- Email: [email protected]
Diamond Based Biosensors: Surface-Fluid Interaction Issues
Available online at www.sciencedirect.com
Procedia Chemistry 6 (2012) 117 – 124
2nd International Conference on Bio-Sensing Technology
Diamond based biosensors: surface-fluid interaction issues M.A. Netoa*, C.M.F. Gomesb, E.L. Silvaa, A.J.S. Fernandesc, F.J. Oliveiraa, R.F. Silvaa, a* a
b
CICECO, Dept. of Ceramics & Glass Engineering, University of Aveiro, Aveiro 3810-193, Portugal. IBILI, Pharmacology and Experimental Therapeutics, Faculty of Medicine, University of Coimbra, Coimbra 3000-548, Portugal. c I3N, Dept. of Physics, University of Aveiro, Aveiro 3810-193, Portugal.
Abstract The interaction of nanocrystalline (NCD) and microcrystalline (MCD) p-type semiconducting diamond surfaces with phosphate buffer saline (PBS), simulated body fluid (SBF) and bovine serum albumin (BSA) solutions under applied electric potentials is the main subject of this work. The effect of these fluids on the electrical resistivity of the diamond surface is evaluated. Furthermore, the interaction effects of these surfaces with BSA containing SBF solution during 30 days, is addressed. The electrical measurements have shown that the electrical current passing through the NCD samples is influenced by the fluid’s ionic composition, whereas no variation was detected for the MCD samples. Both NCD and MCD surfaces favor the formation of a deposit layer after 30 days in contact with SBF+BSA solution. This deposit is mainly composed of Ca, P, Mg and O, containing also a significant concentration of BSA proteins.
© 2011 Published by Elsevier Ltd. Selection and/or peer-review under responsibility of the Institute of Bio-Sensing Technologies, UWE Bristol. Keywords: Diamond surfaces,boron doped, bioinertess, simulated body fluid, albumin.
1. Introduction The increasing need for devices that can continuously monitor certain human pathologies requires the development of reliable long term implantable biosensors. In many cases these devices must stay in contact with blood. This could lead to frequent protein adsorption and/or ionic compound formation on
* Corresponding author. Tel.: +351 234370200; fax: +351 234370985. E-mail address: [email protected].
1876-6196 © 2012 Elsevier B.V. doi:10.1016/j.proche.2012.10.137
118
M.A. Neto et al. / Procedia Chemistry 6 (2012) 117 – 124
their surfaces. Such effects are not desirable and could hamper or even suppress the biological activity of the immobilized moieties on the sensor’s bioactive surface thereby affecting the sensor’s performance. Polycrystalline p-type semiconducting diamond surfaces grown on silicon nitride ceramics are expected to be ideal platform for a number of biomedical applications and devices. Such diamond surfaces are highly biocompatible [1] and can be easily functionalized to achieve immobilization of biological entities through covalent bonding [2, 3]. These and other properties boost up diamond based technology as an effective solution for bio-sensing applications. However, before the functionalization procedure is applied on these surfaces a number of issues have to be addressed. First the background amperometric response under applied dc voltage has to be studied when the material is in contact with physiological fluids. Furthermore, diamond surfaces that support protein adsorption and/or ionic compound formation need to be identified. 2. Experimental 2.1. Diamond growth Six boron-doped diamond surfaces were grown, for 3h, in a hot-filament chemical vapour deposition (HFCVD) reactor, using hydrogen (H2), methane (CH4) and argon (Ar) as the feed gases and boron oxide (B2O3) as the boron doping source. Boron oxide was dissolved in 99.9% pure ethanol (C2H6O) with B/C of 10000 ppm. This solution was placed in a bubbler and then dragged into the reactor chamber by means of a constant argon flow (12 ml/min). Table 1 summarizes the growth parameters used for the diamond surfaces. The chamber pressure and CH4/H2 ratio were selected to yield both NCD and MCD diamond surfaces with different microstructures. Table 1. Main growth parameters for the six diamond surfaces.
