Multiplexed chemiluminescence imaging assay of protein biomarkers using DNA microarray with proximity binding-induced hybridization chain reaction amplification.

A multiplexed chemiluminescence (CL) imaging assay was designed for sensitive screen detection of a panel of protein biomarkers by integrating DNA microarray with proximity binding-induced hybridization chain reaction (HCR) amplification. The DNA microarray was manufactured by printing hairpin DNAs for different analytes detection in an array format onto the sensing cells arranged on an aldehyde-functionalized glass chip. The existence of target proteins induced the formation of sandwich immunocomplexes along with the hybridization of DNA strands labeled on the pair of antibodies via proximity effect, which subsequently unfolded their corresponding spotting hairpin DNAs to expose prelocked primer and trigger HCR assembly. Through the streptavidin-biotin conjugation, numerous horseradish peroxidase (HRP) was coupled on the HCR assemblies to catalyze H2O2-luminol reaction and produce amplified CL signals for sensitive CL imaging assay of protein targets. As a proof of concept, the DNA microarray was used to CL imaging assay of carcinoembryonic antigen, α-fetoprotein and carcinoma antigen 125 in ranges of 0.05-500 ng mL-1, 0.06-600 ng mL-1 and 0.02-200 U mL-1, respectively. The CL microarray imaging assay could also be extended to screen detect other biomarker groups by printing different hairpin DNAs on the microarray and using corresponding antibody-DNA pairs. The proposed CL microarray imaging assay showed advantages of small sample consumption, good cost effectiveness, and multiplex detection performance, exhibiting good applicability in clinical diagnosis.