Surface roughness of the substrateplays 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
Trang 2Erdem, A., H Karadeniz, et al Single-Walled Carbon Nanotubes Modified Graphite
Electrodes for Electrochemical Monitoring of Nucleic Acids and Biomolecular Interactions Electroanalysis, v.21, n.3-5, Feb, p.464-471 2009
Fiorito, P A & S I C De Torresi Glucose amperometric biosensor based on the
co-immobilization of glucose oxidase (GOx) and ferrocene in poly(pyrrole) generated from ethanol/water mixtures Journal of the Brazilian Chemical Society, v.12, n.6, Nov-Dec, p.729-733 2001
Ghadiri, M R., J R Granja, et al Self-Assembling Organic Nanotubes Based on a Cyclic
Peptide Architecture Nature, v.366, n.6453, Nov 25, p.324-327 1993
Godwin, H A & J M Berg A fluorescent zinc probe based on metal-induced peptide
folding Journal of the American Chemical Society, v.118, n.27, Jul 10, p.6514-6515
1996
Guha, S & A Banerjee Self-Assembled Robust Dipeptide Nanotubes and Fabrication of
Dipeptide-Capped Gold Nanoparticles on the Surface of these Nanotubes Advanced Functional Materials, v.19, n.12, Jun 23, p.1949-1961 2009
Guilbault, G G Biosensors Current Opinion in Biotechnology, v.2, n.1, Feb, p.3-8 1991
Hartgerink, J D., J R Granja, et al Self-assembling peptide nanotubes Journal of the
American Chemical Society, v.118, n.1, Jan 10, p.43-50 1996
Hauser, C A E & S G Zhang Designer self-assembling peptide nanofiber biological
materials Chemical Society Reviews, v.39, n.8, p.2780-2790 2010
He, Q., L Duan, et al Microcapsules containing a biomolecular motor for ATP biosynthesis
Advanced Materials, v.20, n.15, Aug 4, p.2933-2937 2008
Hiller, M., C Kranz, et al Amperometric biosensors produced by immobilization of redox
enzymes at polythiophene-modified electrode surfaces Advanced Materials, v.8, n.3, Mar, p.219-& 1996
Hirata, T., F Fujimura, et al A novel polypseudorotaxane composed of cyclic beta-peptide
as bead component Chemical Communications, n.10, p.1023-1025 2007
Kelly, D., K M Grace, et al Integrated optical biosensor for detection of multivalent
proteins Optics Letters, v.24, n.23, Dec 1, p.1723-1725 1999
Khan, F., T E Saxl, et al Fluorescence intensity- and lifetime-based glucose sensing using an
engineered high-K-d mutant of glucose/galactose-binding protein Analytical Biochemistry, v.399, n.1, Apr 1, p.39-43 2010
Kholkin, A., N Amdursky, et al Strong Piezoelectricity in Bioinspired Peptide Nanotubes
Acs Nano, v.4, n.2, Feb, p.610-614 2010
Kim, J., T H Han, et al Role of Water in Directing Diphenylalanine Assembly into
Nanotubes and Nanowires Advanced Materials, v.22, n.5, Feb 2, p.583-+ 2010 Kim, J H., S Y Lim, et al Self-assembled, photoluminescent peptide hydrogel as a versatile
platform for enzyme-based optical biosensors Biosensors and Bioelectronics, v.26, n.5, p.1860-1865 2011
Kobayashi, T., H Okada, et al A digital output piezoelectric accelerometer using a Pb(Zr,
Ti)O-3 thin film array electrically connected in series Smart Materials & Structures, v.19, n.10, Oct, p.- 2010
Krizek, B A., D L Merkle, et al Ligand Variation and Metal-Ion Binding-Specificity in Zinc
Finger Peptides Inorganic Chemistry, v.32, n.6, Mar 17, p.937-940 1993
Trang 3Biosensors Based on Biological Nanostructures 173 Kros, A., W F M Van Hovell, et al Poly(3,4-ethylenedioxythiophene)-based glucose
biosensors Advanced Materials, v.13, n.20, Oct 16, p.1555-+ 2001
Kumar, A Biosensors Based on Piezoelectric Crystal Detectors: Theory and Application
JOM-e 52 2000
Kung, L A., L Kam, et al Patterning hybrid surfaces of proteins and supported lipid
