Orthogonal Chemical Functionalization of Au/SiO2/TiW Patterned Substrates.

Macropatterned and micropatterned gold/silicon dioxide/titanium tungsten (Au/SiO2/TiW) substrates were orthogonally functionalized: three different molecules (monovalent silane, thiol, and phosphonic acid) were used to specifically form organolayers on Au, SiO2, or TiW areas of patterned substrates. The orthogonality of the functionalization (i.e., selective grafting of thiol on Au, phosphonic acid on TiW, and silane on SiO2) was assessed by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), Fourier transform infrared spectroscopy (FTIR), and contact angle measurements. These results are especially promising for the selective anchoring of targets (e.g., biomolecules, nanoparticles, nanowires, nanotubes, or other nano-objects) onto patterned zones of multimaterial substrates, such as nanosensors or other nanodevices.

Oxidized Titanium Tungsten Surface Functionalization by Silane-, Phosphonic Acid-, or Ortho-dihydroxyaryl-Based Organolayers.

Titanium tungsten (TiW) films (200 nm thick) were cleaned by oxygen plasma, and the resulting oxidized surfaces were functionalized by 3-aminopropylphosphonic acid (APPA), 3-ethoxydimethylsilylpropylamine (APDMES), or dopamine (DA) to form three different organolayers. The three resulting organolayers were characterized by X-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectrometry, and Fourier transform infrared spectroscopy analyses. The stability of each organolayer was investigated. Our results suggested that the Si-O-Ti or Si-O-W bonds formed by the reactions of APDMES with surface-oxidized TiW were rather labile, whereas the catechol layer was less labile. The APPA layer was the most stable of all tested surface modifications.

Orthogonal chemical functionalization of patterned Au/TiW substrate for selective immobilization of nanoparticles.

The evolution of nanobiosensors stresses the need for multi-material nanopatterned surfaces to enhance sensing performances. Titanium tungsten (TiW) has been mastered and routinely implemented in nanoelectronic devices, in a reproducible way and at industrial production scales. Such a material may be envisioned for use in (bio)chemical nanoelectronic sensors, but the surface functionalization of such material has yet to be studied. In the present article, the orthogonal chemical functionalization of patterned Au on TiW substrates has been explored for the first time. Surface functionalizations were assessed by x-ray photoelectron spectroscopy, polarization modulation infrared reflection-absorption spectroscopy and time-of-flight secondary ion mass spectrometry imaging. Au/TiW patterned substrates were functionalized with mercapto-undecamine. Thanks to the orthogonality of thiol/Au versus phosphonic acid/TiW reactions, only the Au features were modified leading to the amine derivatized surface. This allowed for the localizing of carboxy-functionalized nanoparticles by electrostatic interaction on Au with a selectivity above 10 when compared to TiW.

Novel Concept of Gas Sensitivity Characterization of Materials Suited for Implementation in FET-Based Gas Sensors.

We propose a novel technique to investigate the gas sensitivity of materials for implementation in field-effect transistor-based gas sensors. Our technique is based on the measurement of the surface charge induced by gas species adsorption, using an electrometer. Platinum sensitivity to hydrogen diluted in synthetic air has been evaluated with the proposed charge measurement technique in the operation temperature range from 80 to 190 °C at constant H2 concentration of 4 % and for different concentrations ranging from 0.5 to 4 % at 130 °C.

pH driven addressing of silicon nanowires onto Si3N4/SiO2 micro-patterned surfaces.

pH was used as the main driving parameter for specifically immobilizing silicon nanowires onto Si3N4 microsquares at the surface of a SiO2 substrate. Different pH values of the coating aqueous solution enabled to experimentally distribute nanowires between silicon nitride and silicon dioxide: at pH 3 nanowires were mainly anchored on Si3N4; they were evenly distributed between SiO2 and Si3N4 at pH 2.8; and they were mainly anchored on SiO2 at pH 2. A theoretical model based on DLVO theory and surface protonation/deprotonation equilibria was used to study how, in adequate pH conditions, Si nanowires could be anchored onto specific regions of a patterned Si3N4/SiO2 surface. Instead of using capillary forces, or hydrophilic/hydrophobic contrast between the two types of materials, the specificity of immobilization could rely on surface electric charge contrasts between Si3N4 and SiO2. This simple and generic method could be used for addressing a large diversity of nano-objects onto patterned substrates.

