Hydrogel‐based biosensing, based on antigen–antibody binding, has been utilized for various biomedical applications such as cancer monitoring. Hydrogels offer highly sensitive detection with the prevention of nonspecific binding because of 3D porous structure and hydrophilicity. However, these hydrogel‐based biosensing platforms require a time scale of hours to complete immunoassays because binding events are diffusion‐limited, where target biomolecules must diffuse into and throughout the 3D porous network. Here, a new rapid microfluidic platform is introduced utilizing a cross‐flow induced advective‐transportation of targets into a hydrogel membrane with fluorescent reporting. This flow enhanced delivery of target analytes significantly reduces their detection time to under 15 min. This flow effect is also numerically investigated on the detection process. Both numerical and experimental results show an exponential decrease in the detection time. More importantly, the cross‐flow configuration in our platform provides an additional size‐based filtration feature that effectively selects against larger components in a blood sample, such as red blood cells, during the detection process. This addition, not seen in conventional biosensing platforms, eliminates the need for blood sample prefiltration.
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