Characteristics of Nanocrystalline Diamond SGFETs Under Cell Culture Conditions

M. Krátká, A. Kromka, and B. Rezek Institute of Physics ASCR, Czech Republic.

A. Brož and M. Kalbáčová 1st Faculty of Medicine, Charles University, Czech Republic.

Abstract. Diamond is an attractive material for bio-electronic systems, from DNA molecules to cells. We characterize electronic properties of protein diamond interface by using microscopic (20 μm) solution-gated field-effect transistors (SGFET) based on H-terminated nanocrystalline diamond films (NCD) on glass. We show that NCD films with grain sizes down to 80 nm and thickness down to 100 nm are operational as SGFETs. We characterize the effects of exposure to HEPES buffer and McCoy’s cell medium with fetal bovine serum (FBS) proteins, rinsing by phosphate buffered saline, UV sterilization and cell culturing process on the SGFET transfer characteristics. We propose a model of bio-electronic interface specific to NCD.

Introduction Unlike other semiconductors, diamond is considered as highly attractive material for bio-

electronic systems, from DNA molecules [Yang et al., 2008; Rezek et al., 2007a] to cells [Dankerl et al., 2009; Rezek et al., 2009a]. This is because diamond exhibits unique combination of electrical, optical and mechanical properties with chemical and biocompatible properties. Diamond films can be easily further functionalized by molecules [Rezek et al., 2007a] and they are suitable for attachment of cells such as osteoblasts, fibroblasts, cervical carcinoma cells (HeLaG) [Kalbacova et al., 2008] or cardiomyocyte cells [Dankerl et al., 2009]. For these cells it is necessary to use serum consisting of proteins and vitamins (medium with fetal bovine serum (FBS)) to provide cells conditions to survive. It was shown by atomic force microscopy (AFM) that proteins in this medium create thin inter-layer on the diamond and they drive the cell selectivity of the surface [Rezek et al., 2009b].

Biological as well as electronic properties of intrinsic diamond can be significantly altered by hydrogen and oxygen atomic surface termination which results in different properties such as electrical conductivity, electron affinity, and surface wettability. Oxygen terminated surfaces are hydrophilic and highly resistive while hydrogen terminated surfaces are hydrophobic and they induce p-type surface conductivity even on undoped diamond [Kawarada et al., 1996; Maier et al., 2000; Chakrapani et al., 2007]. This enables the design of solution gated field effect transistors (SGFET). It is notable that diamond SGFET can operate without a gate oxide layer because the gate is insulated by hydrogen atoms hence it allows direct contact between biomolecules and the surface of FET channel [Rezek et al., 2007b; Rezek et al., 2010]. Transistors enable miniaturization and direct transduction of signals in biosensing. Therefore diamond SGFETs can be employed as cell biosensors for environmental monitoring, biomedicine research or other applications. Employing NCD SGFETs as cell biosensors requires understanding interaction between diamond, proteins and cells or other biomolecules as well as understanding effects of cell incubation process. NCD is complicated system due to presence of sp2 phase and grain boundaries and role of these features for biosensors is not understood yet.

In this work we characterize effects of conditions during cell culturing process and proteins from fetal bovine serum on diamond SGFETs characteristics. However, cell sensor has not been realized yet. The work here represents fundamental study to characterize and understand function of such prospective sensor under cell culture conditions. We employ NCD films of different grain sizes to resolve various interface effects (such as FBS adsorption) and to discuss influence of grain boundaries and sp2 phase on the SGFET function.

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WDS'11 Proceedings of Contributed Papers, Part III, 160–165, 2011. ISBN 978-80-7378-186-6 © MATFYZPRESS

KRÁTKÁ ET AL.: CHARACTERISTICS OF NANOCRYSTALLINE DIAMOND SGFETS

Experimental methods Nanocrystalline diamond films were grown on glass (UQG, 10×10×1 mm3 size) by microwave

plasma chemical vapor deposition (CVD) in Aixtron reactor using gas pressure 30 mbar, 1% CH4 in H2 and power 900–1000 W. The deposition temperature was in the range of 550–600 °C. The periods of deposition were 1 h and 4.3 h to obtain different grain size and thickness. The surfaces of diamond films were hydrogenated in pure H plasma at 600 °C for 10 min. The structure and material composition of NCD films were studied by scanning electron microscopy (SEM), micro-Raman spectroscopy and atomic force microscopy (AFM) (Dimension 3100, Veeco) using standard silicon cantilevers of nominal tip radius 10 nm.

Photolithographic masks were applied on H-terminated NCD films using positive ma-P 1215 photoresist (micro resist technology GmbH; 1.5 μm layer thickness) to define FET channels and contacts. The NCD films were treated in oxygen radio-frequency plasma (300 W, 3 min exposition time) to generate O-terminated areas which surround (and insulate) the channels connecting source and drain. Source and drain gold contacts were prepared by thermal evaporation (10 nm of Ti and 50 nm of Au) followed by lift-off technique. The samples were cleaned in acetone and photoresist stripper (remover mr-REM 660). The area between contacts was covered with positive photoresist ma-P 1240 (thickness 4 μm) and deep UV curing of resist for hardening and biocompatibility was applied. Photolithographic mask exposed openings of 60×60 μm2 to define an active gate area (20 μm channel surrounded by 20 μm O-terminated areas in each side). Cross-sectional scheme of H-diamond SGFET fabrication process is shown in Figure 1. Top view scheme of the device after the step 8 in Figure 1 is shown in Figure 3a.