bilayers Langmuir, v.16, n.17, Aug 22, p.6773-6776 2000
Lakowicz, J R Principles of Fluorescence Spectroscopy: Springer 1999 725 pages p
Li, X J., W Chen, et al Direct measurements of interactions between polypeptides and
carbon nanotubes Journal of Physical Chemistry B, v.110, n.25, Jun 29,
p.12621-12625 2006
Liao, J H., C T Chen, et al A novel phosphate chemosensor utilizing anion-induced
fluorescence change Organic Letters, v.4, n.4, Feb 21, p.561-564 2002
Lu, K., J Jacob, et al Exploiting amyloid fibril lamination for nanotube self-assembly
Journal of the American Chemical Society, v.125, n.21, May 28, p.6391-6393
2003
Mahara, A., R Iwase, et al Bispyrene-conjugated 2 '-O-methyloligonucleotide as a highly
specific RNA-recognition probe Angewandte Chemie-International Edition, v.41, n.19, p.3648-3650 2002
Marvin, J S., E E Corcoran, et al The rational design of allosteric interactions in a
monomeric protein and its applications to the construction of biosensors Proceedings of the National Academy of Sciences of the United States of America, v.94, n.9, Apr 29, p.4366-4371 1997
Massey, M e U J Krull Towards a fluorescent molecular switch for nucleic acid biosensing
Analytical and Bioanalytical Chemistry, v.398, n.4, Oct, p.1605-1614 2010
Mcfarland, S A & N S Finney Fluorescent chemosensors based on conformational
restriction of a biaryl fluorophore Journal of the American Chemical Society, v.123, n.6, Feb 14, p.1260-1261 2001
Merzlyakov, M., E Li, et al Directed assembly of surface-supported bilayers with
transmembrane helices Langmuir, v.22, n.3, Jan 31, p.1247-1253 2006
Motesharei, K e M R Ghadiri Diffusion-limited size-selective ion sensing based on
SAM-supported peptide nanotubes Journal of the American Chemical Society, v.119, n.46, Nov 19, p.11306-11312 1997
Nielsen, K., M Lin, et al Fluorescence polarization immunoassay: Detection of antibody to
Brucella abortus Methods-a Companion to Methods in Enzymology, v.22, n.1, Sep, p.71-76 2000
Pantarotto, D., C D Partidos, et al Synthesis, structural characterization, and
immunological properties of carbon nanotubes functionalized with peptides Journal of the American Chemical Society, v.125, n.20, May 21, p.6160-6164
2003
Poteau, R & G Trinquier All-cis cyclic peptides Journal of the American Chemical Society,
v.127, n.40, Oct 12, p.13875-13889 2005
Reches, M & E Gazit Casting metal nanowires within discrete self-assembled peptide
nanotubes Science, v.300, n.5619, Apr 25, p.625-627 2003
Trang 4Formation of closed-cage nanostructures by self-assembly of aromatic dipeptides Nano
Letters, v.4, n.4, Apr, p.581-585 2004
Controlled patterning of aligned self-assembled peptide nanotubes Nature
Nanotechnology, v.1, n.3, Dec, p.195-200 2006
Ryu, J & C B Park High-Temperature Self-Assembly of Peptides into Vertically
Well-Aligned Nanowires by Aniline Vapor Advanced Materials, v.20, n.19, Oct 2, p.3754-+ 2008a
Solid-phase growth of nanostructures from amorphous peptide thin film: Effect of water
activity and temperature Chemistry of Materials, v.20, n.13, Jul 8, p.4284-4290 2008b
Synthesis of Diphenylalanine/Polyaniline Core/Shell Conducting Nanowires by Peptide
Self-Assembly Angewandte Chemie-International Edition, v.48, n.26, p.4820-4823
2009
High Stability of Self-Assembled Peptide Nanowires Against Thermal, Chemical, and
Proteolytic Attacks Biotechnology and Bioengineering, v.105, n.2, Feb 1, p.221-230
2010
Sackmann, E Supported membranes: Scientific and practical applications Science, v.271,