Sample A B C D E F
Pressure (mbar) 25 25 50 50 100 100
CH4/H2 0.016 0.063 0.016 0.063 0.016 0.063
Ts (ºC) 790 780 780 770 770 760
These surfaces were grown on electrically insulating sintered silicon nitride (Si3N4) ceramic substrates. These substrates were rectangular shaped (10mmx13mm), 1mm thick, and they were prepared using a standard pressureless sintering process optimized in our lab [4]. Mirror like finished surfaces were achieved after the sintered bodies were cut, ground and polished with 15ȝm, 6ȝm diamond grit and colloidal silica (0.05ȝm). Prior to diamond growth, the substrates were ultrasonically seeded for 30 min in a suspension of ethanol and diamond slurry (15ȝm). 2.2. Characterization The interaction of these surfaces with a physiological fluid was carried out by placing them in contact, for 30 days, with a simulated body fluid (SBF) solution containing bovine serum albumin (BSA) at a
M.A. Neto et al. / Procedia Chemistry 6 (2012) 117 – 124
concentration of 40mg/ml. The SBF was prepared using the procedure developed by Oyane et al [5] and the BSA was purchased from Sigma-Aldrich. The coatings’ morphology was characterized by scanning electron microscopy (SEM) equipment, before and after the 30 day period. Energy dispersive X-ray spectroscopy (EDS) was applied on these surfaces to characterize their elemental composition. For the electrical measurements two copper wires, 8 mm apart, were fixed with conductive silver glue to the diamond surfaces. These contacts were then isolated from the surrounding environment using an epoxy based resin. The I-V response of each conducting surface in contact with phosphate buffer saline (PBS), SBF and SBF with BSA was obtained using a specially developed homemade equipment which incorporates a 16bit USB DAQ (National Instruments) controlled by a pc. These measurements were performed in continuous stirred solutions maintained at 37ºC. Finally, protein adsorption on these surfaces was estimated using the bicinchoninic acid (BCA) assay in conjunction with Fourier Transformed Infrared absorption spectroscopy (FT-IR). 3. Results and discussion The SEM micrographs of the six diamond surfaces presented in Fig. 1 confirm the growth of two distinctive morphologies: microcrystalline (MCD) for the A, C and E samples grown with CH4/H2=0.016, and nanocrystalline (NCD) for samples B, D and F grown with CH4/H2=0.063.
A
B
200 nm C
D
200 nm E
F
200 nm Fig.1. SEM micrographs of the six as-grown diamond surfaces obtained at: 25mbar (samples A and B), 50mbar (samples C and D), 100mbar (samples E and F), with two CH4/H2 ratios.
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M.A. Neto et al. / Procedia Chemistry 6 (2012) 117 – 124
It is clearly seen in the micrographs of Fig. 1 that the MCD surfaces have bigger diamond grains than the NCD coatings, resulting in rougher surfaces. Furthermore, for the MCD morphology, the deposition pressure seems to influence the surface roughness, reducing the grain size with increasing pressure. These different microstructures should incorporate boron in different ways and have different electrical behaviour. The typical I-V response of the diamond surfaces in contact with PBS, SBF and SBF with BSA is illustrated by the graphs of Fig. 2. These graphs show that the two surface morphologies behave differently in contact with the physiological media. While the more conductive MCD surfaces are essentially inert, the current passing through the NCD samples is influenced by the fluid’s ionic composition. This is explained by the differential electrical potential created along the 8mm gap on the NCD diamond surface that induces charge transfer at the surface/fluid interface. Due to the higher electrical conductivity of the MCD samples this differential potential is very small as indicated on the axis of the correspondent graph of Fig. 2. Consequently, negligible charge transfer at the surface/fluid interface is expected to occur for such surfaces. Also, the current collected by the NCD surfaces is reduced upon contact with the BSA containing SBF solution when compared to the pure SBF fluid. This could be explained by the binding capability of serum albumin molecules to both negative and positive ions in solution [6]. This, locally, reduces the amount of free ions in solution therein reducing the charge transfer at the surface/fluid interface.
