Nanoporous silicon-based surface patterns fabricated by UV laser interference techniques for biological applications

The fabrication of selectively functionalized micropatterns based on nanostructured porous silicon (nanoPS) by phase mask ultraviolet laser interference is presented here. This single-step process constitutes a flexible method for the fabrication of surface patterns with tailored properties. These surface patterns consist of alternate regions of almost untransformed nanoPS and areas where nanoPS is transformed into Si nanoparticles (Si NPs) as a result of the laser irradiation process. The size of the transformed areas as well as the diameter of the Si NPs can be straightforwardly tailored by controlling the main fabrications parameters including the porosity of the nanoPS layers, the laser interference period areas, and laser fluence. The surface patterns have been found to be appropriate candidates for the development of selectively-functionalized surfaces for biological applications mainly due to the biocompatibility of the untransformed nanoPS regions.Postprint (author's final draft

Impedimetric antimicrobial peptide-based sensor for the early detection of periodontopathogenic bacteria

Peri-implantitis, an inflammation caused by biofilm formation, constitutes a major cause of implant failure in dentistry. Thus, the detection of bacteria at the early steps of biofilm growth represents a powerful strategy to prevent implant-related infections. In this regard, antimicrobial peptides (AMPs) can be used as effective biological recognition elements to selectively detect the presence of bacteria. Thus, the aim of the present study was to combine the use of miniaturized and integrated impedimetric transducers and AMPs to obtain biosensors with high sensitivity to monitor bacterial colonization. Streptococcus sanguinis, which is one of the most prevalent strains in the onset of periodontal diseases, was used as a model of oral bacteria. To this end, a potent AMP derived from human lactoferrin was synthesized and covalently immobilized on interdigitated electrode arrays (IDEA). X-ray photoelectron spectroscopy (XPS) and electrochemical impedance spectroscopy (EIS) were employed to optimize and characterize the method of immobilization. Noteworthy, the interaction of Streptococcus sanguinis with AMP-coated sensors provoked significant changes in the impedance spectra, which were univocally associated with the presence of bacteria, proving the feasibility of our method. In this regard, the developed biosensor permits to detect the presence of bacteria at concentrations starting from 101 colony forming units (CFU) mL-1 in KCl and from 102 CFU mL-1 in artificial saliva. Moreover, the system was devoid of cytotoxicity for human fibroblasts. These results indicate that the proposed approach can be effective in the detection of initial stages of biofilm formation, and may be useful in the early prevention and treatment of peri-implantitisPeer ReviewedPostprint (author's final draft

Biofunctionalization with a TGFβ-1 Inhibitor Peptide in the Osseointegration of Synthetic Bone Grafts: An In Vivo Study in Beagle Dogs

Objectives: The aim of this research was to determine the osseointegration of two presentations of biphasic calcium phosphate (BCP) biomaterial—one untreated and another submitted to biofunctionalization with a TGF- 1 inhibitor peptide, P144, on dental alveolus. The biofunctionalization of the biomaterial’s granules maintains a stable membranous bone formation throughout the experiment timeline, benefitting from the constant presence of vascular structures in the alveolar space, in a more active manner that in the control samples. Better results in the experimental groups were proven both by quantitative and qualitative analysis. Synthetic bone graft biofunctionalization results in slightly better quantitative parameters of the implant’s osseointegration. The qualitative histological and ultramicroscopic analysis shows that biofunctionalization may shorten the healing period of dental biomaterials.The present study was supported entirely by the Research Cathedra Klockner-University of Granada

Cell adhesive peptides functionalized on CoCr alloy stimulate endothelialization and prevent thrombogenesis and restenosis