n.5245, Jan 5, p.43-48 1996
Sadik, O A., A O Aluoch, et al Status of biomolecular recognition using electrochemical
techniques Biosensors & Bioelectronics, v.24, n.9, May 15, p.2749-2765 2009
Sahoo, D., V Narayanaswami, et al Pyrene excimer fluorescence: A spatially sensitive probe
to monitor lipid-induced helical rearrangement of apolipophorin III Biochemistry, v.39, n.22, Jun 6, p.6594-6601 2000
Sanchez, C., H Arribart, et al Biomimetism and bioinspiration as tools for the design of
innovative materials and systems Nature Materials, v.4, n.4, Apr, p.277-288
2005
Sharma, M & N K Gohil Optical features of the fluorophore azotobactin: Applications for
iron sensing in biological fluids Engineering in Life Sciences, v.10, n.4, Aug,
p.304-310 2010
Shklovsky, J., P Beker, et al Bioinspired peptide nanotubes: Deposition technology and
physical properties Materials Science and Engineering B-Advanced Functional Solid-State Materials, v.169, n.1-3, May 25, p.62-66 2010
Sima, V., C Cristea, et al Electroanalytical properties of a novel biosensor modified
with zirconium alcoxide porous gels for the detection of acetaminophen Journal of Pharmaceutical and Biomedical Analysis, v.48, n.4, Dec 1, p.1195-
1200 2008
Singh, G., A M Bittner, et al Electrospinning of diphenylalanine nanotubes Advanced
Materials, v.20, n.12, Jun 18, p.2332-+ 2008
Smallshaw, J E., S Brokx, et al Determination of the binding constants for three
HPr-specific monoclonal antibodies and their fab fragments Journal of Molecular Biology, v.280, n.5, Jul 31, p.765-774 1998
Smith, R T & F S Welsh Temperature Dependence of Elastic, Piezoelectric, and Dielectric
Constants of Lithium Tantalate and Lithium Niobate Journal of Applied Physics, v.42, n.6, p.2219-& 1971
Trang 5Biosensors Based on Biological Nanostructures 175 Song, J., Q Cheng, et al "Smart" materials for biosensing devices: Cell-mimicking
supramolecular assemblies and colorimetric detection of pathogenic agents Biomedical Microdevices, v.4, n.3, Jul, p.213-221 2002
Song, X D., J Shi, et al Flow cytometry-based biosensor for detection of multivalent
proteins Analytical Biochemistry, v.284, n.1, Aug 15, p.35-41 2000
Song, X D & B I Swanson Direct, ultrasensitive, and selective optical detection of protein
toxins using multivalent interactions Analytical Chemistry, v.71, n.11, Jun 1, p.2097-2107 1999
Song, Y J., S R Challa, et al Synthesis of peptide-nanotube platinum-nanoparticle
composites Chemical Communications, n.9, May 7, p.1044-1045 2004
Szmacinski H, L J Lifetime-based sensing New York: Plenum Press, v.4 1994 ( InTopics in
fluorescence spectroscopy (Vol 4))
Terrettaz, S., W P Ulrich, et al Immunosensing by a synthetic ligand-gated ion channel
Angewandte Chemie-International Edition, v.40, n.9, p.1740-1743 2001
Thevenot, D R., K Toth, et al Electrochemical biosensors: Recommended definitions and
classification - (Technical Report) Pure and Applied Chemistry, v.71, n.12, Dec, p.2333-2348 1999
Valeur, B Molecular Fluorescence: Principles and Applications New York: Wiley-VCH
2001
Wang, J Electrochemical glucose biosensors Chemical Reviews, v.108, n.2, Feb, p.814-825
2008
Wang, J & Y H Lin Functionalized carbon nanotubes and nanofibers for biosensing
applications Trac-Trends in Analytical Chemistry, v.27, n.7, Jul-Aug, p.619-626
2008
Wang, J., D L Wang, et al Photoluminescence of water-soluble conjugated polymers: Origin
of enhanced quenching by charge transfer Macromolecules, v.33, n.14, Jul 11, p.5153-5158 2000
Worsfold, O., C Toma, et al Development of a novel optical bionanosensor Biosensors &
