Microfluidic chip electrophoresis for simultaneous fluorometric aptasensing of alpha-fetoprotein, carbohydrate antigen 125 and carcinoembryonic antigen by applying a catalytic hairpin assembly

An aptamer based assay is presented that is making use of a catalytic hybrid assembly and a microfluidic chip electrophoresis format. It enables simultaneous determination of the biomarkers (BMs) α-fetoprotein (AFP), carbohydrate antigen 125 (CA125), and carcinoembryonic antigen (CEA). The respective aptamers were covalently bound to Fe₃O₄@AuNPs (AuMPs) magnetic beads and then used to capture the biomarkers on their surface. Different single-stranded DNA primers were then labeled with various antibodies as encoding and signaling tags. The signal tags reacted with AuMPs-BMs to form different antibody-BM-aptamer complexes. After magnetic separation, three pairs of hairpins as substrates were introduced to trigger catalytic hybrid assembly by the primers in the complex. This will form many duplex DNA products of different length in the supernatant. The products can be magnetically separated by microfluidic chip electrophoresis and determined by fluorometry at excitation/emission wavelengths of 495/525 nm. Several experimental conditions including the hairpin concentration, reaction time and temperature were systemically optimized. The method can simultaneously quantify AFP, CEA and CA125, respectively, with detection limits of 0.1, 0.2, 0.15 pg mL⁻¹ (at S/N = 3). The aptamer functionalized magnetic beads can be reused for at least 20 times with a recovery of up to 80% after heat treatment. The method was employed to simultaneously detect the three BMs in serum samples.

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