Chemoselective fabrication of high density peptide microarray by hetero‐bifunctional tetra(ethylene glycol) linker for click chemistry conjugation

Abstract

A hetero-bifunctional tetra(ethylene glycol) molecule with silane and azide termini was synthesized, and this molecule was used to prepare azide-derivatized glass surface in one step. The resulting glass surface was available for fabricating peptide microarray by the conjugation with alkyne-containing peptide using click chemistry, which proceeded to the completion at low temperature and in aqueous solution. A high density of peptide on the surface was achieved due to concise overall procedure and highly efficient conjugation reaction. Immobilized peptides were highly bio-functional on the surface, as demonstrated by the ability to detect protease activity. Due to the biologically orthogonal manner of conjugation, peptide conjugated by site-specific immobilization was more accessible by protease than that conjugated by random amide conjugation. This site-specific and high efficient immobilization technique could be expanded to large scale development of biocompatible peptide and protein arrays for use in various applications.

Use of Microarrays as a High-Throughput Platform for Label-Free Biosensing

“…To overcome this deficiency, linkers are typically inserted between targets and the solid surface so that the key chemical properties of the targets are kept more or less intact. [49][50][51]…”

Enzymatic reactions on immobilised substrates

“…Slides can be cheaply bought and are available prefunctionalised with various groups such as amines, epoxides and aldehydes for covalent immobilisation. 7,74,128 The advantage of glass slides is their thermal stability making them highly suitable for DNA array applications. Glass slides are also stable to organic and aqueous, and acidic and basic solutions, unlike many synthetic polymers.…”

“…Next in line are enzymes that are involved in posttranslational modifications (PTMs) of immobilised peptides and proteins, catalysing reactions such as phosphorylations, glycosylations, acetylation, ubiquitinations and proteolysis, which provide a rich pipeline of therapeutic targets. [5][6][7][8][9][10][11][12][13][14][15][16][17][18] Following the early studies on nucleic acids and peptides, 10,16,[19][20][21][22] the range of enzyme substrates that have been immobilised to surfaces is now very broad in terms of structures and functionalities, extending to lipids and glycans 12,23 and a broad variety of small molecule substrates. 24 There have been many successful applications in diagnostics, 19 omics studies, 25 sensors, 26 plant sciences, 27 enzyme kinetics, 28 enzyme specificity, 29 cell biology, 30 and organic (synthetic) chemistry.…”

A fluorescent amino acid probe to monitor efficiency of peptide conjugation to glass surfaces for high density microarrays

“…We synthesized a bifunctional peptide with NBD at the C-terminus and an alkyne group at the N-terminus. The alkyne group can be conjugated onto a glass surface bearing an azide group using the click reaction; 25 surface-bound peptide can be detected by a microarray scanner. Furthermore, a fluorescent peptide substrate for trypsin, which can be cleaved at the carboxyl side of the amino acids lysine and arginine, was conjugated to the surface by this procedure.…”