A Strategy for the Generation of Surfaces Presenting Ligands for Studies of Binding Based on an Active Ester as a Common Reactive Intermediate: A Surface Plasmon Resonance Study

Abstract

This paper describes the immobilization of ten proteins and two low-molecular-weight ligands on mixed self-assembled monolayers (SAMs) of alkanethiolates on gold generated from the tri(ethylene glycol)-terminated thiol 1 (HS(CH2)11(OCH2CH2)3OH) (chi(1) = 1.0-0.0) and the longer, carboxylic acid-terminated thiol2(HS(CH2)11(OCH2-CH2)6OCH2CO2H) (chi(2) = 0.0-1.0). The immobilization was achieved by a two-step procedure: generation of reactive N-hydroxysuccinimidyl esters from the carboxylic acid groups of 2 in the SAM and coupling of these groups with amines on the protein or ligand. Because this method involves a common reactive intermediate that is easily prepared, it provides a convenient method for attaching ligands to SAMs for studies using surface plasmon resonance spectroscopy (and, in principle, other bioanalytical methods that use derivatized SAMs on gold, silver, and other surfaces). These SAMs were resistant to nonspecific adsorption of proteins having a wide range of molecular weights and isoelectric points. The pH of the coupling buffer, the concentration of protein, the ionic strength of the solution of protein, and the molecular weight of the protein all influenced the amount of the protein that was immobilized. For the proteins investigated in detail--carbonic anhydrase and lysozyme--the highest quantities of immobilized proteins were obtained when using a low ionic strength solution at a value of pH approximately one unit below the isoelectric point (pI) of the protein, at a concentration of approximately 0.5 mg mL-1. Comparisons of the kinetic and thermodynamic constants describing binding of carbonic anhydrase and vancomycin to immobilized benzenesulfonamide and N-alpha-Ac-Lys-D-Ala-D-Ala groups, respectively, on mixed SAMs (by methods described in this paper) and in the carboxymethyl dextran matrix of commercially available substrates yielded (for these systems) essentially indistinguishable values of Kd, koff, and kon.

Wet chemical routes to the assembly of organic monolayers on silicon surfaces via the formation of Si–C bonds: surface preparation, passivation and functionalization

“…58,59,225 We reason this as the major cause behind the limited number of literature examples on this directive approach, while on the other hand the step-wise NHS/EDC activation scheme (Si-RCOOH to Si-RC(O)NHS) remain widespread in surface science. 50,51,226,227 It is also worth noting that an optimization study to evaluate the optimal range of NHS and EDC concentrations and molar ratios has only very recently been published for porous silicon substrates by Sam et. al.…”

Ligand rebinding: self-consistent mean-field theory and numerical simulations applied to surface plasmon resonance studies

“…The surfaces used for these studies were composed of a mixed self assembled monolayer (mSAM) on gold (500 nm) coated slides (EMF Corporation, Ithaca, N.Y.) prepared as previously described [Lahiri et al 1999]. Briefly, the gold coated slides were immersed in a mixture of 0.2 mM carboxylic acid-terminated thiol and 1.8 mM tri(ethylene glycol)-terminated thiol (Toronto Research Chemicals, Toronto, Canada) for 12 hours.…”

Electrochemical Surface Plasmon Resonance Spectroscopy at Bilayered Silver/Gold Films

“…[3][4][5][6] SPR is attractive owing to several inherent advantages (e.g., labelfree analysis, simplicity, cost-effectiveness, and high sensitivity). [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] The SPR resonance angle variation is dependent on various physicochemical processes occurring at the SPR substrate (a thin metal film or a chemically modified metal surface) and/or the substrate/ solution interface. Thus, when coupled with electrochemistry (EC-SPR), it offers a viable avenue to probe optical and electrochemical properties of adsorbates and a sensitive means to quantify thickness variations of ultrathin films accompanying redox reactions.…”