Controlling Cross‐Pollination of Biosensing Probes in Nanoscale Multiplexed Arrays

Abstract Multi‐target biosensing platforms require immobilization of sensing probes on surfaces with nanoscale precision. A critical challenge is preventing unwanted molecular transfer (cross‐pollination) between different probe patterns, which compromises measurement accuracy. This study investigates cross‐pollination in multiplexed biosensing arrays fabricated using thermal scanning probe lithography (tSPL), a method that offers sub‐10 nm spatial resolution. By systematically optimizing both the tSPL patterning parameters and chemical functionalization protocols, particularly biomolecule incubation times, this work demonstrates the ability to reduce cross‐pollination to undetectable levels (0%). These experimental results establish a clear relationship between processing conditions and cross‐contamination rates, providing a practical framework for reliable nanoscale patterning of multiple biomolecular probes on a single surface. These findings enable the development of high‐fidelity, multi‐target biosensing platforms with enhanced specificity, and therefore sensitivity, for applications in medical diagnostics, environmental monitoring, and biological research.