Analyses of Acceptability Judgments Made Toward the Use of Nanocarrier-Based Targeted Drug Delivery: Interviews with Researchers and Research Trainees in the Field of New Technologies.

The assessment of nanotechnology applications such as nanocarrier-based targeted drug delivery (TDD) has historically been based mostly on toxicological and safety aspects. The use of nanocarriers for TDD, a leading-edge nanomedical application, has received little study from the angle of experts' perceptions and acceptability, which may be reflected in how TDD applications are developed. In recent years, numerous authors have maintained that TDD assessment should also take into account impacts on ethical, environmental, economic, legal, and social (E3LS) issues in order to lead to socially responsible innovation. Semi-structured interviews (n = 22) were conducted with French and Canadian researchers and research trainees with diverse disciplinary backgrounds and involved in research related to emerging technologies. The interviews focussed on scenarios presenting two types of TDD nanocarriers (carbon, synthetic DNA) in two contexts of use (lung cancer, seasonal flu). Content and inductive analyses of interviews showed how facets of perceived impacts such as health, environment, social cohabitation, economy, life and death, representations of the human being and nature, and technoscience were weighed in acceptability judgments. The analyses also revealed that contextual factors related to device (nature of the treatment), to use (gravity of the disease), and to user (culture) influenced the weighting assigned to perceived impacts and thus contributed to variability in interviewees' judgments of acceptability. Giving consideration to researchers' perspective could accompany first steps of implementation and development of nanomedicine by producing a first, but wide, picture of the acceptability of nanocarrier-based TDD.

Effects of disciplinary cultures of researchers and research trainees on the acceptability of nanocarriers for drug delivery in different contexts of use: a mixed-methods study.

The acceptability of nanomedical applications, which have the potential to generate ethical and societal impacts, is a significant factor in the deployment of nanomedicine. A lack of fit between nanomedical applications and society's values may result from a partial consideration of such impacts. New approaches for technological evaluation focused on impact perception, acceptance, and acceptability are needed to go beyond traditional technology assessment approaches used with nanotechnology, which focus mainly on toxicological and safety criteria. Using a new evaluative approach based on perceived impacts of nanotechnology, the objective of this study was to assess perceptions among researchers and research trainees familiar with emergent technologies and from different disciplinary background the scope of acceptability judgments made towards the use of nanocarriers. This mixed-methods study was based on scenarios presenting two types of drug-delivery nanocarriers (carbon, synthetic DNA) in two contexts of use (lung cancer treatment, seasonal flu treatment). Researchers and research trainees in the natural sciences and engineering, and the social sciences and the humanities were invited by email to take part in this project. An online questionnaire followed by semi-directed interviews allowed characterization of disciplinary divergences regarding to impact perception, acceptance, and acceptability of the scenarios. The results suggest that impact perception is influenced by disciplinary culture. Also, trends can be seen between respondents' profiles and variables of acceptance and acceptability, and certain components of the acceptability judgement are specific to each disciplinary culture. The acknowledgment and consideration of these disciplinary divergences could allow, among others, for opening up interdisciplinary dialogue on matters related to the acceptability of nanomedical applications and their developments.

The acceptability of nanocarriers for drug delivery in different contexts of use: perceptions of researchers and research trainees in the field of new technologies.

