Peptide Self-Assembled Monolayers for Label-Free and Unamplified Surface Plasmon Resonance Biosensing in Crude Cell Lysate

Abstract

Short peptides, composed of polar or ionic amino acids, derived with a short organic thiol, significantly reduce nonspecific adsorption of proteins in complex biological matrices such as serum and crude cell lysate, which have nonspecific protein concentrations of 76 and 30-60 mg/mL, respectively. Minimizing these nonspecific interactions has allowed rapid and direct quantification of beta-lactamase in a crude cell lysate using a surface plasmon resonance (SPR) biosensor. A library of short peptides with varying chain length and amino acid composition were synthesized using a solid-phase approach. A 3-mercaptopropionic acid (3-MPA) linker was covalently attached to the amino terminus of the peptides to subsequently form a monolayer on gold in the form of 3-MPA-(AA)(n)-OH, where n is the length of the amino acid chain (n = 2-5). Leu, Phe, Ser, Asp, and His were selected to investigate the effect on nonspecific adsorption with different physicochemical properties of the sidechains; aliphatic, aromatic, polar, acid, and base. Advancing contact angles measured the hydrophobicity of each peptidic self-assembled monolayer (SAM) and showed that hydrophilicity of the gold surface improved as the chain length of the polar or ionic peptides increased, while aromatic and aliphatic peptides decreased the hydrophilicity as the chain length increased. The nonspecific adsorption of undiluted bovine serum on SPR sensors prepared with the library of 3-MPA-(AA)(n)-OH showed that the lowest nonspecific adsorption occurred with polar or ionic amino acids with a chain length of n = 5. We demonstrate that a monolayer composed of 3-MPA-(Ser)(5)-OH has significant advantages, including the following: (1) it minimizes nonspecific adsorption in undiluted bovine serum; (2) it provides a high surface concentration of immobilized antibodies; (3) it shows a great retention of activity for the antibodies; (4) it improves the response from beta-lactamase by approximately 1 order of magnitude, compared to previous experiments; and (5) it allows direct quantification of submicromolar beta-lactamase concentration in a crude cell lysate with a nonspecific protein concentration of 30-60 mg/mL. The use of this peptide-based monolayer offers great advantages for quantitative SPR biosensing in complex biological media.

Short peptides as biosensor transducers

“…Recently, to reproduce the chemical and structural complexity of proteins, a nanoengineering approach was used to design peptides able to cap such surfaces, conferring stability [22], minimizing nonspecific interactions [23], and providing an appropriate template to incorporate several molecular receptors or recognition groups [23,24]. Most of these sequences, such as CALNN and 3-MPA-(Ser) 5 -OH (where MPA is mercaptoproprionic acid), are provided by rational or combinatorial designs.…”

Discrimination and detection of bacteria with a label-free impedimetric biosensor based on self-assembled lectin monolayer

“…For instance, nanoparticles biomimetic interfaces have been employed to immobilize living cells, which allows adsorption based on electrostatic force or weak interaction between nanoparticles and cells membrane [6][7][8][9]. Antibodies, functional peptides, such as arginine-glycine-aspartic acid-serine (RGDS) tetrapeptide, have been used to functionalize electrode for capturing cells via the specific binding between these molecules and cellular surface units [10][11][12][13]. Moreover, aptamer-based interface has also been used for enrichment and detection of multiple cancer cells [14].…”

Electrografted Diazonium Salt Layers for Antifouling on the Surface of Surface Plasmon Resonance Biosensors

“…10 Concurrently, tethered amino acids, such as phenylalanine, have been shown to be resistant to biofouling. 11 Surface plasmon resonance is a well-established surfacesensitive optical technique which allows for the qualitative and quantitative measurements of biomolecular interactions in real time without requiring a labeling procedure. 12 The excellent sensitivity of SPR for detecting proteins tenders the increasing popularity of SPR in bioanalyses.…”

“…17,20 PEG-functionalized surfaces reveal good ability for prevention of nonspecific protein adsorption with only 100 ng/cm 2 nonspecific adsorption. 11 However, their applications have been limited by the need for carboxylic acid functionalization of PEG to immobilize the molecular receptor. Bolduc et al demonstrated that nonspecific adsorption can be substantially reduced by functionalizing gold surfaces with 3-mercaptopropionic acid (3-MPA)-linked different amino acids and peptide sequences.…”