Surface-Specific Interaction of the Extracellular Domain of Protein L1 with Nitrilotriacetic Acid-Terminated Self-Assembled Monolayers

Abstract

We report a study on the interaction of the extracellular domain of trans-membrane proteins N-cadherin and L1 with nitrilotriacetic acid (NTA)-terminated self-assembled monolayers (SAMs) grown on silver and gold surfaces. Quartz crystal microbalance (QCM) and reflection absorption infrared spectroscopy (RAIRS) measurements reveal that upon addition of protein to an NTA-SAM there is a subsequent change in the mass and average chemical structure inside the films formed on the metal substrates. By using vibrational sum frequency generation (VSFG) spectroscopy and making a comparison to SAMs prepared with n-alkanethiols, we find that the formed NTA-SAMs are terminated by ethanol molecules from solution. The ethanol signature vanishes after the addition of L1, which indicates that the L1 proteins can interact specifically with the NTA complex. Although the RAIRS spectra display signatures in the amide and fingerprint regions, the VSFG spectra display only a weak feature at 866 cm(-1), which possibly indicates that some of the abundant phenyl rings in the complex are ordered. Although cell biology experiments suggest the directional complexation of L1, the VSFG experiments suggest that the alpha-helices and beta-sheets of L1 lack any preferential ordering.

Ni(ii)-modified solid substrates as a platform to adsorb His-tag proteins

“…Solid substrates (e.g., electrodes, chips and (nano)particles) modied with metal cations (e.g., Ni(II), Cu(II), Co(II) or Zn(II)) have been used for a wide variety of applications including protein purication, 1 bio-functional surfaces, [2][3][4] cell recognition behavior, 5 controlled drug release 6 and electron transfer reactions in enzymes. 7 The metal cations are usually incorporated by graing a chelating agent on the sorbent surface, such as nitrilotriacetic acid (NTA), iminodiacetic acid (IDA) and EDTA, that partially coordinates the cations.…”

“…10 SAMs on gold substrates are oen used because the distance between the protein and the substrate can be tuned by changing the length of the thiol, and the use of thiols with different terminal groups can change the density of the protein layer. Moreover, gold substrates are widely used in different techniques such as quartz crystal microbalance (QCM), 5,11 surface plasmon resonance (SPR), 6 and electrochemical methods, 7 which are used to study proteins at interfaces, detect bio-recognition events or evaluate enzymatic electron transfer reactions. Two methods are currently used to generate chelating layers on SAM-modied gold substrates: (1) a single-step method based on the assembly of designed and synthesized chelate-terminated thiols [2][3][4][5][6][7]12 and (2) a multi-step method based on graing the chelating moiety onto a pre-assembled SAM with exposed reactive groups.…”

“…Moreover, gold substrates are widely used in different techniques such as quartz crystal microbalance (QCM), 5,11 surface plasmon resonance (SPR), 6 and electrochemical methods, 7 which are used to study proteins at interfaces, detect bio-recognition events or evaluate enzymatic electron transfer reactions. Two methods are currently used to generate chelating layers on SAM-modied gold substrates: (1) a single-step method based on the assembly of designed and synthesized chelate-terminated thiols [2][3][4][5][6][7]12 and (2) a multi-step method based on graing the chelating moiety onto a pre-assembled SAM with exposed reactive groups. 11,13 Although both methods have advantages and disadvantages, multi-step modication does not require tedious purication of the synthesized molecules, is easily applied to micro-and nanoarrays, can be extended to other substrates by changing the rst reactive layer that is assembled, and is easily complemented with other moieties (e.g., oligoethylene glycol) by adding an additional step during the modication process.…”

In vitro observation of dynamic ordering processes in the extracellular matrix of living, adherent cells

“…[29][30][31][32][33][34][35][36][37][38][39][40] In particular, this technique has been applied to the in situ investigation of biomolecules, including peptides, [41][42][43][44][45][46][47][48][49][50][51][52] proteins, and DNA. [75][76][77][78][79] Previous work from our lab has demonstrated the ability of SFG spectroscopy to detect substrate modifications through a layer of adherent, fixed cells 80 and through living, nonadherent cells.…”

A Survey of the 2010 Quartz Crystal Microbalance Literature

“…The dominant effect of the properties of the SAM was also demonstrated with mixtures of bovine serum albumin and gamma globulin (Benesch et al ., ). Nitrilotriacetic acid terminated SAMs were grown on gold and silver surfaces and the binding of the extracellular domain of the trans‐membrane proteins N‐cadherin and L1 was demonstrated by QCM (Fick et al ., ). Biotinylated SAMs and streptavidin binding was investigated for both native streptavidin (Seifert et al ., ) and streptavidin‐coated microspheres (Yuan et al ., ).…”