Immobilization of bioactive peptide sequences on CoCr surfaces is an effective route to improve endothelialization, which is of great interest for cardiovascular stents. In this work, we explored the effect of physical and covalent immoblization of RGDS, YIGSR and their equimolar combination peptides on endothelial cells (EC) and smooth muscle cell (SMC) adhesion and on thrombogenicity. We extensively investigated using RT-qPCR, the expression by ECs cultured on functionalised CoCr surfaces of different genes. Genes relevant for adhesion (ICAM-1 and VCAM-1), vascularization (VEGFA, VEGFR-1 and VEGFR-2) and anti-thrombogenicity (tPA and eNOS) were over-expressed in the ECs grown to covalently functionalized CoCr surfaces compared to physisorbed and control surfaces. Pro-thrombogenic genes expression (PAI-1 and vWF) decreased over time. Cell co-cultures of ECs/SMCs found that functionalization increased the amount of adhered ECs onto modified surfaces compared to plain CoCr, independently of the used peptide and the strategy of immobilization. SMCs adhered less compared to ECs in all surfaces. All studied peptides showed a lower platelet cell adhesion compared to TCPS. Covalent functionalization of CoCr surfaces with an equimolar combination of RGDS and YIGSR represented prevailing strategy to enhance the early stages of ECs adhesion and proliferation, while preventing SMCs and platelet adhesion.Postprint (author's final draft

Study and characterization of modified silicon surfaces with organic molecules

Nanostructured thin films and subsequent biofunctionalization of silicon substrates are essential for the development of biosensors devices. The formation of organic monolayers on silicon substrates via Si-C bound allows specific interactions with biomolecules and presents several advantages like greater detection sensitivity and stability against hydrolytic cleavage.1 In this sense, to control the orientation and spacing between grafted functional moieties on the surface, tripodal oligo (p-phenylene)s have become ideal anisotropic adsorbates due to their shape-persistent and selfstanding characteristics.2 On the other hand, biomolecules such as tehtahydro[3]benzazepines are well-known to contain in their structure a phenetylamine skeleton, which is also present in dopaminergic receptors and drugs, therefore these molecules have a remarkable interest in medicinal chemistry. Here we report the synthesis and characterization of several tetrahydro[3]benzazepines and tripod-shaped oligo(p-phenylene)s which were suitably functionalized for its subsequent adsorption on silicon surfaces by hydrosilylation and/or CuAAc click reaction. X-ray photoemission spectroscopy (XPS) and atomic force microscopy (AFM) analysis were also carried out to reveal the presence of the grafted molecules on the different Si surfaces.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Folate-based single cell screening using surface enhanced Raman microimaging

Recent progress in nanotechnology and its application to biomedical settings have generated great advantages in dealing with early cancer diagnosis. The identification of the specific properties of cancer cells, such as the expression of particular plasma membrane molecular receptors, has become crucial in revealing the presence and in assessing the stage of development of the disease. Here we report a single cell screening approach based on Surface Enhanced Raman Scattering (SERS) microimaging. We fabricated a SERS-labelled nanovector based on the biofunctionalization of gold nanoparticles with folic acid. After treating the cells with the nanovector, we were able to distinguish three different cell populations from different cell lines (cancer HeLa and PC-3, and normal HaCaT lines), suitably chosen for their different expressions of folate binding proteins. The nanovector, indeed, binds much more efficiently on cancer cell lines than on normal ones, resulting in a higher SERS signal measured on cancer cells. These results pave the way for applications in single cell diagnostics and, potentially, in theranostic

Real Time Monitoring of a UV Light-Assisted Biofunctionalization Protocol Using a Nanophotonic Biosensor

