This review describes the progress of the development of surface chemical reactions for the modification of self-assembled monolayers (SAMs) and the fabrication of surface chemical gradients. Various chemical reactions can be carried out on SAMs to introduce new functionalities. "Click" reactions, which are highly efficient and selective, have largely contributed to the development and implementation of surface chemical reactions in the fields of biotechnology, drug discovery, materials science, polymer synthesis, and surface science. Besides full homogeneous functionalization, SAMs can be modified to exhibit a gradual variation of physicochemical properties in space. Surface-confined chemical reactions can be used for the fabrication of surface chemical gradients making the preparation of exceptionally versatile interfaces accessible.

Studying and controlling reactions at surfaces is of great fundamental and applied interest in, among others, biology, electronics and catalysis. Because reaction kinetics is different at surfaces compared with solution, frequently, solution-characterization techniques cannot be used. Here we report solution gradients, prepared by electrochemical means, for controlling and monitoring reactivity at surfaces in space and time. As a proof of principle, electrochemically derived gradients of a reaction parameter (pH) and of a catalyst (Cu(I)) have been employed to make surface gradients on the micron scale and to study the kinetics of the (surface-confined) imine hydrolysis and the copper(I)-catalysed azide-alkyne 1,3-dipolar cycloaddition, respectively. For both systems, the kinetic data were spatially visualized in a two-dimensional reactivity map. In the case of the copper(I)-catalysed azide-alkyne 1,3-dipolar cycloaddition, the reaction order (2) was deduced from it.

We report the fabrication of a patterned protein array using three orthogonal methods of immobilization that are detected exploiting a fluorogenic surface. Upon reaction of thiols, the fluorogenic tether reports the bond formation by an instantaneous rise in (blue) fluorescence intensity providing a means to visualize the immobilization even of nonfluorescent biomolecules. First, the covalent, oriented immobilization of a visible fluorescent protein (TFP) modified to display a single cysteine residue was detected. Colocalization of the fluorescence of the immobilized TFP and the fluorogenic group provided a direct tool to distinguish covalent bond formation from physisorption of proteins. Subsequent orthogonal immobilization of thiol-functionalized biomolecules could be conveniently detected by fluorescence microscopy using the fluorogenic surface. A thiol-modified nitrilotriacetate ligand was immobilized for binding of hexahistidine-tagged red-fluorescing TagRFP, while an appropriately modified biotin was immobilized for binding of Cy5-labeled streptavidin.

Strategies to generate platforms combining tissue targeting and regeneration properties are in great demand in the regenerative medicine field. Here we employ an approach to directly visualize the immobilization of cysteine-terminated peptides on a novel fluorogenic surface. Peptides with relevant biological properties, CLPLGNSH and CLRGRYW, were synthesized to function as peptide binders to transforming growth factor (TGF)-b1 and collagen type II (CII). The selective immobilization of the peptides was directly detected using a fluorogenic surface. Adhered proteins were confined to patterns of these peptides matching with the fluorogenic areas. These results show that the fluorogenic signal can be used to detect the chemo-selective immobilization of non-fluorescent biomolecules and to correlate the cell response with the patterned peptides. After analyzing the sequence specificity and cross-reactivity of the binding of TGF-b1 and CII to the respective peptide regions employing immunofluorescence assays, both peptides were co-immobilized in a step-wise process as detected by the fluorogenic surface. TGF-b1 and CII could be self-sorted from a mixture in a regio-selective manner resulting in a bi-functional protein platform. Surfaces of CLPLGNSH pre-loaded with TGF-b1 showed excellent bioactivity in combination with human articular chondrocytes (HACs) and stimulated expression of chondrogenic markers.

The overwhelming majority of reactions and interactions in biology do not occur in solution but at interfaces. Therefore, the study of how surfaces play a role in the control of biological interactions poses a great challenge. The interface between synthetic biomaterials and cells, among others, is of particular interest owing to its high impact on the design of novel biomaterials for tissue engineering and biomedical applications. [1] A key aspect of the interfacing of biological material with an artificial substrate is the surface chemistry. This has led to the development of various types of surface chemistry, often in the form of reactive monolayers, thereby allowing the controlled immobilization of biomolecules while paying attention to such properties as control over orientation, retention of biological activity, and selectivity and specificity of the biological interaction. [2] The fabrication of fluorogenic monolayers on surfaces can yield fast, simple, sensitive and nondestructive detection of the immobilization products by fluorescence microscopy. For example, reporter monolayers for immobilization of amine-terminated (bio)molecules [3] for microarrays fabrication [4] and for orthogonal surface modification [5] have been published. This method allows faster and cheaper surface imaging and avoids techniques such as X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF SIMS), and scanning probe microscopy (SPM), while retaining high spatial resolution.The important role that thiols play in nature has encouraged scientists to develop molecular probes for their sensing and quantification in biological systems. [6] For this reason, there are several recent examples of fluorescent and colorimetric sensors for the detection of thiols in solution. However, to the best of our knowledge, no examples exist in the area of surface platforms for biological applications. Developing a platform chemistry for thiols has high potential for reasons such as selectivity and orthogonality of immobilization and the controlled orientation of appropriately bioengineered (e.g., cysteine-modified) peptides and proteins.Maleimide chemistry, because of the high selectivity of its reaction with thiols under physiological conditions, is frequently employed for the selective surface immobilization of biomolecules. [2, 7] Therefore, this reaction is a good candidate for the design of rapid methods to visualize in situ covalent binding of thiols on surfaces, with applications in bioconjugation, bioassays, and materials science.Here we report a strategy for the simultaneous anchoring and detection of molecular and biomolecular thiols in a fast manner, by using a fluorogenic reactive monolayer on glass. This monolayer functions as a molecular construction platform in which the fluorogenic response upon immobilization provides spatial identification and coverage assessment of the thiol immobilization. We show its potential in two applications: colocalization of a dye by supramolecular host-guest c...