Bioelectronics, v.19, n.11, Jun 15, p.1505-1511 2004
Yan, X H., Y Cui, et al Organogels based on self-assembly of diphenylalanine peptide and
their application to immobilize quantum dots Chemistry of Materials, v.20, n.4, Feb 26, p.1522-1526 2008
Yang, H., S Y Fung, et al Ionic-Complementary Peptide Matrix for Enzyme Immobilization
and Biomolecular Sensing Langmuir, v.25, n.14, Jul 21, p.7773-7777 2009
Yang, J S., C S Lin, et al Cu2+-induced blue shift of the pyrene excimer emission: A new
signal transduction mode of pyrene probes Organic Letters, v.3, n.6, Mar 22,
p.889-892 2001
Yanlian, Y., K Ulung, et al Designer self-assembling peptide nanomaterials Nano Today,
v.4, n.2, p.193-210 2009
Yeh, J I., A Lazareck, et al Peptide nanowires for coordination and signal transduction of
peroxidase biosensors to carbon nanotube electrode arrays Biosensors & Bioelectronics, v.23, n.4, Nov 30, p.568-574 2007
Yemini, M., M Reches, et al Peptide nanotube-modified electrodes for enzyme-biosensor
applications Analytical Chemistry, v.77, n.16, Aug 15, p.5155-5159 2005 Novel
Trang 6electrochemical biosensing platform using self-assembled peptide nanotubes Nano Letters, v.5, n.1, Jan, p.183-186 2005
Yoo, E H e S Y Lee Glucose Biosensors: An Overview of Use in Clinical Practice Sensors,
v.10, n.5, May, p.4558-4576 2010
Trang 71 Introduction
Understanding the interaction between the biological environment (tissues, cells, proteins,electrolytes, etc.) and a solid surface is crucial for biomedical applications such asbio-sensors, bio-electronics, tissue engineering and the optimization of implant materials.Cells, the cornerstones of living tissue, perceive their surroundings and subsequentlymodify it by producing extracellular matrix (ECM), which serves as a basis to simplifytheir adhesion, spreading and differentiation (Shakenraad & Busscher, 1989) Thisprocess is considerably complex, flexible and strongly depends on the cell cultivationconditions including the type of the substrate Surface roughness of the substrateplays 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 polystyreneand glass In this context, diamond as a technological material can provide a relativelyunique combination of excellent semiconducting, mechanical, chemical as well as biologicalproperties (Nebel et al., 2007) Diamond also meets the basic requirements for large-scaleindustrial application, most notably, it can be prepared synthetically Diamond can besynthesized 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 varioussubstrates including glass and metal (Kromka et al., 2008; Potocky et al., 2007) Moreover,the application of selective nucleation makes it possible to directly grow conductivediamond 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 cm2 or more) usinglinear antennas (Kromka et al., 2011; Tsugawa et al., 2010) The excellent compatibility ofdiamond with biological materials and environment (Bajaj et al., 2007; Grausova et al., 2009;
Diamond as Functional Material for Bioelectronics and Biotechnology
Bohuslav Rezek1, Marie Krátká1, Egor Ukraintsev1, Oleg Babchenko1,
Alexander Kromka1, Antonín Brož2 and Marie Kalbacova2
1Institute of Physics, Academy of Sciences of the Czech Republic, Prague
2Institute of Inherited Metabolic Diseases, First Faculty of Medicine, Charles University
and General Faculty Hospital in Prague
Czech Republic
8
