Polymer Domains Control Diffusion in Protein–Polymer Conjugate Biosensors

Abstract

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.

Intraparticle Macromolecular Migration Alters the Structure and Function of Proteins Reversibly Immobilized on Porous Microbeads

“…implants), industrial biocatalysts, or even protein arrays for drug screening. [1][2][3][4][5] In this context, biomolecules such as proteins have been successfully immobilized on a plethora of solid supports to enhance their functional properties. [6] Therefore, a deep understanding of the interactions between proteins and solid supports is crucial to gaining control over the immobilization process.…”

“…Protein adsorption and immobilization on solid surfaces have been a scientific and industrial strive for the past decades to fabricate biomaterials with biotechnological applications (i.e., surface‐based biosensors, medical devices, and biomedical implants), industrial biocatalysts, or even protein arrays for drug screening. [ 1–5 ] In this context, biomolecules such as proteins have been successfully immobilized on a plethora of solid supports to enhance their functional properties. [ 6 ] Therefore, a deep understanding of the interactions between proteins and solid supports is crucial to gaining control over the immobilization process.…”

On the Use of Surface Plasmon Resonance Biosensing to Understand IgG-FcγR Interactions

“…This practice also minimizes the risks of aggregation, steric hindrance, and rebinding artifacts [ 69 ]. Further work on a better understanding of the transport in protein-polymer thin films and the development of nanopore arrays are also potential avenues to help overcome mass transport limitation artifacts [ 70 , 71 , 72 , 73 ]. In fact, the use of nanopore arrays has been shown to enhance transport and sensitivity, thus becoming a growing niche within SPR-based biosensing novel methods [ 70 , 71 , 73 ].…”

Estrogen Receptor β-Targeted Near-Infrared Inherently Fluorescent Probe: A Potent Tool for Estrogen Receptor β Research

“…Diffusion coefficient is an important content of protein dynamics, it enhances the understanding of protein activity, function and is beneficial for the screening of protein-targeted drugs. Fluorescence recovery after photobleaching (FRAP) is a widely used means for protein dynamics, in which the diffusion coefficient of probe-bound protein can reflect its mobility to a certain extent. − Given that there is no research for ERβ dynamic motions, we carried out the FRAP experiment to explore the diffusion coefficient of ERβ for the first time (Figure ). DU-145 cells were incubated with P5 (10 μM) for 30 min, then the ROI was bleached and the recovery process was observed.…”