Recent Advances in Controlled Immobilization of Proteins onto the Surface of the Solid Substrate and Its Possible Application to Proteomics

Abstract

Proteome analysis plays a key role in the elucidation of the functions and applications for numerous proteins. For proteome analyses, various microplate-and microarray-based techniques have been developed by a number of researchers. Their intent was to immobilize proteins on the surface of a solid substrate in a site-directed manner while retaining structure and native biological function. In this review, we focus on recent advances in immobilization methodology for proteins/enzymes on a surface, including those using the affinity peptides screened by random peptide library systems. We also discuss applications of the affinity peptide-mediated immobilization method in fields related to proteome analysis, particularly our recent work concerning immunoassay and protein-protein interaction analysis.

Real-time analysis of dual-display phage immobilization and autoantibody screening using quartz crystal microbalance with dissipation monitoring

“…9 As recently reported, the oriented immobilization of bacteriophages on a sensing platform resulted in a higher sensitivity in the detection of autoantibodies in serum samples. 6 A main drawback of bacteriophages-based diagnostic assays is the nonspecific adsorption of proteins, leading to a high background signal and decreasing sensitivity.…”

Protein immobilization techniques for microfluidic assays

“…Random surface immobilization relying on multiple anchoring points can cause a protein to be denatured and lose native activity. 106,107 Also, active sites can orient towards the immobilization surface, resulting in reduced activity. 106 Even though the diffusion length within microfluidic channel is short and thus the overall reaction can be faster than macroscopic counterparts, a single monolayer of protein may not provide sufficiently high analytical signal in immunoassays or a sufficiently high conversion rate in enzyme reactors.…”

“…The intermolecular forces are highly dependent on environmental condition such as pH, ionic strength, temperature, and surface condition. 107 Therefore, immobilization of proteins-in a reproducible mannercan be difficult using physisorption. As protein immobilization is randomly oriented on the surface, some fraction of the binding sites within a population of immobilized proteins are likely not accessible.…”

“…106 Further, protein can be immobilized to the surface via multiple binding sites, which may result in conformational change and reduction of protein activity. 106,107 Immobilization density depends on protein size, as well as physicochemical surface properties. If the immobilization density is too high, active sites could be sterically blocked.…”

“…A bioaffinity interaction yields relatively stronger, highly specific, and oriented protein immobilization. 11,106,107 Therefore, protein leakage can be minimized and immobilized protein offers better accessibility to binding partners than random orientation strategies. Additionally, bioaffinity immobilization can be reversed using chemical treatment, pH change, or heat treatment.…”