Trang 8Kalbacova, Kalbac, Dunsch, Kromka, Vanecek, Rezek, Hempel & Kmoch, 2007; Tang et al.,1995) is of immense importance for its application in medicine This bio-compatibility stemsfrom the fact that diamond is a crystalline form of carbon that is mechanically, chemicallyand 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 thusopen 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 maindifference arises from the opposite dipoles of C–H and C–O bonds Oxygen-terminateddiamond is insulating, whereas the hydrogen-terminated surface causes the emergence
of two-dimensional hole surface conductance on otherwise insulating diamond Theseproperties 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 anyother 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-terminateddiamond surface is also an ideal starting point for covalent bonding of other moleculessuch 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 theoxidized 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-terminateddiamond 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 atomictermination 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 cultivationmedium, the dimensions and shape of microstructures, and surface roughness We show thatthe 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 theFBS layers adsorbed on differently terminated diamond substrates The influence of proteinsand cells on the electronic properties of diamond is demonstrated by employing a field-effecttransistor 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 aswell as the prospects in medicine
2 Preparation of nanocrystalline diamond layers
The growth of thin-film nanocrystalline-diamond layers (NCD) was realized on silicon orglass substrates using microwave plasma enhanced chemical vapor deposition (MW-CVD)(Kromka et al., 2008; Potocky et al., 2007) The substrates were 10×10 mm2 large and had
Trang 9Diamond as Functional Material for Bioelectronics and Biotechnology 3
Fig 1 Schematic depiction of the preparation procedure of thin-film diamond on glass orsilicon substrates: (a) nucleation of the substrates carried out in an ultrasonic bath withultra-dispersed diamond (UDD), (b) the resulting nucleation layer and (c) the nanocrystallinediamond 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 anultrasonic bath with a colloidal suspension of a diamond powder (UDD – ultra-disperseddiamond; NanoAmando, New Metals and Chemicals Corp Ltd., Kyobashi) with nominalparticle 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 microwaveplasma-enhanced chemical vapor deposition (MW-CVD) of diamond films The depositionconditions were: temperature of substrates 600–800°C, 1% CH4 in H2, microwave power1.4–2.5 kW, gas pressure 30–50 mbar, duration approximately 4 hours, the thickness oflayers reaches 100–500 nm The same conditions, only with methane gas switched off andprocess 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 isschematically shown in Figure 1 This figure also depicts the photos of the set-ups for thelarge-area diamond growth (linear plasma) with high deposition rate (focused plasma).NCD layer were chemically cleaned in acids (97.5% H2SO4+ 99% KNO3powder in the ratio
of 4:1) at 200°C for 30 minutes This process ensures high quality of the hydrogen-terminatedsurface (surface conductance in the order of 10−7 S/sq) (Kozak et al., 2009) The surfacemorphology and chemical quality of NCD layers were characterized by AFM, scanning
179
Diamond as Functional Material for Bioelectronics and Biotechnology
Trang 10Fig 2 Basic characteristics of a typical NCD layer on Si: (a) morphology by SEM and (b) atypical Raman-scattering spectrum.
