Characterization of Three Amino-Functionalized Surfaces and Evaluation of Antibody Immobilization for the Multiplex Detection of Tumor Markers Involved in Colorectal Cancer

Antibody microarrays are powerful and high-throughput tools for screening and identifying tumor markers from small sample volumes of only a few microliters. Optimization of surface chemistry and spotting conditions are crucial parameters to enhance antibodies' immobilization efficiency and to maintain their biological activity. Here, we report the implementation of an antibody microarray for the detection of tumor markers involved in colorectal cancer. Three-dimensional microstructured glass slides were functionalized with three different aminated molecules ((3-aminopropyl)dimethylethoxysilane (APDMES), Jeffamine, and chitosan) varying in their chain length, their amine density, and their hydrophilic/hydrophobic balance. The physicochemical properties of the resulting surfaces were characterized. Antibody immobilization efficiency through physical interaction was studied as a function of surface properties as well as a function of the immobilization conditions. The results show that surface energy, steric hindrance, and pH of spotting buffer have great effects on protein immobilization. Under optimal conditions, biological activities of four immobilized antitumor marker antibodies were evaluated in multiplex immunoassay for the detection of the corresponding tumor markers. Results indicated that the chitosan functionalized surface displayed the highest binding capacity and allowed to retain maximal biological activity of the four tested antibody/antigen systems. Thus, we successfully demonstrated the application of amino-based surface modification for antibody microarrays to efficiently detect tumor markers.

Stability and physical properties of alkylsilane and alkylphosphonate self-assembled monolayer (SAM) films on metal oxide substrates characterized at the micro- and nanoscale

Enamul Hoque

Self-assembled monolayer (SAM) films have attracted immense attention for both fundamental and applied research. A SAM is composed of a large number of molecules with a head group that chemisorbs onto a substrate, a tail group that interacts with the outer surface of the film, and a spacer (backbone) chain group that connects the head and tail groups resulting in a coating. Interactions between spacer groups of different molecules, such as van der Waals forces and/or hydrogen bonding, hasten SAM film formation and contribute to its stability. In this dissertation, SAM and thin films have been formed onto copper and aluminum oxide surfaces by reaction with 1H,1H,2H,2H-perfluorodecyldimethylchlorosilane (PFMS), 1H,1H,2H,2H-perfluorodecyltrichlorosilane (PFTS), 1H,1H,2H,2H-perfluorodecylphosphonic acid (PFDP), octylphosphonic acid (OP), decylphosphonic acid (DP), and octadecylphosphonic acid (ODP). The properties and stability of the films were investigated employing complementary surface analysis techniques: X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), friction force microscopy (FFM), a derivative of AFM, contact angle measurements (CAMs), and Fourier transform infrared reflection/absorption spectroscopy (FT-IRRAS). The perfluoroalkylsilane SAM on Cu is found to be extremely hydrophobic typically having sessile drop static contact angles of more than 130° for pure water and a surface energy of 14 mJ/m2 (mN/m). FFM showed a significant reduction in the adhesive force and friction coefficient of PFMS modified Cu (PFMS/Cu) compared to unmodified Cu. Treatment by exposure to harsh conditions showed that PFMS/Cu SAM can withstand boiling nitric acid (pH=1.8), boiling water, and warm sodium hydroxide (pH=12, 60 °C) solutions for at least 30 minutes. Furthermore, no SAM degradation was observed when PFMS/Cu was exposed to warm nitric acid solution for up to 70 min at 60 °C or 50 min at 80 °C. XPS and FT-IRRAS data reveal a coordination of the PFMS silicon (Si) atom with a cuprate (CuO) molecule present on the oxidized copper substrate. The data give good evidence that the stability of the SAM film on the PFMS modified oxidized Cu surface is largely due to the formation of a siloxy-copper (-Si-O-Cu-) bond via a condensation reaction between silanol (-Si-OH) and copper hydroxide (CuOH). For a PFTS modified Cu surface (PFTS/Cu), the sessile drop static contact angle of pure water has been measured to be more than 125° and the surface energy to be typically less than 16 mJ/m2. Stability tests show that the PFTS/Cu film can survive in boiling pure water for one hour, boiling nitric acid (pH 1.5 or 1.8) for 30 minutes, sodium hydroxide solution (pH 12, 70 °C) for 30 minutes, and autoclave conditions (steam at 134 °C and 3 atmospheres) for 15 minutes. The more commonly used self-assembled monolayer (SAM) modifications of Cu surfaces, e.g. thiol compounds, are significantly less stable than PFTS/Cu. Extremely hydrophobic (low surface energy) and stable PFMS/Cu SAMs and PFTS/Cu films could be useful as corrosion inhibitors in micro/nanoelectronic devices and/or as promoters for anti-wetting, low adhesion surfaces or drop-wise condensation on heat exchange surfaces. XPS analysis confirmed the presence of perfluorinated and non-perfluorinated alkylphosphonate molecules on the PFDP, DP, and ODP SAMs deposited at the aluminum oxide coated silicon (Al/Si) surfaces. The sessile drop static contact angle of pure water on PFDP SAMs was typically more than 130° and on DP and ODP typically more than 125° indicating that the phosphonic acid SAMs reacted with Al samples were very hydrophobic. The surface roughness for PFDP/Al, DP/Al, ODP/Al, and bare Al was approximately 35 nm, as determined by AFM. The surface energy for PFDP/Al was determined to be approximately 11 mJ/m2 by the Zisman plot method compared to 21 mJ/m2 and 20 mJ/m2 for DP/Al and ODP/Al, respectively. PFDP/Al gave the lowest adhesion and friction force while bare Al gave the highest. The adhesion and friction forces for ODP/Al and DP/Al SAMs were in between. ODP, DP, and OP SAMs have been studied in detail on relatively flat aluminum oxide surfaces. The rms surface roughness for ODP/Al, DP/Al, OP/Al, and bare Al was less than 15 nm, as determined by AFM. The sessile drop static contact angle of pure water on ODP/Al and DP/Al was typically more than 115° and on OP/Al typically less than 105°. The surface energy for ODP/Al and DP/Al was determined to be approximately 21 mJ/m2 and 22 mJ/m2, respectively, compared to 26 mJ/m2 for OP/Al. ODP/Al and OP/Al were studied by FFM to better understand their micro-/nano-tribological properties. ODP/Al gave the lowest coefficient of friction values while bare Al gave the highest. The adhesion forces for ODP/Al and OP/Al were comparable. The chemical stability of ODP/Al, PFDP/Al, DP/Al, OP/Al, and PFMS/Al SAMs has been inspected by exposure to warm nitric acid (pH 1.8, 30 min, 60-95 °C). The XPS data and stability against harsh chemical conditions indicate that a type of bond forms between a phosphonic acid (PA) or silane molecule and the oxidized Al surface. Stability tests using warm nitric acid (pH 1.8, 30 min, 60-95 °C) show ODP/Al SAMs to be most stable followed by PFDP/Al, DP/Al, PFMS/Al, and OP/Al SAMs. For PFTS/Al, stability tests demonstrate that modified aluminum is able to survive exposure to warm nitric acid (pH = 1.8, 60 °C, 30 min) indicating some degree of robustness. Hydrophobic, low adhesion, and robust aluminum surfaces have useful applications for micro/nano-electromechanical systems (MEMS/NEMS), such as digital micro-mirror devices (DMDs). These studies are expected to aid in the design and selection of proper lubricants for MEMS/NEMS.