[EN] A protocol for the covalent biofunctionalization of silicon-based biosensors using a UV light-induced thiol-ene coupling (TEC) reaction has been developed. This biofunctionalization approach has been used to immobilize half antibodies (hIgG), which have been obtained by means of a tris(2-carboxyethyl)phosphine (TCEP) reduction at the hinge region, to the surface of a vinyl-activated silicon-on-insulator (SOI) nanophotonic sensing chip. The response of the sensing structures within the nanophotonic chip was monitored in real time during the biofunctionalization process, which has allowed us to confirm that the bioconjugation of the thiol-terminated bioreceptors onto the vinyl-activated sensing surface is only initiated upon UV light photocatalysis.This work was supported by the Horizon 2020 Programme of the European Union under the project H2020-PHC-634013 (PHOCNOSIS).Sabek, J.; Torrijos-Morán, L.; Griol Barres, A.; Díaz-Betancor, Z.; Bañuls Polo, M.; Maquieira Catala, Á.; García-Rupérez, J. (2018). Real Time Monitoring of a UV Light-Assisted Biofunctionalization Protocol Using a Nanophotonic Biosensor. Biosensors. 9(1):1-9. https://doi.org/10.3390/bios90100061991Chin, C. D., Linder, V., & Sia, S. K. (2007). Lab-on-a-chip devices for global health: Past studies and future opportunities. Lab Chip, 7(1), 41-57. doi:10.1039/b611455eWu, J., Dong, M., Santos, S., Rigatto, C., Liu, Y., & Lin, F. (2017). Lab-on-a-Chip Platforms for Detection of Cardiovascular Disease and Cancer Biomarkers. Sensors, 17(12), 2934. doi:10.3390/s17122934Estevez, M. C., Alvarez, M., & Lechuga, L. M. (2011). Integrated optical devices for lab-on-a-chip biosensing applications. Laser & Photonics Reviews, 6(4), 463-487. doi:10.1002/lpor.201100025Vestergaard, M., Kerman, K., & Tamiya, E. (2007). An Overview of Label-free Electrochemical Protein Sensors. Sensors, 7(12), 3442-3458. doi:10.3390/s7123442Johnson, B. N., & Mutharasan, R. (2012). Biosensing using dynamic-mode cantilever sensors: A review. Biosensors and Bioelectronics, 32(1), 1-18. doi:10.1016/j.bios.2011.10.054Jonkheijm, P., Weinrich, D., Schröder, H., Niemeyer, C. M., & Waldmann, H. (2008). Chemical Strategies for Generating Protein Biochips. Angewandte Chemie International Edition, 47(50), 9618-9647. doi:10.1002/anie.200801711Phaner-Goutorbe, M., Dugas, V., Chevolot, Y., & Souteyrand, E. (2011). Silanization of silica and glass slides for DNA microarrays by impregnation and gas phase protocols: A comparative study. Materials Science and Engineering: C, 31(2), 384-390. doi:10.1016/j.msec.2010.10.016Escorihuela, J., Bañuls, M.-J., Grijalvo, S., Eritja, R., Puchades, R., & Maquieira, Á. (2014). Direct Covalent Attachment of DNA Microarrays by Rapid Thiol–Ene «Click» Chemistry. Bioconjugate Chemistry, 25(3), 618-627. doi:10.1021/bc500033dEscorihuela, J., Bañuls, M. J., Puchades, R., & Maquieira, Á. (2012). DNA microarrays on silicon surfaces through thiol-ene chemistry. Chemical Communications, 48(15), 2116. doi:10.1039/c2cc17321bGonzález-Lucas, D., Bañuls, M.-J., García-Rupérez, J., & Maquieira, Á. (2017). Covalent attachment of biotinylated molecular beacons via thiol-ene coupling. A study on conformational changes upon hybridization and streptavidin binding. Microchimica Acta, 184(9), 3231-3238. doi:10.1007/s00604-017-2310-4Alonso, R., Jiménez-Meneses, P., García-Rupérez, J., Bañuls, M.-J., & Maquieira, Á. (2018). Thiol–ene click chemistry towards easy microarraying of half-antibodies. Chemical Communications, 54(48), 6144-6147. doi:10.1039/c8cc01369aGonzález-Guerrero, A. B., Alvarez, M., Castaño, A. G., Domínguez, C., & Lechuga, L. M. (2013). A comparative study of in-flow and micro-patterning biofunctionalization protocols for nanophotonic silicon-based biosensors. Journal of Colloid and Interface Science, 393, 402-410. doi:10.1016/j.jcis.2012.10.040Povinelli, M. L., Johnson, S. G., & Joannopoulos, J. D. (2005). Slow-light, band-edge waveguides for tunable time delays. Optics Express, 13(18), 7145. doi:10.1364/opex.13.007145Ruiz-Tórtola, Á., Prats-Quílez, F., González-Lucas, D., Bañuls, M.-J., Maquieira, Á., Wheeler, G., … García-Rupérez, J. (2018). High sensitivity and label-free oligonucleotides detection using photonic bandgap sensing structures biofunctionalized with molecular beacon probes. Biomedical Optics Express, 9(4), 1717. doi:10.1364/boe.9.001717Golas, A., Parhi, P., Dimachkie, Z. O., Siedlecki, C. A., & Vogler, E. A. (2010). Surface-energy dependent contact activation of blood factor XII. Biomaterials, 31(6), 1068-1079. doi:10.1016/j.biomaterials.2009.10.03

