Paper-based multiplex analytical device for simultaneous detection of Clostridioides difficile toxins and glutamate dehydrogenase

We report a new paper-based multiplex analytical device (mPAD) for simultaneous screening of three analytes (glutamate dehydrogenase, toxin A, and toxin B) known as biomarkers for Clostridioides difficile infection (CDI). To overcome the limitation of common rapid assays (e.g. lateral flow immunochromatographic and enzyme immunoassays) in terms of multiplexing, sensitivity, simplicity, and ease-of-use, the mPAD is constructed with a three dimensional (3D) configuration of paper components with a multi-channel design. Multiple fluidic paths developed with wax-patterned paper allow the simultaneous detection of glutamate dehydrogenase, toxin A, and toxin B without any cross-reactivity. The 3D fluidic network on the mPAD facilitates a self-operating test procedure for the mixing and addition of amplification reagents with a one-step sliding operation. The results of the multiplex CDI assay can be easily interpreted by the naked eye within 10 min, and are visually intensified over time resulting in up to 3-fold signal amplification. Our device exhibited remarkable analytical performances for the simultaneous detection of three CDI biomarkers, providing a sensitivity of 97%, specificity of 88%, accuracy of 95%, and limits of detection for glutamate dehydrogenase, toxin A, and toxin B of 0.16 ng mL⁻¹, 0.09 ng mL⁻¹, and 0.03 ng mL⁻¹, respectively. These results indicate the high applicability and feasibility of mPAD for multiplex testing for CDI with the advantages of being simple, sensitive, inexpensive, user-friendly, and equipment-free.