BACKGROUND: Despite marked optimism in the field of nanomedicine about the use of drug-delivery nanocarriers, uncertainties exist concerning nanocarriers' possible unintended impacts and effects. These uncertainties could affect user acceptance and acceptability. "Acceptance" refers to the intention to put a technology or a device to a specified use. "Acceptability" refers to a value judgment that accounts for acceptance. The objectives of this study were to characterize impact perception, acceptance, and acceptability in relation to drug-delivery nanocarriers in different contexts of use, and to explore relationships among these concepts. METHODS: A sample of European and Canadian researchers and graduate research trainees active in the field of new technologies was recruited by targeted email invitation for participation in a web-based questionnaire study. The questionnaire presented scenarios for two contexts of use (lung cancer, seasonal flu) of drug-delivery nanocarriers with two compositions (carbon, synthetic DNA). Respondents' impact perception, acceptance, and acceptability judgment in relation to each kind of nanocarrier in each context of use were measured with Likert scale questions and scored using categorical values. RESULTS: Two hundred and fourteen researchers and graduate research trainees completed the questionnaire. The results showed that nanocarrier composition influenced impact perception: as compared with the carbon nanocarrier impact perception, the positive impacts of the synthetic DNA nanocarrier were perceived as more significant and more likely to occur than its negative impacts. Composition did not influence acceptance or acceptability. Context of use significantly influenced acceptance and acceptability of both kinds of nanocarriers: researchers were more likely to accept the use of nanocarriers to treat lung cancer than the seasonal flu. The results also showed a significant relationship between acceptance and the perceived usefulness of the treatments. CONCLUSION: Nanocarrier composition does not appear to influence acceptance or acceptability. On the other hand, the nanocarriers' perceived usefulness and context of use are both major factors in accounting for acceptance and acceptability.

Nanoparticles selectively immobilized onto large arrays of gold micro and nanostructures through surface chemical functionalizations.

Latex nanoparticles (100nm and 200nm diameter) were precisely located onto the gold regions of micro and nanopatterned gold/silica substrates through surface chemical functionalizations. The gold patterns were selectively functionalized with alkylthiols bearing biotin or amine headgroups. This selective functionalization allowed the trapping of streptavidin- or carboxy-functionalized latex nanoparticles onto the gold structures with very little non-specific adsorption onto the surrounding silica. Quantitative data of nanoparticle capture on gold and silica, obtained through SEM image analysis, showed a one to two order of magnitude increase on gold with a similar low coverage on silica (non-specific adsorption) thanks to chemical functionalizations. Single nanoparticles were captured at the gap of dimer gold nanostructures.

Orthogonal chemical functionalization of patterned gold on silica surfaces.

Single-step orthogonal chemical functionalization procedures have been developed with patterned gold on silica surfaces. Different combinations of a silane and a thiol were simultaneously deposited on a gold/silica heterogeneous substrate. The orthogonality of the functionalization (i.e., selective grafting of the thiol on the gold areas and the silane on the silica) was demonstrated by X-ray photoelectron spectroscopy (XPS) as well as time-of-flight secondary ion mass spectrometry (ToF-SIMS) mapping. The orthogonal functionalization was used to immobilize proteins onto gold nanostructures on a silica substrate, as demonstrated by atomic force microscopy (AFM). These results are especially promising in the development of future biosensors where the selective anchoring of target molecules onto nanostructured transducers (e.g., nanoplasmonic biosensors) is a major challenge.

Carbodiimide/NHS derivatization of COOH-terminated SAMs: activation or byproduct formation?

COOH-terminated self-assembled monolayers (SAMs) are widely used in biosensor technology to bind different amine-containing biomolecules. A covalent amide bond, however, can be achieved only if the carboxylic acids are activated. This activation process usually consists of forming an N-hydroxysuccinimidyl ester (NHS-ester) by consecutively reacting carboxylic acids with a carbodiimide and NHS. Though many papers report using this method,1-8 the experimental conditions vary greatly between them and chemical characterization at this stage is often omitted. Evidence of an efficient activation is therefore rarely shown. Furthermore, recent publications9-11 have highlighted the complexity of this process, with the possible formation of different byproducts. In this paper, we have conducted a study on NHS activation under different conditions with chemical characterization by polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS) and time-of-flight secondary ion mass spectroscopy (ToF-SIMS). Our results indicate that the nature of the solvent and carbodiimide and the reactant concentrations play crucial roles in activation kinetics and efficiency.

