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Introduction Understanding the interaction between the biological environment (tissues, cells, proteins, electrolytes, etc.) and a solid surface is crucial for biomedical applications such as bio-sensors, bio-electronics, tissue engineering and the optimization of implant materials. Cells, the cornerstones of living tissue, perceive their surroundings and subsequently modify it by producing extracellular matrix (ECM), which serves as a basis to simplify their adhesion, spreading and differentiation (Shakenraad & Busscher, 1989). This process is considerably complex, flexible and strongly depends on the cell cultivation conditions including the type of the substrate. Surface roughness of the substrate plays an important role (Babchenko et al., 2009; Kalbacova et al., 2009; Kromka et al., 2009; Zhao et al., 2006), other influential factors include both the porosity (Tanaka et al., 2007) and the wettability of the substrate, the latter influencing protein conformation (Browne et al., 2004; Rezek, Ukraintsev, Michalíková, Kromka, Zemek & Kalbacova, 2009) as well as the adsorption and viability of cells (Grausova et al., 2009; Kalbacova, Kalbac, Dunsch, Kromka, Vanecek, Rezek, Hempel & Kmoch, 2007). Materials which are commonly employed as substrates for in vitro testing are polystyrene and glass. In this context, diamond as a technological material can provide a relatively unique combination of excellent semiconducting, mechanical, chemical as well as biological properties (Nebel et al., 2007). Diamond also meets the basic requirements for large-scale industrial application, most notably, it can be prepared synthetically. Diamond can be synthesized either as a bulk material under high-pressure and high-temperature conditions, or in the form of thin films by chemical vapor deposition of methane and hydrogen on various substrates including glass and metal (Kromka et al., 2008; Potocky et al., 2007). Moreover, the application of selective nucleation makes it possible to directly grow conductive diamond microstructures, which operate e.g. as transistors or pH sensors (Kozak et al., 2010). Nowadays, it is possible to deposit diamond even on large areas (600 cm 2 or more) using linear antennas (Kromka et al., 2011; Tsugawa et al., 2010). The excellent compatibility of diamond with biological materials and environment (Bajaj et al., 2007; Grausova et al., 2009; Diamond as Functional Material for Bioelectronics and Biotechnology Bohuslav Rezek 1 , Marie Krátká 1 , Egor Ukraintsev 1 , Oleg Babchenko 1 , Alexander Kromka 1 , Antonín Brož 2 and Marie Kalbacova 2 1 Institute of Physics, Academy of Sciences of the Czech Republic, Prague 2 Institute of Inherited Metabolic Diseases, First Faculty of Medicine, Charles University and General Faculty Hospital in Prague Czech Republic 8 2 Will-be-set-by-IN-TECH Kalbacova, Kalbac, Dunsch, Kromka, Vanecek, Rezek, Hempel & Kmoch, 2007; Tang et al., 1995) is of immense importance for its application in medicine. This bio-compatibility stems from the fact that diamond is a crystalline form of carbon that is mechanically, chemically and physically very stable. Despite the general chemical stability, diamond surface can be terminated by different atomic species (Rezek et al., 2003) and organic molecules (Rezek, Shin, Uetsuka & Nebel, 2007), which can alter diamond’s natural properties and thus open the door for countless new applications. For example, electrical conductance and electron affinity are both significantly influenced by surface termination of diamond by hydrogen or oxygen atoms (Chakrapani et al., 2007; Kawarada, 1996; Maier et al., 2001; Rezek et al., 2003; Ri et al., 1995). The main difference arises from the opposite dipoles of C–H and C–O bonds. Oxygen-terminated diamond is insulating, whereas the hydrogen-terminated surface causes the emergence of two-dimensional hole surface conductance on otherwise insulating diamond. These properties can be exploited for the fabrication of a planar field-effect transistor (FET), whose gate is formed solely by