Characterizing protein–surface and protein–nanoparticle conjugates: Activity, binding, and structure

Abstract

Many sensors and catalysts composed of proteins immobilized on inorganic materials have been reported over the past few decades. Despite some examples of functional protein-surface and protein-nanoparticle conjugates, thorough characterization of the biological-abiological interface at the heart of these materials and devices is often overlooked in lieu of demonstrating acceptable system performance. This has resulted in a focus on generating functioning protein-based devices without a concerted effort to develop reliable tools necessary to measure the fundamental properties of the bio-abio interface such as surface concentration, biomolecular structure, and activity. In this Perspective we discuss current methods used to characterize these critical properties of devices that operate by integrating a protein into both flat surfaces and nanoparticle materials. We highlight the advantages and drawbacks of each method as they relate to understanding the function of the protein-surface interface, and explore the manner in which an informed understanding of this complex interaction leads directly to the advancement of protein-based materials and technology.

Nanoparticle‐Based Bioaffinity Assays: From the Research Laboratory to the Market

“…Furthermore, the optimization work may be slowed down by the challenges associated with the characterization of the NP-antibody bioconjugates. [228,229] The binding reactivity of antibody-coated NP bioconjugates depends on the density of antibodies on the surface. A higher coating density and the proper orientation of antibodies improves their binding properties until a certain limit is reached.…”

Quantifying Bound Proteins on Pegylated Gold Nanoparticles Using Infrared Spectroscopy

“…9−12 We recently surveyed the currently available methods to determine this quantity on NPs and the few available methods often require expertise and instrumentation that are not readily available. 9 Though measuring protein concentration in solution is straightforward and well established, 13 the presence of NPs makes this measurement significantly more challenging as NPs (particularly metal NPs) tend to interfere with the most common optical techniques used to determine protein concentration, UV−visible and fluorescence spectroscopies. NPs often absorb in the visible range of the electromagnetic spectrum (400−800 nm), thus interfering with common assays of protein concentration, structure, and function.…”

“…Nanoparticles (NPs) have been shown to be effective scaffolds on which stable and active proteins and enzymes can be immobilized. − Characterization of proteins grafted onto NPs is crucial to the development of these protein-based nanomaterials. Some common and important characterization parameters include activity, stability, and amount of bound protein. , Out of these important characteristics, the quantification of the amount of bound protein is one of the most important since it determines the efficiency of binding and the effects of binding on protein activity. − We recently surveyed the currently available methods to determine this quantity on NPs and the few available methods often require expertise and instrumentation that are not readily available . Though measuring protein concentration in solution is straightforward and well established, the presence of NPs makes this measurement significantly more challenging as NPs (particularly metal NPs) tend to interfere with the most common optical techniques used to determine protein concentration, UV–visible and fluorescence spectroscopies.…”