Polymer Domains Control Diffusion in Protein–Polymer Conjugate Biosensors

Although surface-based biosensors have been widely used in diagnostic applications, these sensors experience reduced sensitivity in the most common detection fluids because of nonspecific binding effects from nonanalyte molecules. Protein–polymer conjugate thin films have been demonstrated to overcome many of these nonspecific binding issues because of their ability to restrict transport of impurity molecules into the film, but the transport mechanism within these films is not understood. Herein, the diffusion coefficients of 15 different proteins and dextran molecules are measured within protein gels and polymer solutions that mimic the phase-separated protein and polymer domains of the conjugate thin films using fluorescence recovery after photobleaching. Although most molecules have diffusivities that are consistent with size-based diffusion models, several protein diffusivities deviate significantly from these model predictions in polymer solutions. Mixtures of monomeric streptavidin (mSA2), an analyte for the thin-film biosensors, with proteins that diffuse faster than mSA2 in the polymer solutions give greater biosensor sensitivity for mSA2 than solutions of mSA2 alone. Furthermore, a mixture containing several of the proteins in this work is also found to result in greater sensitivity for mSA2 than a pure mSA2 solution. These findings suggest that the polymer domains are primarily responsible for transport in the thin films and, unlike other surface-based biosensors, protein–polymer conjugate biosensors can exhibit enhanced sensitivity in complex sensing mixtures.