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Query: UNIPROT:P30536 (PBS)
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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.
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PMID:Monolayers of 3-mercaptopropyl-amino acid to reduce the nonspecific adsorption of serum proteins on the surface of biosensors. 1882 86

Near-zero fouling monolayers based on binary patterned peptides allow low nanomolar detection of the matrix metalloproteinase-3 (MMP-3) directly in crude bovine serum, without sample pretreatment, secondary antibody detection or signal amplification. The peptide 3-MPA-HHHDD-OH (3-MPA, 3-mercaptopropionic acid) was found optimal compared to other binary patterned peptides based on 3-MPA-A(x)-B(y)-OH, where 0 <or= x, y <or= 5, and x + y = 5, and compared to PEG. In this study, amino acid A was His, Asp, Ser, or Leu, and amino acid B was His, Asp, or Ser. Zwitterionic peptides and other peptides exhibited excellent resistance to nonspecific adsorption. Binary patterned peptides were capped with 3-MPA on the N-terminus providing a monolayer with the C-terminus carboxylic acid available to subsequently immobilize antibodies. Thereby, an IgG biosensor demonstrated the efficiency of binary patterned peptides in SPR biosensing with a detection limit of 1-10 pM in PBS, similar to other optical or electrochemical techniques. This protocol was applied to establish a calibration curve for MMP-3, an analyte of clinical interest for many pathologies and a potential indicator of cancer. The LOD for MMP-3 was 0.14 nM in PBS, with a linearity of up to 50 nM. With the use of PBS calibration, MMP-3 was quantified at low nanomolar in undiluted bovine serum. The SPR response in serum was statistically the same as in PBS. A sensor exposed to blank serum exhibited negligible nonspecific adsorption. Hence, binary patterned peptides are suitable for biosensing directly in complex biological matrixes.
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PMID:SPR Biosensing in crude serum using ultralow fouling binary patterned peptide SAM. 2035 64