Monolayers of 3-Mercaptopropyl-amino Acid to Reduce the Nonspecific Adsorption of Serum Proteins on the Surface of Biosensors

Abstract

Monolayers prepared with polar or ionic amino acids with short side chains have a reduced nonspecific adsorption of serum proteins compared to that of hydrophobic amino acids and organic monolayers immobilized on the gold surface of surface plasmon resonance (SPR) biosensors. Proteins contained in biological samples adsorb on most surfaces, which in the case of biosensors causes a nonspecific response that hinders the quantification of biomarkers in these biological samples. To circumvent this problem, self-assembled monolayers (SAM) of N-3-mercaptopropyl-amino acids (3-MPA-amino acids) were prepared from 19 natural amino acids. These SAM were investigated to limit the nonspecific adsorption of proteins contained in biological fluids and to immobilize molecular receptors (i.e., antibodies) that are necessary in the construction of biosensors. SPR and Ge attenuated total reflection (GATR) FTIR spectroscopy were employed to characterize the formation of the amino acid SAMs. Monolayers of 3-MPA-amino acids densely packed on the surface of the SPR biosensors result in a surface concentration of approximately 10 (15) molecules/cm (2). SPR also quantifies the surface concentration of serum proteins nonspecifically adsorbed on 3-MPA-amino acids following the exposure of the biosensor to undiluted bovine serum. The concentration of nonspecifically bound proteins ranged from approximately 400 ng/cm (2) with polar and ionic amino acids to approximately 800 ng/cm (2) with amino acids of increased hydrophobicity. The nonspecific adsorption of serum proteins on the 3-MPA-amino acids increases in the following order: Asp < Asn < Ser < Met < Glu < Gln < Thr < Gly < His < Cys < Arg < Phe < Trp < Val < Pro < Ile < Leu < Ala < Tyr. The analysis of the adsorption and desorption curves for serum proteins on the SPR sensorgram has demonstrated the strong irreversibility of the protein adsorption on each surface. The effective hydrophilicity of the SAMs was measured from the contact angle with a saline buffer and has demonstrated that surfaces minimizing the contact angle with PBS performed better in serum. The antibody for beta-lactamase was immobilized on a 3-MPA-glycine SAM, and beta-lactamase was detected in the nanomolar range. The presence of beta-lactamase is an indicator of antibiotic resistance.

Oxinate-Aluminum(III) Nanostructure Assemblies Formed via In-situ and Ex-situ Oxination of Gold-Self-Assembled Monolayers Characterized by Electrochemical, Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy, and X-ray Photoelectron Spectroscopy Methods

“…The results (Fig. 5) indicate that the electrochemical signal backgrounds observed on the Au electrode modified by oxinate functional group depends highly on the nature of the spacers, so that the backgrounds were minimized upon using azo spacer [14,86] formed by ATP and maximized upon using two amide spacers formed by glycine [87] (Supplementary Data, Fig. S9).…”

SPR-MS: from identifying adsorbed molecules to image tissues

“…Our previous studies investigated the relationship of organic and peptide surface chemistries as a function of nonspecifically bound material: proteins from serum [5][6][7][8] and lipids from cell lysate [15]. It was found that nonspecifically bound proteins were more abundant on organic surfaces than peptide surfaces.…”

“…However, plasmonic biosensors remain largely susceptible to nonspecific adsorption, limiting their applications in biofluids [1,2]. Recently, new surface chemistries based on zwitterionic polymers [3,4] or peptide chemistry [5][6][7] demonstrated the capacity of plasmonic biosensors for the detection of biomarkers in biofluids. While these surface chemistries effectively suppress a large fraction of the nonspecific adsorption of biofluids, their mechanism of action remains poorly described due to the difficulty of identifying nonspecifically adsorbed molecules with surface plasmon resonance (SPR) [8].…”

Surface plasmon resonance biosensing toward real biological sample analysis

“…A peptide-like polymer was synthesized to limit cell adhesion to a surface for up to two weeks [20]. Amino acid monolayers are capable of low nonspecific adsorption and immobilize antibodies to detect sub-µM protein concentration [21]. Short peptides demonstrated low self-aggregation on gold nanoparticle, demonstrating low interactions between the peptides [22; 23].…”