hydrogen surface atoms without the employment of any other insulating layers and which is sensitive to the pH of a solution (Dankerl et al., 2007; Nebel et al., 2006; Rezek, Shin, Watanabe & Nebel, 2007). The hydrogen-terminated diamond surface is also an ideal starting point for covalent bonding of other molecules such as DNA or proteins (Härtl et al., 2004; Rezek, Shin, Uetsuka & Nebel, 2007; Yang et al., 2002). On the other hand, the hydrogen-terminated diamond surface is generally less favorable for the adhesion, spreading and viability of cells than the oxidized surface (Kalbacova, Kalbac, Dunsch, Kromka, Vanecek, Rezek, Hempel & Kmoch, 2007). This difference is due to the hydrophillicity of oxygen-terminated diamond (O-diamond) in contrast to the hydrophobicity of the hydrogen-terminated diamond (H-diamond). As a result, the combination of both hydrogen- and oxygen-terminated diamond surface is very interesting for bio-electronics (Dankerl et al., 2009; Rezek, Krátká, Kromka & Kalbacova, 2010) as well as for tissue engineering (Kalbacova et al., 2008; Rezek, Michalíková, Ukraintsev, Kromka & Kalbacova, 2009). In this chapter we present the influence of micro-structuring morphology and atomic termination of diamond surfaces on cell growth and assembly. We investigate the influence of key parameters such as the seeding concentration of cells, the type of the applied cells, the duration of cultivation, the concentration of fetal bovine serum (FBS) in the cultivation medium, the dimensions and shape of microstructures, and surface roughness. We show that the adsorption of proteins from the FBS serum is the key factor. Atomic force microscopy (AFM) both in solution and in air is applied in order to characterize the morphology of the FBS layers adsorbed on differently terminated diamond substrates. The influence of proteins and cells on the electronic properties of diamond is demonstrated by employing a field-effect transistor on hydrogen-terminated diamond, whose gate is exposed to a solution (SG-FET). These results are discussed from the point of view of fundamental physics and biology as well as the prospects in medicine. 2. Preparation of nanocrystalline diamond layers The growth of thin-film nanocrystalline-diamond layers (NCD) was realized on silicon or glass substrates using microwave plasma enhanced chemical vapor deposition (MW-CVD) (Kromka et al., 2008; Potocky et al., 2007). The substrates were 10 × 10 mm 2 large and had 178 New Perspectives in Biosensors Technology and Applications Diamond as Functional Material for Bioelectronics and Biotechnology 3 Fig. 1. Schematic depiction of the preparation procedure of thin-film diamond on glass or silicon substrates: (a) nucleation of the substrates carried out in an ultrasonic bath with ultra-dispersed diamond (UDD), (b) the resulting nucleation layer and (c) the nanocrystalline diamond layer after the microwave-plasma deposition. The deposition machines for (d) large-area growth of diamond (linear plasma) and (e) high-speed growth (focused plasma). surface roughness < 1 nm. Before the deposition, the substrates were ultrasonically cleaned in isopropanol and deionized water and were subsequently immersed for 40 min into an ultrasonic bath with a colloidal suspension of a diamond powder (UDD – ultra-dispersed diamond; NanoAmando, New Metals and Chemicals Corp. Ltd., Kyobashi) with nominal particle size of 5 nm. This process leads to the formation of a 5- to 25-nm-thin layer of nanodiamond powder. This nucleation procedure was followed by a microwave plasma-enhanced chemical vapor deposition (MW-CVD) of diamond films. The deposition conditions were: temperature of substrates 600–800°C, 1% CH 4 in H 2 , microwave power 1.4–2.5 kW, gas pressure 30–50 mbar, duration approximately 4 hours, the thickness of layers reaches 100–500 nm. The same conditions, only with methane gas switched off and process time 10 min, were used for H-termination of the diamond surface. In some cases, the nucleation and growth were repeated on the other side of the substrate, which leads to the hermetical encapsulation