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Magnetic nanoparticles (MNPs) are used in many biomedical applications, including sentinel lymph node biopsy (SLNB) and magnetic particle imaging (MPI). DiffMag handheld probe ,based upon nonlinear detection principles has been developed by University of Twente for SLNB with MNPs. This method uses an excitation coil to activate the MNPs, and a detection coil to acquire the consequent magnetization of the MNPs. The hydrodynamic size and surface properties of MNPs are changed when introduced to new environment with different viscosity. Consequently, the number of mechanical degrees of freedom for rotation and translation in MNPs is reduced. This alteration in mechanical behavior has an effect on their magnetic behavior and particles Brownian relaxation time. In this study, we optimize the protocol settings for the nonlinear handheld detection probe during the SLNB procedure and investigate the detection depth in two environments.