Novel chemiluminescent imaging microtiter plates for high-throughput detection of multiple serum biomarkers related to Down's syndrome via soybean peroxidase as label enzyme.

Novel chemiluminescent (CL) imaging microtiter plates with high-throughput, low-cost, and simple operation for detection of four biomarkers related to Down's syndrome screening were developed and evaluated. To enhance the sensitivity of CL immunosensing, soybean peroxidase (SBP) was used instead of horseradish peroxide (HRP) as a label enzyme. The microtiter plates were fabricated by simultaneously immobilizing four capture monoclonal antibodies, anti-inhibin-A, anti-unconjugated oestriol (anti-uE3), anti-alpha-fetoprotein (anti-AFP), and beta anti-HCG (anti-β-HCG), on nitrocellulose (NC) membrane to form immunosensing microtiter wells. Under a sandwiched immunoassay, the CL signals on each sensing site of the microtiter plates were collected by a charge-coupled device (CCD), presenting an array-based chemiluminescence imaging method for detection of four target antigens in a well at the same time. The linear response to the analyte concentration ranged from 0.1 to 40 ng/mL for inhibin-A, 0.075 to 40 ng/mL for uE3, 0.2 to 400 ng/mL for AFP, and 0.4 to 220 ng/mL for β-HCG. The proposed microtiter plates possessed high-throughput, good stability, and acceptable accuracy for detection of four antigens in clinical serum samples and demonstrated potential for practical applicability of the proposed method to Down's syndrome screening. Graphical Abstract Schematic evaluation of the microtiter plater for simultaneous detection of the four biomarkers.