Gating of SG-FET was realized by immersing H-terminated channel into electrolyte solutions which were in contact with Ag/AgCl gate electrode. Types of used solutions were: 1) McCoy´s 5A medium supplemented with heat inactivated 15% fetal bovine serum (FBS; Biowest) and HEPES, 2) HEPES buffer alone.

SAOS-2 cells (sarcoma osteogenic, human osteoblast-like cell line; DSMZ GmbH) were grown in McCoy’s 5A medium (BioConcept) supplemented with heat inactivated 15% FBS (Biowest), penicillin (20 U/ml) and streptomycin (20 μg/ml). Cells were plated in the densities of 10000 cells/cm2 using a droplet technique: substrate surface was covered by 10 μl droplet of cell suspension in the medium. After 1 hour incubation 1.4 ml of the medium was added and cells were cultivated for 2 days in 5% CO2 at 37 °C. All transistor characteristics were measured after removing the cells. The cells were let to delaminate and were washed by PBS and DIW. The only difference between culturing process with or without cells is the presence or absence of cells during the culturing process. Other-wise all conditions are the same for both processes. Sterilization was performed by UV-C for 10 min.

We characterize the effects of exposure to HEPES buffer and McCoy’s cell medium with fetal bovine serum (FBS) proteins, rinsing by phosphate buffered saline, UV sterilization and cell culturing process with or without cells on the SGFET transfer characteristics and we used the HEPES buffer solutions to independently probe these effects.

The transistor characteristics were measured using Keithley K327 source-measure units and custom software. The software was made using a measurement and control software package developed under Delphi by A. Fejfar, Institute of Physics. We show always the fifth characteristic, which is stabilized. The sweeping rate was 50 mV/s and initial delay time was 5 s. All experiments were performed at room temperature.

Results The SEM images of nanocrystalline diamond films are shown in Figure 2. The average grain

sizes, estimated from SEM images, are 250 ± 50 nm and 80 ± 50 nm. SEM images show facets on the grains in particular on sample A. The thicknesses of the diamond films, calculated from AFM measured across a scratch in the film are 445 ± 27 μm and 108 ± 19 μm for large and small grain sample respectively (error bars correspond to rms roughness of the films as obtained by AFM in the area of 5 × 5 μm).

Raman spectra confirm the presence of a diamond phase (peak at 1332 cm–1) and reveal also graphitic (sp2) phase (band at 1580 cm–1). In spite of graphitic phase both types of diamond exhibit surface conductivity related with H-termination and they are operational as SGFET.

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1) hydrogenation of NCD

H HH HH HH H

NCD

2) photoresist 3) litography 4) oxygen plasma

5) removing photoresist 6) contact deposition 7) covering resist 8) opening in resist

H HH HH HH H

NCD

O HO OH HH O

NCD

H HH HH HH H

NCD

HH HH

NCD

O HO OH HH O

NCD

HH HH

NCD

HH HH

NCD

Au

Figure 1. Cross-sectional scheme of H-diamond SGFET fabrication process.

Figure 2. SEM images of NCD films with grain size about 250 nm (left) and 80 nm (right).

covering resistgoldcontact

C-H

1 cm

a) b) c)

C-O

opening

Figure 3. Top view scheme (a), optical image (b) and SEM image (c) of NCD SGFET with 5 H-terminated channels.

Figure 3 shows a top view scheme, optical image, and SEM image of NCD sample with 5 integrated SGFET devices. The optical image of SGFET shows that NCD is transparent and only encapsulation and gold contacts are visible (Figure 3b). H-terminated channels are visible in the SEM image of SGFET (Figure 3c).

The effects of protein adsorption, rinsing and cell culture process with cells on diamond SGFET characteristics are evidenced in Figure 4. The transfer characteristics were measured in amplification regime at Uds = − 0.6 V in HEPES buffer. FET is open at Ug 0 V and the channels can be closed at Ug 0.3 V, which corresponds to p-type conductivity of the channel. The SGFET conductivity decreased after application of FBS as reflected by the shift of SGFET transfer characteristics by –50 mV and by the decreased slope. We found that this shift is permanent, even after rinsing using deionized water and phosphate buffered saline (PBS). Additional shift of transfer characteristics was observed after the cell culturing process with cells. We obtained similar transfer characteristics from cell culturing process without cells.

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Figure 4. Transfer characteristics at Uds = – 0.6 V of pristine device (black), after adsorption of FBS (blue) and after rinsing by deionized water (gray) and after the cell culturing process (green) for large (a) and small grains (b) measured in HEPES buffer.