electron microscopy (SEM) and Raman spectroscopy Roughness evaluated in the tappingAFM regime is 15−30 nm rms (1×1 μm2area), 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 thelayers (see Figure 2(b)) With a small alteration of the deposition conditions, grain sizes ofeven 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 nmdiamond nanoparticles using the ultrasonic treatment in UDD colloidal suspension, ii) 30 nmnickel 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 diamondnanostructures 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 CF4and 50 sscm of O2 Remainingnickel masks were then removed by a wet etching process Finally, the diamond surfaces weretreated in r.f oxygen plasma to obtain hydrophilic character of the surface that is suitable forcellular adhesion (Kalbacova, Kalbac, Dunsch, Kromka, Vanecek, Rezek, Hempel & Kmoch,2007)
Scanning electron microscopy (SEM) images of the NCD films with nanoparticle masks andafter 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) notenough 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 ofhuman osteoblast-like cells Human osteoblast-like cells (SAOS-2; DSMZ, Germany) wereplated on the samples in 25,000 cells/cm2 concentration and grown in the McCoy’s 5Amedium without phenol red (BioConcept) supplemented with 15% heat-inactivated fetal
Trang 11Diamond as Functional Material for Bioelectronics and Biotechnology 5
Fig 3 Scanning electron microscopy (SEM) image of the NCD layer (a) with diamondnanoparticle mask and (b) resulting nanostructured surface (nano-cones) after plasmaetching (c) Fluorescence microscopy image of stained focal adhesions (vinculin) of
osteoblast-like cells on the surface with nano-cones (d) Schematic drawing of cellularadhesion on the nano-cones (focal adhesions – red, nucleus – blue, cytoskeleton – green).Same measurements for the case on nickel nanoparticle mask and diamond nanorods areshown in (e-h)
bovine serum (PAA), 20 U penicillin and 20 μg/ml streptomycin in a humidified 5%
CO2 atmosphere at 37°C Resulting morphology of focal adhesions of SAOS-2 cells wascharacterized by immunofluorescent staining of vinculin (1:150, Sigma, anti-mouse Alexa 568)and imaging in the epi-fluorescence microscope (Nikon E-400)
The fluorescence images are shown in Figure 3c and 3g, next to the SEM images Based onthe fluorescence images, osteoblasts exhibit generally well spread fibroblast-like morphology
on both substrates During the 48h incubation the cells went through one cell cycle Thisalso indicates general substrate suitability However, the size and shape of highlighted focaladhesions differ on each type of the nanostructures Osteoblasts cultivated on relatively shortand broad nano-cones form well pronounced large focal adhesions with intensive vinculinstaining indicating bigger surface available for adhesion and thus stronger adhesion contactsbetween cell and diamond On the other hand, cells cultivated on relatively high andthin nanorods form very thin and fine focal adhesions indicating weaker adhesion This isschematically shown in Figure 3d and 3h Another crucial role in cell-diamond interactionplay atoms terminating the diamond surface
4 Cell growth on diamond with atomic micro-patterns
To characterize influence of diamond surface atoms on the arrangement of cells, NCDlayers with hydrogen and oxygen surface atoms forming microscopic patterns of widthsfrom 30 to 200 μm were fabricated as follows Positive photoresist ma-P1215 (micro resisttechnology GmbH, Germany) was spin-coated on the NCD surface an micro-patterned by
181
Diamond as Functional Material for Bioelectronics and Biotechnology
Trang 12Fig 4 SEM image of a nanocrystalline-diamond layer with 200-μm-wide stripes with
alternating hydrogen and oxygen termination Light stripes correspond to the hydrogensurface due to its low electron affinity The cross in the upper part of the image is made up of
a thin layer of gold and serves as a mark for the differentiation of particular stripes Typicalmeasurements of wetting angle on the two types of diamond surfaces (uniformly
terminated) are shown along the left side of the SEM image
optical lithography Afterwards, the NCD layers were exposed with a photolithographic mask
in high-frequency oxygen plasma (power 300 W, duration 3 minutes), which gives rise to theoxidation of the surface, and, consequently, to the formation of hydrophilic patterns Thewetting angle of water on oxygen-terminated diamond was<20◦, in contrast to about 80◦
