Chemiluminescence Immunoassays For Multiplex Analysis Based On Microfluidic Paper-based Analytical Devices

Chemiluminescence immunoassay is a combination of chemiluminescence and immunoassay.The reaction process is simple and does not require complicated operations,and it can directly and quantitatively analyze a variety of biomarker samples.Because of the advantages of wide linear range,fast detection speed,low background interference and high recovery rate,it has been accepted by many people and widely used in various fields.The microfluidic paper chip successfully integrates liquid injection,chemical reaction,signal detection and other operations together.Its advantages are low manufacturing cost,miniaturization,multiplexed simultaneous analysis,no pollution,and low rejection of biomarkers.In this paper,a new chemiluminescence immunoassay technique was developed and a multichannel chemiluminescence immunoassay device based on microfluidic paper chip was successfully applied to the quantitative detection of various myocardial infarction markers and cancer markers.The main research is as follows:A chemiluminescence immunoassay device based on time-resolved microfluidic paper-based device was prepared.The simultaneous determination of cardiac troponin I?c Tn I?,cardiac fatty acid binding protein?H-FABP?,and peptide?Copeptin?three myocardial infarct biomarkers was first achieved.A dual-signal amplification strategy was introduced including by employing primary antibody functionalized gold nanoparticles?Ab₁-GNPs?immobilized on the detection zone as amplified capture probes,and Co?II?catalyst,secondary antibody,luminol multifunctionalized gold nanoparticles?Co?II?-Ab₂-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 Ab₁-GNPs,antigen,and Co?II?-Ab₂-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.Finally,three AMI biomarkers including heart-type fatty acid-binding protein?H-FABP?,cardiac troponin I?c Tn I?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 from0.1 pg/m L to 1?g/m L,0.5 pg/m L to 1?g/m L and 1 pg/m L to 1 mg/m L with the detection limits down to 0.06 pg/m L,0.3 pg/m L and 0.4 pg/m L for H-FABP,c Tn I and copeptin detection,respectively.The developed?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.Chemiluminescence?CL?immunoassay detection device was proposed based on microfluidic paper-based analytical device??PAD?as detection platform and a smartphone as signal readout.Label-free multicolored CL immunoassays were innovatively developed for multiplex analysis of three cancer biomarkers including carcinoembryonic antigen?CEA?,?-fetoprotein?AFP?,and prostate specific antigen?PSA?as model analytes.To fabricate the CL immunoassays,firstly,mixed solution of luminol and fluorescein,luminol alone,or mixed solution of luminol and acriflavine,then specific capture antibody anti-CEA,anti-AFP or anti-PSA,were co-immoblized on three detection zone of a fabricated double-layered?PAD,respectivley.In the presence of corresponding antigens,immunocomplex could be formed on the paper sensing interface.Then,Co?II?-based zeolitic imidazolate frameworks ZIF-67 as novel CL catalyst were further added.Finally,H₂O₂ solution was automatically transported to the three detection zones sequentially through the double-layered?PAD to initiate the multicolored CL and CL resonance energy transfer?CRET?reactions in the detection zones.Temporal-spatial resolved CL images were captued by a smartphone through its camera with photographing detection mode or video detection mode.The formed immunocomplex on the sensing interface could restricted the transport and catalytic effect ZIF-67,leading to a decrease in CL signals in an antigen concentration-dependent model.Under the photographing detection mode,the CL immunoassays could detect CEA,AFP and PSA down to 2.1 pg/m L,0.4 pg/m L,3.9pg/m L,respectively.A self-compiled smartphone application?APP?was also developed for convenient and intelligent detection.The developed smartphone based temporal-spatial resolved CL imaging detection method exhibited ultra-wide detection ranges,good selectivity,promising portability and high sensitiviey,which offered a promising platform for early cancer diagnostics and point-of-care testings.