of the substrate by the NCD layer (Kalbacova et al., 2008; Rezek, Michalíková, Ukraintsev, Kromka & Kalbacova, 2009). The preparation procedure is schematically shown in Figure 1. This figure also depicts the photos of the set-ups for the large-area diamond growth (linear plasma) with high deposition rate (focused plasma). NCD layer were chemically cleaned in acids (97.5% H 2 SO 4 + 99% KNO 3 powder in the ratio of 4:1) at 200°C for 30 minutes. This process ensures high quality of the hydrogen-terminated surface (surface conductance in the order of 10 −7 S/sq) (Kozak et al., 2009). The surface morphology and chemical quality of NCD layers were characterized by AFM, scanning 179 Diamond as Functional Material for Bioelectronics and Biotechnology 4 Will-be-set-by-IN-TECH Fig. 2. Basic characteristics of a typical NCD layer on Si: (a) morphology by SEM and (b) a typical Raman-scattering spectrum. electron microscopy (SEM) and Raman spectroscopy. Roughness evaluated in the tapping AFMregimeis15 −30 nm rms (1 ×1μm 2 area), grain size as measured by SEM is 50 −150 nm (see Figure 2(a)). The grains exhibit clear facets that evidences their crystalline diamond form. Raman spectroscopy (excitation wavelength 325 nm) confirmed the diamond character of the layers (see Figure 2(b)). With a small alteration of the deposition conditions, grain sizes of even several hundreds of nanometers can be reached. 3. Cell growth on diamond with surface nanostructures To produce nanostructured diamond surfaces the NCD films were first masked with: i) 5 nm diamond nanoparticles using the ultrasonic treatment in UDD colloidal suspension, ii) 30 nm nickel particles prepared by deposition of 3 nm nickel layer on diamond and its treatment in hydrogen plasma for 5 min (Babchenko et al., 2009). Subsequent etching of diamond nanostructures was performed by reactive ion etching (RIE) system (Phantom LT RIE System, Trion Technology) at about 100°C for 300 s using 2 sccm of CF 4 and 50 sscm of O 2 . Remaining nickel masks were then removed by a wet etching process. Finally, the diamond surfaces were treated in r.f. oxygen plasma to obtain hydrophilic character of the surface that is suitable for cellular adhesion (Kalbacova, Kalbac, Dunsch, Kromka, Vanecek, Rezek, Hempel & Kmoch, 2007). Scanning electron microscopy (SEM) images of the NCD films with nanoparticle masks and after the RIE process are shown in Figure 3a-b and 3e-f. Diamond nanoparticle mask resulted in a formation of isolated cone-like structures (height 5–100 nm, diameter up to 80 nm) randomly spread on the remaining NCD film. Mask made of the nickel nanoparticles resulted in a formation of upright, densely packed diamond nanorods with the height of 120–200 nm and diameter 20–40 nm. Diamond nanoparticles are obviously (and expectably) not enough resistant to the plasma etching process. Therefore, the surface exhibit lower density of cone-like structures. Nickel nanoparticles were able to withstand the whole etching period, hence the nanorods were formed. These nanostructured diamond surfaces were used as artificial substrates for growth of human osteoblast-like cells. Human osteoblast-like cells (SAOS-2; DSMZ, Germany) were plated on the samples in 25,000 cells/cm 2 concentration and grown in the McCoy’s 5A medium without phenol red (BioConcept) supplemented with 15% heat-inactivated fetal 180 New Perspectives in Biosensors Technology and Applications [...]... cells 192 16 New Perspectives in Biosensors Technology and Applications Will-be-set-by -IN- TECH Fig 11 Schematic sketch of the interface between the surface-condutive SG-FET channel and cell medium containing proteins and cells The electric field and its reach over the interface is depicted on the right It demonstrates that the interaction is limited to distances of several tens of nm using the interface... developing, constructing and manufacturing of new sensing devices to get more efficient and reliable information (Figure 1.); (Mohanty and Kougianos, 2006) Engineering Biochemical Chemical Materials Science Fundamental Sciences Physics Chemistry