Current (A)
1,0x10
-4
-4
H2O PBS SBF SBF+BSA
Current (A)
2,0x10
0,0
-1,0x10
-2,0x10
-4
MCD
-4
-0,2
-0,1
0,0
Voltage (V)
0,1
0,2
1,0x10
-4
8,0x10
-5
6,0x10
-5
4,0x10
-5
2,0x10
-5
H2O PBS SBF SBF+BSA
0,0 -2,0x10
-5
-4,0x10
-5
-6,0x10
-5
-8,0x10
-5
-1,0x10
-4
-1,2x10
-4
-12 -10
NCD -8
-6
-4
-2
0
2
4
6
8
10
12
Voltage (V)
Fig.2. Typical I-V response of the MCD and NCD diamond surfaces in contact with distilled water, PBS, SBF and SBF with BSA.
All of these surfaces were then rinsed in distilled water and placed in contact with a SBF solution containing BSA (40mg/ml) for 30 days at 37ºC. After this period, the samples were rinsed again in distilled water in an ultrasonic bath for 30 minutes. The new SEM micrographs of the diamond surfaces are presented in Fig. 3 and illustrate radical changes. The typical MCD and NCD morphologies are no longer observed. Rather, the diamond surfaces are now covered by a more or less uniform deposit with small variations between the samples. This suggests a marked interaction between the diamond surfaces and the biological fluid.
M.A. Neto et al. / Procedia Chemistry 6 (2012) 117 – 124
To identify the chemical composition of the deposit, EDS spectra were obtained on every diamond surface. The data presented by the graph of Fig. 4 for samples A and B, prove that Ca, P, Mg, O and Si are the main elements present in the deposited layer. While the detection of Si could be attributed to the silicon nitride substrate or to silicon impurities incorporated in the diamond surfaces during their growth, the other elements can only originate from the physiological fluid.
A
B
10 μm C
10 μm D
10 μm E
10 μm F
10 μm
10 μm
Fig.3. SEM micrographs of the diamond surfaces after 30 days in contact with the SBF solution containing BSA.
The amount of albumin adsorbed to the surface of diamonds after 30 days incubation period with SBF solution containing BSA was estimated using the BCA assay, that measure the concentration of the protein in fluid based on a calibration curve. Knowing the initial amount of BSA in the solution it was possible to estimate the concentration of adsorbed protein on the samples by subtracting the amount of albumin left in the fluid (Fig. 5a). It is clear that the concentration of adsorbed BSA is influenced by the surface morphology. For MCD, the data shows a concentration increase of adhered protein with decreasing grain size. This observation though, well perceptible for the MCD surfaces it is less pronounced for the NCD morphology. To confirm the presence of BSA proteins on the deposit layer, FT-IR spectra of the diamond surfaces, before and after the 30 day test, were obtained (Fig. 5b). The Amide I (~1649cm-1) and Amide II
121
122
M.A. Neto et al. / Procedia Chemistry 6 (2012) 117 – 124
(~1539cm-1) IR bands are clearly detected, which arise from the amide bonds that link the amino acids of the protein: while the Amide I band is due to carbonyl stretching vibrations, the Amide II is due primarily to NH bending vibrations. This result effectively proves that the BSA protein adheres to both MCD and NCD diamond surfaces. However the data presented in this work do not allow a definitive clarification on the mechanism that explains the adhesion of BSA to the diamond surfaces. In fact, the albumin protein is a very reactive molecule and can easily bind to small ions present in solution (i.e. Ca2+, Mg2+) [6, 7]. Consequently the precipitation of ionic compounds (i.e. hydroxyapatite) on the surface is affected by the presence of BSA. 2000
MCD NCD
1800 1600 1400
Counts
SBF+BSA 30 days
O
1200
P
1000
Ca
800
Si
600
Mg
400 200 0 0
2
4
6
Energy (keV) Fig.4. Typical EDS spectra of the MCD (sample E) and NCD (sample D) surfaces after 30 days in contact with the physiological fluid (SBF+BSA). 0,6
200
Absorbance
100 mbar
50 mbar
0,3
Amide I
0,2 0,1 0,0
Absorbance
160
NCD
1500
2000
3000
3500
4000
MCD as grown MCD SBF+BSA 30 days
0,15 0,10
Amide II
Amide I
0,05
1000
MCD
NCD as grown NCD SBF+BSA 30 days
Amide II
0,4
1000
25 mbar
50 mbar
180
25 mbar
100 mbar
-2
Adsorbed proteinμ(g.m m )
0,5
1500
2000
3000
3500
4000
-1
Wavenumber (cm )
Fig.5. a) Comparative graph for the estimated adsorbed BSA protein concentration on the six diamond surfaces; b) Typical FT-IR spectra of the MCD (sample E) and NCD (sample D) diamond surfaces with the highest BSA adsorption.
