Adhered-3D paper microfluidic analytical device based on oxidase-mimicking activity of Co-doped carbon dots nanozyme for point-of-care testing of alkaline phosphatase

Paper-based microfluidic analytical devices (μPADs) have become promising alternatives to clinical laboratory-based methods for point-of-care testing (POCT) of biomarkers in family care and resource-limited communities. Here, Co-doped carbon dots (Co-CDs) nanozyme with outstanding oxidase-mimicking catalytic activity and red fluorescent emission were prepared, and combined adhered-3D μPAD (A-3D μPAD) to monitor facilely alkaline phosphatase (ALP) level in whole blood samples. Co-CDs catalyzed the oxidization of nonfluorescent o-phenylenediamine (OPD) into 2,3-diaminophenazine (oxOPD) with yellow fluorescent emission due to the generation of tremendous O₂^•- species. With addition of ALP, ALP hydrolyzed l-ascorbic acid 2-phosphate into ascorbic acid, and the latter was oxidized by Co-CDs, then reacted with OPD to form blue fluorescent emission 3-(dihydroxyethyl)furo [3,4-b]quinoxaline-1-one (DFQ). Both DFQ and oxOPD quenched the fluorescence intensity of Co-CDs via inner-filter effect. The cascade reaction of ALP/Co-CDs was incorporated into A-3D μPAD based on above sensing principles. A-3D μPAD enabled sample pretreatment, cascade reaction and signal output, and integrated portable minimized device and smartphone for visual ALP detection. The linear range and limit of detection for ALP were 0.5–150 U L⁻¹ and 0.1 U L⁻¹, respectively, and the color varied from red, yellow to blue. The detection results for whole blood samples were consistent with biochemical detector. The efficiency, disposability, practicality and low-cost of A-3D μPAD can be extended to determine various biomarkers, and provided technical support for nanozyme applications in POCT environments.