silicon and maleimide

A new nanostructure initiator mass spectrometry (NIMS) methodology is presented that uses the strategy of fluorous-phase immobilization and capture by a maleimide-functionalized affinity tag to selectively enrich peptide subsets containing cysteine residues. This surface-based approach allows complex protein digests to be analyzed. The proposed platform makes use of a chemically unmodified porous silicon (pSi)-based NIMS chip. Unlike matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS), the approach described in this paper does not require analytes to be incorporated or cocrystallized with an initiator. The mass spectra generated by the approach in this work are characterized by low background noise and, therefore, high analyte detection sensitivity. Experiments were also conducted that show the potential the approach described in this work has for generating simplified mass spectra for MS/MS analyses.

Topics: Affinity Labels; Cysteine; Fluorocarbons; Maleimides; Microarray Analysis; Nanostructures; Peptide Fragments; Serum Albumin, Bovine; Silicon; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tandem Mass Spectrometry

The availability of metal mesh device sensors has been investigated using surface-modified nickel mesh. Biotin was immobilized on the sensor surfaces consisting of silicon and nickel via a thiol-ene click reaction, known as the Michael addition reaction. Biotinylation on the maleimidated surface was confirmed by X-ray photoelectron spectroscopy. The binding of streptavidin to the biotinylated surfaces was evaluated using a quartz crystal microbalance and a metal mesh device sensor, with both techniques providing similar binding constant value. The recognition ability of the biotin immobilized using the thiol-maleimide method for streptavidin was comparable to that of biotin immobilized via several other methods. The adsorption of a biotin conjugate onto the streptavidin-immobilized surface via the biotin-streptavidin-biotin sandwich method was evaluated using a fluorescent microarray, with the results demonstrating that the biological activity of the streptavidin remained.

Topics: Adsorption; Biosensing Techniques; Biotin; Biotinylation; Gold; Immobilized Proteins; Maleimides; Nanostructures; Nickel; Silicon; Streptavidin; Surface Properties

In the present study we investigated the preparation of biofunctionalized surfaces using the direct electrochemical grafting of maleimidophenyl molecules with subsequent covalent immobilization of specific peptide to detect target antibody, thereby extending the application of the biosensing systems towards immunodiagnostics. Para-maleimidophenyl (p-MP) functional groups were electrochemically grafted on gold and silicon surfaces from solutions of the corresponding diazonium salt. A specially synthesized peptide modified with cysteine (Cys-peptide) was then immobilized on the p-MP grafted substrates by cross-linking between the maleimide groups and the sulfhydryl group of the cysteine residues. Accordingly, the Cys-peptide worked as an antigen that was able to bind specifically the target antibody (anti-GST antibody), while it was non-sensitive to a negative contrast antibody (i.e. anti-Flag β). The immobilization of both specific and non-specific antibodies on the Cys-peptide-modified surfaces was monitored by infrared spectroscopic ellipsometry, a quartz crystal microbalance integrated in flow injection analysis system and potentiometric response. The results obtained clearly demonstrated that the direct modification of a surface with maleimidophenyl provides a very simple and reliable way of preparing biofunctionalized surfaces suitable for the construction of immunological biosensors.

Topics: Antibodies; Biosensing Techniques; Cysteine; Electrochemical Techniques; Electrodes; Gold; Immobilized Proteins; Maleimides; Peptides; Silicon; Spectrophotometry, Infrared; Surface Properties