A portable microfluidic chip-based fluorescent and colorimetric aptasensor combining recombinase polymerase amplification for bovine pregnancy-associated glycoproteins detection.

A portable dual-mode PDMS-based microfluidic chip aptasensor was developed to detect bovine pregnancy-associated glycoproteins (bPAG) in bovine milk. Reagents within the chip chambers underwent reactions driven by gravity, where pre-encoded rich C sequences on the complementary strand of the aptamer facilitated the generation of abundant G-quadruplexes via subsequent RPA reaction, which activated the chromogenic substrates and fluorogenic precursors in the chip, producing distinct colorimetric and fluorescent signals. These signals were captured by our developed smartphone application and converted into RGB values, further enabling the quantification of bPAG with detection limits of 0.079 ng/mL and 0.024 ng/mL for colorimetric and fluorescent modes, respectively, over a linear range of 0.1-100 ng/mL. Bovine milk and other animal source milk were evaluated in the proposed assay, accurate identification results were obtained, indicating significant potential in bovine milk monitoring. The work further provided a valuable reference for point-of-care testing of non-nucleic acid targets in food samples.