Biosensors that can determine protein concentration and structure are highly desired for biomedical applications. For the development of such biosensors, the use of Fourier transform infrared (FTIR) spectroscopy with the attenuated internal total reflection (ATR) configuration is particularly attractive, but it requires appropriate surface functionalization of the ATR optical element. Indeed, the surface has to specifically interact with a target protein in close contact with the optical element and must display antifouling properties to prevent nonspecific adsorption of other proteins. Here, we report robust monolayers of calix[4]arenes bearing oligo(ethylene glycol) (oEG) chains, which were grafted on germanium and gold surfaces via their tetradiazonium salts. The formation of monolayers of oEGylated calix[4]arenes was confirmed by AFM, IR, and contact angle measurements. The antifouling properties of these modified surfaces were studied by ATR-FTIR spectroscopy and fluorescence microscopy, and the nonspecific absorption of bovine serum albumin was found to be reduced by 85% compared to that of unmodified germanium. In other words, the organic coating by oEGylated calix[4]arenes provides remarkable antifouling properties, opening the way for the design of germanium- or gold-based biosensors.

Labile ligands such as thiols and carboxylates are commonly used to functionalize AuNPs, though little control over the composition is possible when mixtures of ligands are used. It was shown recently that robustly functionalized AuNPs can be obtained through the reductive grafting of calix[4]arenes bearing diazonium groups on the large rim. Here, we report a calix[4]arene-tetradiazonium decorated by four oligo(ethylene glycol) chains on the small rim, which upon grafting gave AuNPs with excellent stability thanks to the C-Au bonds. Mixtures of this calixarene and one with four carboxylate groups were grafted on AuNPs. The resulting particles were analyzed by infrared spectroscopy, which revealed that the composition of the ligand shell clearly reflected the ratio of calixarenes that was present in solution. This strategy opens the way to robustly protected AuNPs with well-defined numbers of functional or postfunctionalizable groups.

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Many in vivo and in vitro applications using gold nanoparticles (AuNPs) require (i) their PEGylation, as it increases their stability and prevents nonspecific protein adsorption, and (ii) their conjugation to biomolecules, that provides them with specific recognition properties. Currently, the functionalization of AuNPs is based on thiol chemistry that suffers from two major drawbacks: (i) the Au–S bond is labile and confers limited chemical robustness to the organic layer, and (ii) control over the bioconjugation density is highly challenging. We report here a novel functionalization strategy based on calix[4]­arene-tetradiazonium platforms for the coating of AuNPs with a robust PEG layer and their controlled bioconjugation. AuNPs were first modified with a functional calix[4]­arene-diazonium salt bearing three PEG chains ended by a methoxy group and one by a carboxyl group. The resulting particles showed excellent chemical and colloidal stabilities, compared to similar systems obtained via a classical thiol chemistry, and could even be dispersed in human serum without degrading or aggregating. In addition to that, the carboxyl groups protruding from the PEG layer allowed their conjugation via amide bond formation with amine-containing biomolecules such as peptides. The control of the bioconjugation was obtained by grafting mixed layers of functional and nonfunctional PEGylated calix[4]­arenes, that allowed varying the number of functional groups carried by the AuNPs and subsequently their bioconjugation capacity while preserving their dense protective PEG shell. Finally, we used these nanomaterials, modified with peptide aptamers, for the in vitro biosensing of a cancer biomarker, Mdm2.

Germanium is particularly suitable for the design of FTIR-based biosensors for proteins. The grafting of stable and thin organic layers on germanium surfaces remains, however, challenging. To tackle this problem, we developed a calix[4]arene−tetradiazonium salt decorated with four oligo(ethylene glycol) chains and a terminal reactive carboxylic group. This versatile molecular platform was covalently grafted on germanium surfaces to yield robust readyto-use surfaces for biosensing applications. The grafted calixarene monolayer prevents nonspecific adsorption of proteins while allowing bioconjugation with biomolecules such as bovine serum albumin (BSA) or biotin. It is shown that the native form of the investigated proteins was maintained upon immobilization. As a proof of concept, the resulting calix[4]arene-based germanium biosensors were used through a combination of ATR-FTIR spectroscopy and fluorescence microscopy for the selective detection of streptavidin from a complex medium. This study opens real possibilities for the development of sensitive and selective FTIR-based biosensors devoted to the detection of proteins. 47 surfaces (i.e., oxidation and passivation) and lead usually to a 48 weak reproducibility. Moreover, once modified, the surfaces 49 present a poor stability, limiting their use in the field of 50 biosensing. Another strategy relies on the reductive grafting of 51 aryldiazonium salts, which leads to a strong and durable 52 attachment of an organic layer onto the germanium sur-53 face. 18,19 However, because of the high reactivity of the radicals 54 produced upon the reduction of diazonium cations, disordered 55 multilayers are generally obtained through this method. The 56 preparation of well-ordered monolayers with diazonium 57 chemistry remains thus highly challenging, 20−24 though this 58 is a key point when immobilizing receptors to warrant an 59 efficient and sensitive sensing. 25,26 60 In this context, we have recently developed a general 61 strategy for surface modification, which consists in the covalent 62 grafting of molecular platforms based on calix[4]arene− 63 tetradiazonium salts. 27−31 As demonstrated on various 64 materials or nanomaterials, the unique macrocyclic structure 65 of calix[4]arenes enables the formation of robust, dense, and

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