Figure 5. Transfer characteristics at Uds = –0.6 V of pristine device (black), after adsorption of FBS (blue), after sterilization (orange) and after the cell culturing process (green) for large grains measured in HEPES buffer.

The slope of transfer characteristics (transconductance) also decreased like in case of transfer characteristics after adsorption of FBS. Figure 5 indicates that UV sterilization, an important step prior to cell culture process, does not cause another shift after FBS adsorption.

Discussion The possible reasons of the decreased slope and decreased conductivity after adsorption of

proteins from FBS were discussed in previous paper [Rezek et al., 2010]. Briefly proteins can modify original equilibrium of the surface conductive layer system. They may replace ions in the very vicinity of the diamond surface. Thereby the conductivity can decrease in spite of that main FBS proteins have negative charge at physiological pH = 7.4 so their presence on the gate of p-type SGFET should increase the channel current. Hence this can not be explained purely as field effect.

Potential shifts after exposure H-terminated channel to FBS are permanent in both SGFETs with different grain size even after rinsing using PBS and other agents. This is due to adsorption of a 2–4 nm primary protein layer from FBS which remains on diamond irrespective of rinsing as evidence by AFM [Rezek et al., 2009b; Ukraintsev et al., 2009]. Similar characteristics after FBS adsorption and sterilization indicate that protein layer does not change during sterilization.

As further potential negative shift of transfer characteristics is observed after the cell culturing process in incubator even without cells, this shift is not caused by the cells. Considering that samples

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are kept at fixed pH all times and the transfer characteristics after removing the cells did not get back to FBS adsorbed state, it can be caused by re-arrangement of proteins on the diamond surface during cell culturing process. Another possibility is degradation of H-termination. AFM showed that proteins are not changing. Thus surface degradation is more likely although H-terminated diamond is generally considered as stable in the long term. These effects need further study.

To explain comparable performance of NCD SGFET independent of grain sizes, we present schematic model of the interface between cell medium containing proteins and H-terminated NCD with grain boundary (Figure 6) and we discuss effects in this interface depending on different grain size. In this model there is indicated that proteins are stuck directly to the diamond surface independently of grain boundaries due to hydrophobic-hydrophobic interaction of H-diamond and protein “core.” This is in agreement with AFM studies of FBS proteins on diamond [Rezek et al., 2009b]. Electronic transport in the surface conductive channel take places in the grain interior where it also behaves as in monocrystal. The carrier (hole) densities are also comparable to the monocrystal surface conductive layer [Hubik et al., accepted]. Overall lower currents on NCD compared to MCD are due to limited mobility of charge carriers across grain boundaries. Nevertheless this does not influence the device/sensor function. Comparison of SGFETs with various grain sizes shows that tunneling transport across grain boundaries is not influenced by protein adsorption and only the interior of diamond grains controls the interaction of proteins with surface conductive layer in diamond.

Conclusion We have shown that intrinsic NCD films on glass with average grain sizes from 250 nm down to

80 nm and thickness down to 100 nm are operational as SGFETs employing surface conductivity of H-terminated surface. We observed shift (– 50 mV) and decreased slope of transfer characteristics after adsorption of FBS in both SGFETs with different grain size. Another shift of transfer characteristics of both SGFETs was observed after 2-day cell culturing process. These shifts are permanent even after rinsing using deionized water and other agents because proteins from the cell medium create a thin primary inter layer which remains on diamond irrespective of rinsing. We proposed the model of the interface between H-terminated nanocrystalline diamond and cell medium containing proteins from fetal bovine serum and we suggested that proteins can modify original equilibrium of the surface conductive layer system. Comparable performance of SGFETs with various grain sizes shows that sensor function is controlled by interaction with diamond grains not sp2 phase between the grains. It is obvious that cell culturing process influences diamond surface conductive layer system, hence it is necessary to consider these effects in application of diamond SGFETs as biosensors.

Figure 6. Schematic model of the interface between H-terminated nanocrystalline diamond and cell medium containing proteins from fetal bovine serum.

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List of abbreviations SGFET—solution gated field effect transistor; NCD—nanocrystalline diamond; FBS—fetal

bovine serum; AFM—atomic force microscopy; CVD—chemical vapor deposition; SEM—scanning electron microscopy; PBS—phosphate buffered saline; Uds—drain-source voltage; Ug—gate voltage.

Acknowledgments. Technical support of Vlastimil Jurka, Karel Hruška, Martin Ledinský and Zdenka

Poláčková is gratefully appreciated. Antoníın Fejfar is kindly acknowledged for development of measurement and control software package. This research was financially supported by the projects KAN400100701 (AVČR), P204/10/0212 (GACR), IAAX00100902 (GAAV), LC510 (MŠMT), LC06040 (MŠMT), AV0Z10100521, MSM0021620806 (MŠMT), 202/09/H041, by the Fellowship J.E. Purkyně (BR, AK) and by the Fellowship 2010 L´Oreal-UNESCO for Women in Science (MK).

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