M.A. Neto et al. / Procedia Chemistry 6 (2012) 117 – 124
Previously published work has shown that NCD surfaces are essentially bioinert when in contact with SBF [8]. Such study did not report any deposition of Ca, P, and Mg on the diamond surfaces. Another work also showed that such diamond surfaces are not prone to unspecific interaction with BSA protein leading to negligible adsorption [9]. Nevertheless, these works were carried out on non doped diamond surfaces and for a very short period time (10 days). Consequently, their main results cannot be compared with the results of the present study. 4. Conclusions This work clearly demonstrates that the amperometric response of low boron doped NCD surfaces is much more influenced by the physiological fluids than the heavy boron doped MCD surfaces where no change is detected. This could be explained by a charge transfer between the ionic media and the diamond surface when a higher electrical potential is build up on the NCD surfaces. The contact of both boron doped MCD and NCD surfaces, for 30 days, with the SBF containing BSA solution, demonstrates the bioactivity of these surfaces. A deposit layer is formed on every NCD and MCD surface, and it is mainly composed of Ca, P, Mg, O and BSA. Acknowledgements This work was supported by project PTDC/CTM-MET/113645/2009, funded by FEDER through COMPETE programmme- Operacional Factors for Competitivity and by national funds through FCT Portuguese Science and Technology Foundation. M.A. Neto and E.L. Silva would like to acknowledge, respectively, the grants SFRH/BPD/45610/2008 and SFRH/BD/61675/2009 from FCT - Fundação para a Ciência e a Tecnologia. References [1] Tang L, Tsai C, Gerberich WW, Kruckebeu L, Kania DR. Biocompatibility of chemical-vapour-deposited diamond. Biomaterials 1995;16:483-8. [2] Härtl A, Schmich E, Garrido JA, Hernando J, Catharino SCR, Walter S, Feulner P, Kromka A, Steinmüller D, Stutzmann M. Protein-modified nanocrystalline diamond thin films for biosensor applications. Nature Mater 2004;3:736-42. [3] Yang W et al. DNA-modified nanocrystalline diamond thin-films as stable, biologically active substrates. Nature Mater 2002;1:253-7. [4] Neto MA, Silva EL, Ghumman CA, Teodoro OM, Fernandes AJS, Oliveira FJ, Silva RF. Composition profiles and adhesion evaluation of conductive CVD diamond coatings on dielectric ceramics. Thin Solid Films 2012; Sumited to publication. [5] Oyane A, Kim H-M, Furuya T, Kokubo T, Miyazaki, Nakamura Tl. Preparation and assessmet of revised simulated body fluids. J Biomed Mater Res A. 2003 May 1;65(2):188-95 [6] Deland EC, Heirschfeldt R. Protein binding of small ions – A mathematical model of serum albumin. Memorandum 1967; RM-5254-PR. [7] Fogh-Andersen N, Bjerrum PJ, Siggaard-Andersen O. Ionic Binding, Net Charge, and Donnan Effect of Human Serum Albumin as a Function of pH. Clin. Chem. 1993;39/1:48-52. [8] Popov C, Kulisch W, Jelinek M, Bock A, Strnad J. Nanocrystalline diamond/amorphous carbon composite films for
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Procedia Chemistry 6 (2012) 117 – 124
2nd International Conference on Bio-Sensing Technology
Diamond based biosensors: surface-fluid interaction issues M.A. Netoa*, C.M.F. Gomesb, E.L. Silvaa, A.J.S. Fernandesc, F.J. Oliveiraa, R.F. Silvaa, a* a
b
CICECO, Dept. of Ceramics & Glass Engineering, University of Aveiro, Aveiro 3810-193, Portugal. IBILI, Pharmacology and Experimental Therapeutics, Faculty of Medicine, University of Coimbra, Coimbra 3000-548, Portugal. c I3N, Dept. of Physics, University of Aveiro, Aveiro 3810-193, Portugal.
