Although vertical flow assays (VFAs) have a number of advantages compared to lateral flow assays (LFAs) such as a short analysis time, no line interference, and no Hook effect, VFAs are not preferred as LFAs because of their complicated operation principle. In this study, we demonstrated VFAs with multistep reactions for the detection of C-reactive protein (CRP) based on the programmed reagent loading in a pressed region integrated 3D paper-based microfluidic device. The flow order of all reagents in a 3D paper-based microfluidic device was programmed based on the delayed flow caused by the pressed region as well as the geometry modification of the paper channel. After simultaneous loading of all the reagents required for assays, they are sequentially loaded into the analysis region with a programmed sequence. As a proof of concept, a high-sensitivity CRP (hs-CRP) detection with signal amplification was performed to predict the riskiness of cardiovascular disease within 15 min. The detection limit was improved from 0.01 to 0.005 mu g/mL via a maximum 3.47-fold signal amplification. Additionally, the upper limit of hs-CRP detection increased to 5 mu g/mL without Hook effect. Finally, we successfully detected hs-CRP in a clinically relevant range (0.005-5 mu g/mL), while LFAs cannot cover due to the Hook effect. (C) 2017 Elsevier B.V. All rights reserved.