Surface functionalization and analysis thereof by ambient mass spectrometry

A challenge in the global healthcare is the lack of suitable diagnostic tools for early disease detection. One possible solution is the use of biosensors in diagnostic tests. By definition, a biosensor is a bioanalytical device that detects the presence of a compound (analyte) in the sample. The detection relies on the specific interactions between the ligands that are attached onto the biosensor surface and the analytes in the sample. This PhD dissertation is focused on developing an optimal protocol for attachment of ligands onto the biosensing surface. A step-wise approach was established for the versatile and reproducible modification and functionalization of a silicon nitride-based biosensor. This approach included the application of bioorthogonal copper-free reactions as a useful tool for oriented attachment of biomolecules. Additionally, a novel surface sensitive analytical method was developed for the identification of covalently bound molecules in monolayers. The method, which is fast and easy to apply, uses DART ionization coupled to a high-resolution mass spectrometer. The nm-thin layers were analysed, and interpretation rules for the obtained mass spectra were formulated. The method was applied in the identification of commercially available nm-thin coatings and biochips.</p

Surface Functionalization by Strain-Promoted Alkyne-Azide Click Reactions

Clicks without Cu: There is a growing demand for reproducible site-specific functionalization of surfaces with biomolecules without introduction of unwanted groups or catalysts, as they may interfere with later applications. The title reactions (see picture) could fulfill these requirements, and four recent applications are discussed

Copper-Free Click Biofunctionalization of Silicon Nitride Surfaces via Strain-Promoted Alkyne-Azide Cycloaddition Reactions

Cu-free "click" chemistry is explored on silicon nitride (Si3N4) surfaces as an effective way for oriented immobilization of biomolecules. An omega-unsaturated ester was grafted onto Si3N4 using UV irradiation. Hydrolysis followed by carbodiimide-mediated activation yielded surface-bound active succinimidyl and pentafluorophenyl ester groups. These reactive surfaces were employed for the attachment of bicyclononyne with an amine spacer, which subsequently enabled room temperature strain-promoted azide alkyne cycloaddition (SPAAC). This stepwise approach was characterized by means of static water contact angle, X-ray photoelectron spectroscopy, and fluorescence microscopy. The surface-bound SPAAC reaction was studied with both a fluorine-tagged azide and an azide-linked lactose, yielding hydrophobic and bioactive surfaces for which the presence of trace amounts of Cu ions would have been problematic. Additionally, patterning of the Si3N4 surface using this metal-free click reaction with a fluorescent azide is shown. These results demonstrate the ability of the SPAAC as a generic tool for anchoring complex molecules onto a surface under extremely mild, namely ambient and metal-free, conditions in a clean and relatively fast manner

Ambient Mass Spectrometry of Covalently Bound Organic Monolayers

Detailed molecular analysis by Direct Analysis in Real Time High Resolution Mass Spectrometry (DART-HRMS) of ester and amide-terminated monolayers is demonstrated. The structural information obtained allowed monitoring of the progress of a 4-step surface modification

Ambient Surface Analysis of Organic Monolayers using Direct Analysis in Real Time Orbitrap Mass Spectrometry

A better characterization of nanometer-thick organic layers (monolayers) as used for engineering surface properties, biosensing, nanomedicine, and smart materials will widen their application. The aim of this study was to develop direct analysis in real time high-resolution mass spectrometry (DART-HRMS) into a new and complementary analytical tool for characterizing organic monolayers. To assess the scope and formulate general interpretation rules, DART-HRMS was used to analyze a diverse set of monolayers having different chemistries (amides, esters, amines, acids, alcohols, alkanes, ethers, thioethers, polymers, sugars) on five different substrates (Si, Si3N4, glass, Al2O3, Au). The substrate did not play a major role except in the case of gold, for which breaking of the weak Au–S bond that tethers the monolayer to the surface, was observed. For monolayers with stronger covalent interfacial bonds, fragmentation around terminal groups was found. For ester and amide-terminated monolayers, in situ hydrolysis during DART resulted in the detection of ions characteristic of the terminal groups (alcohol, amine, carboxylic acid). For ether and thioether-terminated layers, scission of C–O or C–S bonds also led to the release of the terminal part of the monolayer in a predictable manner. Only the spectra of alkane monolayers could not be interpreted. DART-HRMS allowed for the analysis of and distinction between monolayers containing biologically relevant mono or disaccharides. Overall, DART-HRMS is a promising surface analysis technique that combines detailed structural information on nanomaterials and ultrathin films with fast analyses under ambient conditions