Biology BIOSENSORS MEDICINE Fig 1 Biosensors; an excellent example of multi and interdisciplinary research area * Dr Bora Garipcan is currently working at Institute of... DNA-modified nanocrystalline diamond thin films as stable, biologically active substrates, Nature Mat 1: 253 196 20 New Perspectives in Biosensors Technology and Applications Will-be-set-by -IN- TECH Zhao, G., Zinger, O., Schwartz, Z., Wieland, M., Landolt, D & Boyan, B D (2006) Osteoblast-like cells are sensitive to submicron-scale surface structure, Clin Oral Implant Res 17: 258 9 New Generation Biosensors Based... molecules and diseases in medicine (Sharma, 1994; D`Orazio, 2003; Mohanty and Kougianos, 2006) Of course, this demand is not restricted only in the field of medicine; for environmental pollutant monitoring, detection of food borne pathogens and potential danger of bioterrorism Therefore, researchers from various fields such as; physics, chemistry, biology, engineering and medicine interested in the developing,... adhesion and conformation of FBS layers on diamond were studied using AFM (Ntegra, NTMDT) The AFM measurements were carried out in air and in solution both in contact and tapping regimes Doped silicon cantilevers (Multi75Al, BudgetSensors) with typical spring constant of 3 N/m, resonant frequency 75 kHz in air and 30 kHz in solution and nominal tip radius < 10 nm were used Polished monocrystalline diamond... working at Institute of Biomedical Engineering as a Part time instructor 198 New Perspectives in Biosensors Technology and Applications Biosensors are the most impressive and useful devices which correspond to these purposes In the literature about biosensors, there are two common articles which describe the term biosensor as “a biosensor is a chemical sensing device in which a biologically derived recognition... of the conformation of proteins on hydrogen- and oxygen-terminated diamond surface Hydrophobic core in green, the black spheres represent polar groups surrounding the core in aqueous environment The red line denotes the height of the protein as detected by AFM in solution 188 12 New Perspectives in Biosensors Technology and Applications Will-be-set-by -IN- TECH subsequent growth of cells on the electronic... technology GmbH, Germany) was spin-coated on the NCD surface an micro-patterned by 182 6 New Perspectives in Biosensors Technology and Applications Will-be-set-by -IN- TECH Fig 4 SEM image of a nanocrystalline-diamond layer with 200-μm-wide stripes with alternating hydrogen and oxygen termination Light stripes correspond to the hydrogen surface due to its low electron affinity The cross in the upper part. .. ligament fibroblasts (HPdLF; Lonza) and human cervical carcinoma cells (HeLaG; DSMZ GmbH) Adhesion and morphology of cells were characterized by fluorescent staining of actin stress fibers (in green) and cell nuclei (in blue) using the protocol described in (Kalbacova, Roessler, Hempel, Tsaryk, Peters, Scharnweber, Kirkpatrick & Dieter, 20 07) The staining was visualized using the E-400 epifluorescence microscope... which correlates with round structures in topography In air AFM experiments in air, such differences in topography and phase channel were not observed (Rezek, Ukraintsev, Michalíková, Kromka, Zemek & Kalbacova, 186 10 New Perspectives in Biosensors Technology and Applications Will-be-set-by -IN- TECH 2009) This discrepancy results from the fact that FBS layers are not in their natural environment (solution) . Potocky et al., 20 07) . The substrates were 10 × 10 mm 2 large and had 178 New Perspectives in Biosensors Technology and Applications Diamond as Functional Material for Bioelectronics and Biotechnology. reached even in NCD layers as thin as 100 nm with the average grain size of (80 ±50) nm. The in uence of the adsorption of proteins and 186 New Perspectives in Biosensors Technology and Applications Diamond. Immobilization and Biomolecular Sensing. Langmuir, v.25, n.14, Jul 21, p .77 73 -77 77. 2009. Yang, J. S., C. S. Lin , et al. Cu2+-induced blue shift of the pyrene excimer emission: A new signal transduction