EPFL2007

Revealing the Surface Chemistry for CO2 Hydrogenation on Cu/CeO2–x Using Near-Ambient-Pressure X-ray Photoelectron Spectroscopy

Young Jun Ko, Mo Li, Wen Luo, Emadeddin Oveisi, Thi Ha My Pham, Andreas Züttel

Catalytic reduction of CO2 to valuable products is an attractive route for CO2 recycling. CeO2-supported Cu catalysts have shown high activity and selectivity for the hydrogenation of CO2 to CO. To uncover the origin of their high performance, we prepared a practical and well-defined model of Cu/CeO2–x catalysts with Cu nanoparticles dispersed on a CeO2–x support. We studied the structure and catalytic activity of the practical catalyst and the evolution of the active phase and surface intermediates using near-ambient-pressure X-ray photoelectron spectroscopy (NAP-XPS) over the model catalyst under CO2 hydrogenation conditions. For both model and practical catalysts, metallic copper and partially reduced ceria with oxygen vacancies were found to be active sites for the reduction of CO2 to CO. Over the model catalyst, partial covering of Cu by CeO2–x and diffusion of Cu into CeO2–x was observed, suggesting that a strong interaction between Cu and CeO2–x was favored during CO2 hydrogenation. The surface analysis results indicated that the CO2 hydrogenation proceeded through the following steps: activation of CO2 in the form of carbonate on the surface of CeO2–x, hydrogenation of carbonate to formate on the surface of Cu by the dissociated H2, and conversion of formate to CO. This work provides direct experimental evidence on the surface properties of Cu and ceria during the RWGS reaction and the activation and hydrogenation processes of CO2 over the Cu/CeO2–x surface for a high RWGS catalytic performance.

