A label-free colorimetric 3D paper-based device for ochratoxin A detection using G-quadruplex/hemin DNAzyme with a smartphone readout

The colorimetric sensor usually depends on enzyme-mediated signal amplification to achieve trace analysis of ochratoxin A (OTA) residues in food samples. However, the enzyme labeling and manual addition of reagents steps increased assay time and operation complexity, restricting their application in point-of-care testing (POCT). Herein, we report a label-free colorimetric device integrating a 3D paper-based analytical device and a smartphone as handheld readout for rapid and sensitive detection of OTA. Using vertical-flow design, the paper-based analytical device enables the specific recognition of target and self-assembly of G-quadruplex (G4)/hemin DNAzyme to be performed, then employs DNAzyme for transducing the OTA binding event signal into a colorimetric signal. The design of independent functional units, including biorecognition unit, self-assembly unit and colorimetric units, which can address crowding and disorder of biosensing interfaces and improve the recognition efficiency of aptamer (apta). In addition, we eliminated signal losses and nonuniform coloring by introducing carboxymethyl chitosan (CMCS) to obtain perfectly focused signals on colorimetric unit. On the basis of parameter optimization, the device exhibited a detection range of 0.1-500 ng/mL and a detection limit of 41.9 pg/mL for OTA. Importantly, good results were obtained in spiked real samples, indicating applicability and reliability of developed device.