplexed analysis. The CL signal obtained by using Co(II)-Abe-luminol-GNPs as signal probe (10576 a.u.) were about 20-fold higher than that by using conventional horseradish peroxidase labeled antibody modified luminol-GNPs as signal probe (531 a.u.). Finally, three AMI biomarkers including heart-type fatty acid-binding protein (H-FABP), cardiac troponin I (cTnI) and copeptin were quantitatively analyzed in one CL detection run by reading the CL intensity of the obtained three CL emission peaks. The detection range were ultra-wide ranged from 0.1 pg/mL to 1 mu g/mL, 0.5 pg/mL to 1 mu g/mL and 1 pg/mL to 1 mg/mL with the detection limits down to 0.06 pg/mL, 0.3 pg/mL and 0.4 pg/mL for H-FABP, cTnI and copeptin detection, respectively. The developed mu PAD based immunoassay performing multiplexed analysis ability, high sensitivity, ultra-wide dynamic range, favorable selectivity, accessible accuracy and reproducibility, have great application potential for the early diagnosis of AMI.