High-resolution temporally resolved chemiluminescence based on double-layered 3D microfluidic paper-based device for multiplexed analysis

In this work, a double-layered three-dimensional (3D) microfluidic paper-based analytical device (μPAD) with high resolution temporally resolved chemiluminescence (CL) emissions were designed for multiplexed CL analysis. The temporally resolved CL emissions were obtained by virtue of the 3D branched microfluidic channel design, which create time delays for luminol transported from one detection zone to another. The peak intensity and peak shape of the temporally resolved CL emissions were quite stable and base-line separated with resolution as high as 21.2-24.4. Then, the fabricated μPAD was applied to multiplexed determination of glucose, lactate, cholesterol, and choline as model analytes. The sample was added to four detection zone modified with CL catalyst cobalt ion and different oxidase by virtue of chitosan. When luminol flowed to μPAD, four temporally resolved CL peaks were successively generated from the cobalt ion catalyzed CL reactions between luminol and generated H₂O₂ from the specific enzymatic reactions between the oxidase and the analytes. The generated four CL emission peaks in the CL kinetic curve increased in proportion to the concentrations of glucose, lactate, cholesterol, and choline, respectively. Finally, four linear calibration curves were obtained for the detection of glucose (0.01-1.0 mmol/L), lactate (0.02-5.0 mmol/L), cholesterol (0.01-0.4 mmol/L), and choline (0.001-1.0 mmol/L). The detection limits were as low as 8 μmol/L, 15 μmol/L, 6 μmol/L, and 0.07 μmol/L for glucose, lactate, choline, and cholesterol detection, respectively. The present work provides a new strategy for the fabrication of simple and sensitive 3D μPAD with high resolution temporally resolved CL emissions for multiplexed CL analysis, which holds great application potential for point-of-care diagnosis.