Near ambient pressure X-ray photoelectron spectroscopy monitoring of the surface immobilization cascade on a porous silicon-gold nanoparticle FET biosensor

Porous silicon (PSi) offers extremely attractive optical, electronic and biofunctional properties for the development of biosensors. In the present work, we have studied the step by step sandwich biofunctionalization cascade of a PSi platform by near ambient pressure X-ray photoelectron spectroscopy (NAP-XPS) and, in parallel, we have developed a three electrode PSi device sensitive to changes in surface conductance. Prior to the NAP-XPS characterization, the organosilanization with glycidyloxy-propyl-trimethoxy-silane, the bioconjugation, and the gold nanoparticle (AuNP) sensitization layer were monitored by spectroscopic ellipsometry. The NAP-XPS analysis revealed outstanding results: a) the NAP-XPS chamber allows detecting the pristine PSi with negligible adventitious carbon contamination, b) the single oxygen bonded carbon component of the Glycidyl group dominates the C1s core level after organosilanization, c) the good progress of the biofunctionalization/recognition is confirmed by the increase of the silica to silicon component ratio in the Si2p core level and, d) the N1s core level describes identical features from the presence of aminoacid sequences in the capture/detection steps. A FET sensing of a prostate specific antigen (PSA) marker was performed through conjugation with AuNPs. For a given concentration of PSA (and AuNPs) the conductance increased with the increase of the gate voltage. For a given gate voltage, the conductance was observed to increase for increasing concentration of PSA. This allowed proposing a calibration line for the biosensor, which is valid from a clinically relevant range of 0.1 ng/mLWe acknowledge MSC funding provided by the European Commission through FP7 grant THINFACE (ITN GA 607232) and from Ministerio de Economía y Competitividad, Spain, through grant SPECTRASENSE (RTC-2017-6311-1

Albumin biofunctionalization to minimize the Staphylococcus aureus adhesion on solid substrates

Staphylococcus aureus has become the most common opportunistic microorganism related to nosocomial infections due to the bacteria capacity to form biofilms on biomedical devices and implants. Since bacterial adhesion is the first step in this pathogenesis, it is evident that inhibiting such a process will reduce the opportunity for bacterial colonization on the devices. This work is aimed at optimizing a surface biofunctionalization strategy to inhibit the adhesion of S. aureus on solid substrates. The first part of the work deals with the albumin adsorption-desorption process, studied by a factorial design of experiments to explore a wide range of experimental factors (protein concentration, pH, flow rate and adsorption time) and responses (initial adsorption rate, adsorbed amount, desorbed extent) for hydrophilic and hydrophobic substrates, with a reduced number of experiments. This approach allows the simultaneous evaluation of the factors affecting the albumin adsorption-desorption process to find a qualitative correlation with the amount of alive S. aureus adhered on albumin biofunctionalized substrates. The results of this work point to a relationship between bacterial adhesion and the degree of albumin relaxation on the solid substrate. In fact, the inhibition of bacterial adhesion on albumin biofunctionalized substrates is due to the surface perturbation on the native structure of the protein. On this base, a biofunctionalization strategy was designed using a solution of thermally treated albumin molecules (higher β-sheet or unordered secondary structure elements) to biofunctionalize solid substrates by dipping. With these albumin biofunctionalized substrates S. aureus adhesion was minimized.Fil: Martín, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; ArgentinaFil: Pfaffen, Maria Valeria del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; ArgentinaFil: Valenti, Laura Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; ArgentinaFil: Giacomelli, Carla Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentin