Enhancing Thermodynamic and Kinetic Performance of Microfluidic Interface-Based Circulating Fetal Cell Isolation for Noninvasive Prenatal Testing

Multivalent strategies have been widely applied in the microfluidic interface to boost the capture efficiency of target cells. However, achieving a balance between binding kinetics and thermodynamics in existing multivalent affinity interfaces remains challenging. Here, we propose a synergistic <b>A</b>ptamer-nanobody hetero-<b>M</b>ultivalency <b>P</b>rogrammable magnetic fluid microfluidic chip (<b>AMP-chip</b>) which utilizes the combined advantages of ligands to enhance both thermodynamic and kinetic properties of the capture interface. The AMP-chip integrates two distinct noninterfering recognition molecules: one with high affinity and another with rapid binding capability, both of which are assembled onto nanomagnetic beads. This integration achieves intermolecular complementarity, effectively enhancing the binding kinetics and thermodynamic stability. We chose mutually noninterfering CD71 recognition targets, a high-affinity nanobody (NB) and a rapid-binding aptamer (XQ 2d), and fully utilized the respective advantages of these ligands to facilitate rapid and tight recognition of the CD71 receptor on target cells. By integrating a herringbone microarray into an AMP-chip to further increase the cell-ligand interaction, we significantly improved the sensitivity and accuracy of circulating nucleated red blood cell (cNRBC) isolation from the peripheral blood mononuclear cells (PBMCs) of pregnant women. Additionally, the ligands were primarily fixed to the chip by magnetic force without chemical bonding, enabling nondestructive cell release and preserving high cell viability for subsequent molecular analyses. Overall, this approach offers a novel thermodynamic-kinetic synergistic heteromultivalency interface with significant potential for clinical applications.