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This article describes the device design and fabrication of two different configurations (flow-through and stopped-flow) of a sequential fluid delivery platform on a microfluidic paper-based device. The developed device is capable of storing and transporting reagents sequentially to the detection channel without the need for external power. The device comprises two components: an origami folding paper (oPAD) and a movable reagent-stored pad (rPAD). This 3D capillary-driven device eliminates the undesirable procedure of multiple-step reagent manipulation in a complex assay. To demonstrate the scope of this approach, the device is used for electrochemical detection of biological species. Using a flow-through configuration, a self-calibration plot plus real sample analysis using a single buffer introduction are established for ascorbic acid detection. We further broaden the effectiveness of the device to a complex assay using a stopped-flow configuration. Unlike other electrochemical paper-based sensors in which the user is required to cut off the device inlet or rest for the whole channel saturation before measurement, herein a stopped-flow device is carefully designed to exclude the disturbance from the convective mass transport. As a proof of concept, multiple procedures for electrode modification and voltammetric determination of serotonin are illustrated. In addition, the research includes an impedimetric label-free immunosensor for α-fetoprotein using the modified stopped-flow device. The beneficial advantages of simplicity, low sample volume (1 μL), and ability to perform a complex assay qualify this innovative device for use with diverse applications.