Multiplexed binding determination of seven glycoconjugates for Pseudomonas aeruginosa lectin I (PA-IL) using a DNA-based carbohydrate microarray.

The binding of seven multivalent glycoconjugates displaying linear or antenna-like structures and different electronic environments were evaluated towards PA-IL on a DNA-based carbohydrate microarray. The affinity can be modulated by the charge and the topology of the galactosylated derivatives.

Evanescent wave fluorescence biosensor combined with DNA bio-barcode assay for platelet genotyping.

An evanescent wave fluorescence biosensor was combined with a DNA bio-barcode assay to resolve problems met in detection of poor biologic samples. Human platelet antigen (HPA) genotyping was used as a demonstrator. Our bio-barcode assay was based on magnetic carboxylatex particles and non-magnetic carboxylatex particles, both functionalized with oligonucleotides. It was assessed for detecting 84mer synthetic oligonucleotides as targets. The assay allows to specifically detect single nucleotide polymorphism with a detection limit of 2 pM of target nucleic acids. The fluorescence detection is achieved in 150 s.

A generic surface chemistry for peptide microarrays implementation: application to the detection of anti-H3 antibody.

Peptide microarray can be implemented by immobilization of peptides on a solid support or by direct on-chip peptide synthesis (OCPS). In the first case, peptide primary sequences can be ensured prior to their immobilization but structural diversity is achieved at high cost in terms of reagents. In the second case, high diversity is achieved with low amount of reagents but the primary and secondary structures cannot be ascertained. In both cases, the immobilization step will influence the overall biological activity. We proposed a strategy where direct peptide on-chip synthesis and peptide immobilization are viewed as complementary approaches. In a first step, OCPS is envisioned for the screening and selection of biologically relevant peptides. In a second step, selected peptides would be synthesized on resin, qualified and immobilized for implementing microarrays (i.e. for diagnosis). A versatile surface chemistry for both OCPS and peptide immobilization was developed allowing for an identical physico-chemical environment for both implementation strategies. In the present report, a 16 mer peptide corresponding to the human histone H3 epitope was synthesized on an amino-functionalized support. Surface stability (including upon deprotection steps) and peptide primary and secondary structures were assessed with Cy3-streptavidine conjugates and with immunoassays. Peptides, either on-chip synthesized or immobilized, exhibited a similar biological activity.

Development of miniaturized immunoassay: influence of surface chemistry and comparison with enzyme-linked immunosorbent assay and Western blot.

Protein microarray technology provides a useful approach for the simultaneous serodetection of various antibodies in low sample volumes. To implement functional protein microarrays, appropriate surface chemistry must be designed so that both the protein structure and the biological activity can be retained. In the current study, two surface chemistries for protein microarrays and immunofluorescent assays were developed. Glass slides were functionalized with N-hydroxysuccinimide (NHS) ester via a monofunctional silane or maleic anhydride-alt-methyl vinyl ether (MAMVE) copolymer to allow covalent grafting of histone proteins. Analytical performance of these microarrays was then evaluated for the detection of anti-histone autoantibodies present in the sera of patients suffering from a systemic autoimmune disease, namely systemic lupus erythematosus (SLE), and the results were compared with those of the classical enzyme-linked immunosorbent assay (ELISA) and Western blot. The detection limit of our MAMVE copolymer microarrays was 50-fold lower than that of the classical ELISA. Furthermore, 100-fold less volume of biological samples was required with these miniaturized immunoassays.

Acid deprotection of covalently immobilized peptide probes on glass slides for peptide microarrays.