on the hydrogen-terminated diamond The morphology of the surface remains unchangedduring this procedure Figure 4 shows how the microscopic stripe patterns look look like
in an electron microscope (hydrogen and oxygen stripes have different SEM contrast due todifferent electron affinity)
Before cell plating, the NCD layers were sterilized using either UV irradiation or 70%ethanol treatment for 10 minutes In most experiments, the cell line of human bone cells(osteoblasts – SAOS-2 cells; DSMZ GmbH) were used The cells were plated on diamond inthe concentrations ranging from 2,500 (sub-confluent coverage) to 10,000 cells/cm2(confluentcoverage, when the cells are in direct contact with each other) and immersed in the McCoy’s5A (BioConcept) medium, which contains penicillin (20 U/ml) and streptomycin (20μg/ml)
and different concentrations of FBS (0–15%) Then, the cells were cultivated in an incubator at37°C in 5% CO2for 48h We used osteoblasts because SAOS-2 is a standard cell line, whoseproperties are stable even for long timespans This is why we are able to compare the results
of different experiments, as well as our results with the literature Other cell types were alsoapplied for comparison: human periodontal ligament fibroblasts (HPdLF; Lonza) and humancervical carcinoma cells (HeLaG; DSMZ GmbH)
Adhesion and morphology of cells were characterized by fluorescent staining of actinstress fibers (in green) and cell nuclei (in blue) using the protocol described in(Kalbacova, Roessler, Hempel, Tsaryk, Peters, Scharnweber, Kirkpatrick & Dieter, 2007) Thestaining was visualized using the E-400 epifluorescence microscope (Nikon); digital imageswere acquired with a DS-5M-U1 Color Digital Camera (Nikon)
Trang 13Diamond as Functional Material for Bioelectronics and Biotechnology 7
Fig 5 Microscopic fluorescence image illustrates how osteoblastic cells (SAOS-2)
preferentially self-assemble on oxygen-terminated diamond after a 48h cultivation in
McCoy’s 5A medium with 15% FBS on H-/O-diamond stripes of 60 μm width Starting cellconcentration was 2,500 cells/cm2 Fluorescence microscopy shows actin filaments in greenand cell nuclei in blue The scheme under the image further clarifies the situation
When the osteoblastic cells were plated and grown on the H-/O-terminated microstructures,they self-assembled preferably on the oxygen-terminated diamond surface A scheme andfluorescence image shown in Figure 5 give an example of such behavior for the case
of 60-μm-wide stripes The cells’ preference is independent of the width of the stripesbetween 30 and 200 μm (Rezek, Michalíková, Ukraintsev, Kromka & Kalbacova, 2009) and
of the surface roughness between 20 and 500 nm rms (Michalíková et al., 2009) However,the shape of cells was found to be influenced by surface roughness (Kalbacova et al.,2009; Kromka et al., 2009) and the width of microstructures (Kalbacova et al., 2008;Rezek, Michalíková, Ukraintsev, Kromka & Kalbacova, 2009) Cells grown on narrowO-stripes (30 μm i.e comparable with the size of the cell) are elongated and form chain-likestructures On the other hand, cells growing on wider stripes (60, 100 a 200 μm – larger thanthe typical cell size) spread over the whole width of the stripe The H-/O-diamond boundaryforms a sharp interface for cell adhesion
Figure 6 confirms that other types of cells are also able of controlled self-assembly onH-/O-diamond stripes Human fibroblasts (HPdLF) and cervical carcinoma cells (HeLaG)were plated on NCD samples with 30-μm-wide stripes and were cultivated for 48h Cellsexhibit a different morphology, however, their preference for O-diamond remains unchanged.Selective growth of cells on H-/O-diamond is also influenced by the seeding concentration,which is illustrated in Figure 7 At low concentrations (2,500 cells/cm2), the cells growpredominantly on the oxygen-terminated surface, where the cells have enough room to
183
Diamond as Functional Material for Bioelectronics and Biotechnology
Trang 14Fig 6 Fluorescence image of (a) fibroblasts (HPdLF) and (b) cervical-carcinoma cells
(HeLaG), which were cultivated for 48h on 30-μm-wide H-/O-diamond stripes Starting cellconcentration was 2,500 cells/cm2, medium was supplemented with 15% FBS Fluorescencemicroscopy shows actin filaments in green and cell nuclei in blue
