A Novel ATR‐FTIR Approach for Characterisation and Identification of Ex Situ Immobilised Species

Abstract

We demonstrate a novel method to analyse ex situ prepared protein chips by attenuated total reflection Fourier IR spectroscopy (ATR‐FTIR), which circumvents tedious functionalisation steps of internal reflection elements (IREs), and simultaneously allows for complementary measurements by other analytical techniques. This concept is proven by utilising immobilised metal affinity capture (IMAC™) chips containing about 10 μm thick films of copolymers coated with nitrilotriacetic acid (NTA) groups, which originally was manufactured for surface enhanced laser desorption ionisation (SELDI) spectrometry. Three immobilisation steps were analysed by ATR‐FTIR spectroscopy: 1) NTA complexation with nickel(II) ions 2) binding of two histidine (His)‐tagged synthetic peptides of 25 (25‐His6) and 48 (48‐His6) amino acids to the NTA‐groups and 3) attachment of a ligand, mesyl amide, to the surface‐bound 48‐His6. Despite interference from H2O, both amide I and II were well resolved. Utilising peptide adsorption in the thick copolymer matrix yields a high saturation peptide concentration of ≈100 mg mL−1 and a dissociation constant of 116±11 μM, as determined by a detailed analysis of the Langmuir adsorption isotherm. The mesyl amide ligand was directly seen in the raw ATR‐FTIR spectrum with specific peaks in the fingerprint region at 1172 and 1350 cm−1. Several aspects of the fine structure of the amide I band of the peptide were analysed: influences from secondary structure, amino side chains and competing contamination product. We believe that this approach has great potential as a stand‐alone or complementary analytical tool for determination of the chemical composition of functionalised surfaces. We emphasise further that with this approach no chemical treatment of IREs is needed; the chips can be regenerated and reused, and applied in other experimental set‐ups.

Nanocrystalline diamond sensor targeted for selective CRP detection: an ATR-FTIR spectroscopy study

“…Significant peaks arise due to amide I and II bands of the binder (Fig. 1), centered at 1652 and 1545 cm -1 , respectively, in good agreement with reported ATR-FTIR spectra of related synthetic peptides ex-situ immobilized on functionalized surfaces [8,9]. It is likely that the binder binds covalently, since the same spectral pattern was reproducibly obtained after careful washing of the substrates.…”

“…In more elaborate schemes, the IRE itself is functionalized and acts as a simulated sensing layer [7]. We have previously reported a novel method to analyze ex-situ immobilization steps of functionalized sensor chips by pressing them with a force-controlled anvil-type diamond IRE [8,9]. In that study, discrimination between the copolymer, peptide and target substance, an organophosphate, was established without loss of functionality.…”

Multistep Anchoring Route of Luminescent (5‐Amino‐1,10‐phenanthroline)tris(dibenzoylmethane)europium(III) on Si(100)

“…This suggests that the β-diketonate coordination environment is maintained after the surface anchoring. The band at 1650 cm -1 , absent in both spectra of NHS-Si and 1 powder, www.eurjic.orgis assigned to the typical ν(C=O) stretches of the amide bond, [25,30] formed from the reaction of 1 with the carboxylic groups on the surface. The wide absorption band centred at 1100 cm -1 is assigned to SiO x species, formed during chemical manipulations.…”

Synthesis and characterization of new branched magnetic nanocomposite for loading and release of topotecan anti-cancer drug

“…The peak at 1090 cm −1 was represent in -C-O alcoholic hydroxyl stretching and at 550 cm −1 Fig. 5 Magnetization curve versus the magnetic field (a) and XPS wide scan spectrum (b) for the MD 3 nanocomposite was demonstrated the characteristic Fe-O peak for MD 3 nanocomposite (Aguirre et al 2014;Andersson et al 2007;Kaya and Mr 2012;Souaya et al 2000). TEM images were taken to get information about the morphology and dimensions of the synthesized nanoparticles and the nanocomposite.…”