Protein microarray technology has shown great advancements in the field of biomedical research and diagnosis, it allows to study and understand protein activities and protein - ligand interactions (e.g. detection of antigen-autoantibody interaction in autoimmune diseases. Autoantibodies are frequently targeted against antigens of the cell nucleus (double and single stranded DNA, histones, and nuclear antigens). The biological activities of proteins (e.g. enzymes, antibodies...) are controlled by peptides sequences of the active site. Consequently, we were interested in the investigation of peptide microarrays in order to further implement in situ peptide synthesis, in particular, deprotection reaction on glass supported peptides. In this work, a protected and biotinylated synthetic peptide was covalently immobilized onto amino functionalized glass surface by activation of its the C-terminus; this allows to orientate the peptide onto the surface. The peptide contains a fragment of the C-terminal end of the human histone H3 protein. The immobilized peptide was then deprotected by using concentrated trifluoroacetic acid solution. After the deprotection, surface stability and peptide grafting density were evaluated by indirect labelling of the immobilized peptide using Cy3 streptavidin conjugates. We also studied biological interaction of IgG polyclonal anti-histone H3 antibody with the immobilized peptide epitope to insure the efficiency of the acid deprotection. The specificity of the antibody interaction with the protected versus non protected peptides. This approach may be applied to in situ synthetic and prototected peptides, in order to elaborate a micro-immunoassay prototype for measurement of peptide-protein interactions on high density microarrays, and detection of antibodies in biological fluids such as serum.

Label-free detection of bacteria by electrochemical impedance spectroscopy: comparison to surface plasmon resonance.

The low but known risk of bacterial contamination has emerged as the greatest residual threat of transfusion-transmitted diseases. Label-free detection of a bacterial model, Escherichia coli, is performed using nonfaradic electrochemical impedance spectroscopy (EIS). Biotinylated polyclonal anti-E. coli is linked to a mixed self-assembled monolayer (SAM) on a gold electrode through a strong biotin-neutravidin interaction. The binding of one antibody molecule for 3.6 neutravidin molecules is determined using the surface plasmon resonance (SPR). The detection limit of E. coli found by SPR is 10(7) cfu/mL. After modeling the impedance Nyquist plot of E. coli/anti-E. coli/mixed SAM/gold electrode for increasing concentrations of E. coli (whole bacteria or lysed bacteria), the main parameter that is modified is the polarization resistance RP. A sigmoid variation of RP is observed when the log concentration of bacteria (whole or lysed) increases. A concentration of 10 cfu/mL whole bacteria is detected by EIS measurements while 103 cfu/mL is detected for lysed E. coli.

Microwave assisted "click" chemistry for the synthesis of multiple labeled-carbohydrate oligonucleotides on solid support.

A versatile approach has been developed for the multiple labeling of oligonucleotides. First, three linkers as a H-phosphonate monoester derivative were condensed on a solid-supported T12 to introduce H-phosphonate diester linkages which were oxidized in the presence of propargylamine. Second, three galactosyl azide derivatives were conjugated to the solid-supported three-alkyne-modified T12 by a 1,3-cycloaddition so-called "click chemistry" in the presence of Cu(I) assisted by microwaves.

Assessment of 35mer amino-modified oligonucleotide based microarray with bacterial samples.

Parallel quantification of a large number of messenger RNA transcripts, using microarray technology, promises to provide unsuspected information about many cellular processes. Although experimental protocols on microarray applications are available, only limited methodological information on glass-slide manufacturing and signal interpretation has been published. The aim of this paper is to provide new insights into the practical aspects of the construction and hybridization of oligonucleotide-based microarrays. The intracellular symbiotic bacterium of aphids, Buchnera aphidicola, is used here as a model organism. The first part of the work is devoted to the optimization of procedures for printing slides, labeling of cDNA targets and hybridization. In the second part, based on a statistical analysis of the results, we discuss the influence of the probe attachment chemistry, of the labeling method, of the oligonucleotide position and of the concentration of a spotted oligonucleotide on signal intensity. The problem of signal specificity is also addressed, based on the calculation of the fluorescent ratio for each probe to its corresponding mismatch control probe. Lastly, the selection of internal spiked RNAs appropriate to our bacterial samples and useful for the data normalization step is presented.