Fig 7 Fluorescence images of osteoblasts, which were cultivated for 48h on 100-μm-wideH-/O-diamond stripes with starting cell concentrations: (a) 2,500 cells/cm2, (a) 10,500cells/cm2 Fluorescence microscopy shows actin filaments in green and cell nuclei in blue.spread on a hydrophilic area (Figure 7(a)) On the other hand, cells plated at high seedingconcentrations (10,000 cells/cm2) colonize also the hydrogen-terminated surface (Figure 7(b)).The FBS serum is another factor which has impact on the selective growth of cells Figure
8 depicts the influence of FBS in the cultivation medium on the arrangement of cells on theH-/O-diamond terminated stripes The range of concentrations between 5% and 15% doesnot significantly influence the cell adhesion (image for 15% FBS concentration is shown).Nevertheless, cells plated in a medium without FBS assemble of the surface independently
of the surface termination The cells’ preference for a particular type of surface is thuspresumably determined by the FBS proteins and not by a direct interaction between diamondsurface dipoles and the cells This is why the properties of FBS layers adsorbed on differenttypes of diamond surfaces were investigated
5 Morphology of protein layers on H-/O-diamond
Adsorption, adhesion and conformation of FBS layers on diamond were studied usingAFM (Ntegra, NTMDT) The AFM measurements were carried out in air and insolution 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 Polishedmonocrystalline diamond was used as a substrate to minimize the influence of its surface
Trang 15Diamond as Functional Material for Bioelectronics and Biotechnology 9
Fig 8 Fluorescence images of osteoblasts, which were cultivated for 48h on 100-μm-wideH-/O-diamond stripes with different starting concentrations of fetal bovine serum: (a) 0%,(a) 15% Fluorescence microscopy shows actin filaments in green and cell nuclei in blue Inthe 0% case, the cells were plated without the serum, however, the serum was added after 2hours to allow cells to grow for next 48 h
morphology on the layers Surface terminations were prepared in the same way as inthe case of NCD films The thickness of the protein layer was determined using thenanoshaving method, in which a part of the protein layer is removed by means of theAFM tip in contact mode and subsequently the profile of the resulting step in height ismeasured in oscillating (tapping) mode (Rezek et al., 2006; Rezek, Shin, Uetsuka & Nebel,2007; Rezek, Ukraintsev, Michalíková, Kromka, Zemek & Kalbacova, 2009) Polishedmonocrystalline diamond is an ideal substrate for this method, because it is flat and hard.Proteins were adsorbed on the surface of diamond from 15% FBS solution (Biowest) inMcCoy’s 5A medium (BioConcept) Two adsorption methods were applied: (i) either
a drop of the solution was deposited on the substrate by a pipette, the substrate wasthen kept in a humid chamber for 10 minutes and was subsequently rinsed with water,
or (ii) the adsorption was carried out directly in a fluid cell of AFM microscope with a
subsequent in-situ measurement Both methods yielded comparable results The protein
monolayer formed on the diamond surface within several seconds after the application(Rezek, Ukraintsev, Michalíková, Kromka, Zemek & Kalbacova, 2009)
AFM nanoshaving experiments showed that the thickness of the protein layer adsorbedfrom the solution is (4±2) nm on O-diamond and (1.5±2) nm on H-diamond(Rezek, Ukraintsev, Michalíková, Kromka, Zemek & Kalbacova, 2009) Thus, FBS layersformed on both types of diamond surfaces Figure 9 presents a detailed topography and aphase map of the protein layers measured in AFM Standard deviation values (i.e RMS –root-mean-square) of the height and phase signals together with the characteristic lateral size
of the features (L x) determined by means of the autocorrelation function are shown belowthe images In the case of the topography, RMS value corresponds to surface roughness.Roughness of the FBS layer on H-diamond (0.6 nm) is approximately 3× smaller whencompared to the O-diamond layer (1.7 nm) Besides, the features on the surface are of differentshapes and sizes (12 and 18 nm, respectively) The phase signal exhibits an even morepronounced difference Whereas in the H-diamond case the phase image of the FBS layerconsists of dark dots correlating with the protrusion in topography, the O-diamond phaseimage is characterized by much larger light areas, which correlates with round structures
in topography In air AFM experiments in air, such differences in topography and phasechannel were not observed (Rezek, Ukraintsev, Michalíková, Kromka, Zemek & Kalbacova,
185
Diamond as Functional Material for Bioelectronics and Biotechnology