ACS2021

Functional polymer brushes via surface-initiated controlled radical polymerizations

Stefano Tugulu

The term polymer brush refers to a well defined arrangement of polymer chains, which are tethered with one end to an interface and usually the surface of a solid substrate. Due to steric repulsion the polymer chains furthermore adopt a defined, stretched chain conformation, which significantly differs from the random walk conformation of free polymer chains in solution or in conventionally solution casted polymer coatings. From a structural point of view polymer brushes can therefore be regarded as the most defined type of polymer coating that can currently be realized. Especially the combination of surface-initiated polymerizations (SIP) with modern, controlled polymerization methods thereby allows tailoring the structure of the polymer brushes almost on the molecular level. Specifically precise control over the thickness, composition, chain architecture, grafting density and via the use of lithographic techniques also over the topography of the brushes can be achieved. This high degree of control over the structural and implicated (physico-)chemical properties of the brush allows at the same time tailoring and fine tuning the interfacial properties of the brush. This thesis describes the systematic exploitation of well-defined polymer brushes as functional coatings with fine-tuned physicochemical properties to direct and control the interaction with and the response of specific biological, bioanalytical and even reactive chemical environments. In detail the use of surface-initiated atom transfer radical polymerization (SI-ATRP) for the fabrication of functional polymer brushes will be presented. These brushes were used as highly defined coatings in various applications ranging from biomedical and bioanalytical applications to biomimetic materials fabrication (Figure 1). Figure 1 Numerous synthetic approaches for the preparation of polymer brushes have been described in the literature. Chapter 2 will therefore present a summary of the most important types of surface-initiated polymerizations, focusing especially on controlled SIP methods and SI-ATRP. Chapter 3 describes a chemoselective and specific immobilization strategy to decorate intrinsically bioinert polymer brushes with proteins of interest in a defined orientation and density. Specifically the resulting substrates represent attractive candidates for the realization of protein microarrays. In view of this application protein-small molecule and protein-protein interactions as well as posttranslational modifications were analyzed within a feasibility study. Due to their well-defined structure polymer brushes represent also attractive candidates for the realization of molecularly defined cell adhesion substrates for tissue engineering or as highly defined biophysical model system to analyze cell adhesion and mechanics. Chapter 4 describes the decoration of intrinsically bioinert polymer brushes with small peptide ligands with defined density to induce integrin specific cell adhesion on the brushes. Peptide functionalized polymer brushes could be readily endothelialized and adhering cell layers have shown to preserve their homeostatic ability to respond to an applied mechanical stimulus in a physiological manner. In Chapter 5 the limitations of high density polymer brushes in biomedical applications were assessed by analyzing their stability under in vitro cell culture conditions. Intrinsically bioinert high density polymer brushes were found to degraft from glass or silicon substrates upon swelling in good solvents, which lead to deterioration of the bioinert properties of the substrate. The degrafting of the brushes might be connected to swelling induced mechanical activation of chemical bonds, which link the brushes to the substrate. Chapter 6 describes the transfer of the developed SIP approach onto flexible PDMS substrates by exploiting the formation of interpenetrating networks of ATRP-initiator siloxane and PDMS. The modification of the PDMS substrates with hydrophilic brushes effectively reduces the unspecific protein adsorption on the substrates. This approach might therefore represent an attractive and facile route for the surface modification of miniaturized PDMS devices for biomedical or bioanalytical applications. In Chapter 7 the systematic development of a reliable and robust protocol for the aqueous SI-ATRP of sodium methacrylate is presented, which gives access to poly(methacrylic acid) (PMAA) brushes with defined molecular weight and grafting density. The resulting brushes were assessed for their ability to direct the mineralization of calcium carbonate. Chapter 8 describes the use of photolithographically patterned poly(methacrylic acid) brushes as biomimetic, acidic macromolecular matrix to fabricate microstructured calcite thin films that are an exact 3D replica of the PMAA brush. The presented strategy relies on three key elements: (i) the use of photolithographic techniques to prepare microstructured PMAA brushes; (ii) the ability of PMAA brushes to stabilize amorphous calcium carbonate (ACC) and (iii) the possibility to convert the metastable ACC phase into a polycrystalline calcite film via a thermal treatment.

EPFL2007

Stability and physical properties of alkylsilane and alkylphosphonate self-assembled monolayer (SAM) films on metal oxide substrates characterized at the micro- and nanoscale

Enamul Hoque

EPFL2007

Functional polymer brushes via surface-initiated controlled radical polymerizations

Stefano Tugulu

EPFL2007