Abstract The interaction of nanocrystalline (NCD) and microcrystalline (MCD) p-type semiconducting diamond surfaces with phosphate buffer saline (PBS), simulated body fluid (SBF) and bovine serum albumin (BSA) solutions under applied electric potentials is the main subject of this work. The effect of these fluids on the electrical resistivity of the diamond surface is evaluated. Furthermore, the interaction effects of these surfaces with BSA containing SBF solution during 30 days, is addressed. The electrical measurements have shown that the electrical current passing through the NCD samples is influenced by the fluid’s ionic composition, whereas no variation was detected for the MCD samples. Both NCD and MCD surfaces favor the formation of a deposit layer after 30 days in contact with SBF+BSA solution. This deposit is mainly composed of Ca, P, Mg and O, containing also a significant concentration of BSA proteins.
© 2011 Published by Elsevier Ltd. Selection and/or peer-review under responsibility of the Institute of Bio-Sensing Technologies, UWE Bristol. Keywords: Diamond surfaces,boron doped, bioinertess, simulated body fluid, albumin.
1. Introduction The increasing need for devices that can continuously monitor certain human pathologies requires the development of reliable long term implantable biosensors. In many cases these devices must stay in contact with blood. This could lead to frequent protein adsorption and/or ionic compound formation on
* Corresponding author. Tel.: +351 234370200; fax: +351 234370985. E-mail address: [email protected].
1876-6196 © 2012 Elsevier B.V. doi:10.1016/j.proche.2012.10.137
118
M.A. Neto et al. / Procedia Chemistry 6 (2012) 117 – 124
their surfaces. Such effects are not desirable and could hamper or even suppress the biological activity of the immobilized moieties on the sensor’s bioactive surface thereby affecting the sensor’s performance. Polycrystalline p-type semiconducting diamond surfaces grown on silicon nitride ceramics are expected to be ideal platform for a number of biomedical applications and devices. Such diamond surfaces are highly biocompatible [1] and can be easily functionalized to achieve immobilization of biological entities through covalent bonding [2, 3]. These and other properties boost up diamond based technology as an effective solution for bio-sensing applications. However, before the functionalization procedure is applied on these surfaces a number of issues have to be addressed. First the background amperometric response under applied dc voltage has to be studied when the material is in contact with physiological fluids. Furthermore, diamond surfaces that support protein adsorption and/or ionic compound formation need to be identified. 2. Experimental 2.1. Diamond growth Six boron-doped diamond surfaces were grown, for 3h, in a hot-filament chemical vapour deposition (HFCVD) reactor, using hydrogen (H2), methane (CH4) and argon (Ar) as the feed gases and boron oxide (B2O3) as the boron doping source. Boron oxide was dissolved in 99.9% pure ethanol (C2H6O) with B/C of 10000 ppm. This solution was placed in a bubbler and then dragged into the reactor chamber by means of a constant argon flow (12 ml/min). Table 1 summarizes the growth parameters used for the diamond surfaces. The chamber pressure and CH4/H2 ratio were selected to yield both NCD and MCD diamond surfaces with different microstructures. Table 1. Main growth parameters for the six diamond surfaces.
Sample A B C D E F
Pressure (mbar) 25 25 50 50 100 100
CH4/H2 0.016 0.063 0.016 0.063 0.016 0.063
Ts (ºC) 790 780 780 770 770 760
These surfaces were grown on electrically insulating sintered silicon nitride (Si3N4) ceramic substrates. These substrates were rectangular shaped (10mmx13mm), 1mm thick, and they were prepared using a standard pressureless sintering process optimized in our lab [4]. Mirror like finished surfaces were achieved after the sintered bodies were cut, ground and polished with 15ȝm, 6ȝm diamond grit and colloidal silica (0.05ȝm). Prior to diamond growth, the substrates were ultrasonically seeded for 30 min in a suspension of ethanol and diamond slurry (15ȝm). 2.2. Characterization The interaction of these surfaces with a physiological fluid was carried out by placing them in contact, for 30 days, with a simulated body fluid (SBF) solution containing bovine serum albumin (BSA) at a
M.A. Neto et al. / Procedia Chemistry 6 (2012) 117 – 124
concentration of 40mg/ml. The SBF was prepared using the procedure developed by Oyane et al [5] and the BSA was purchased from Sigma-Aldrich. The coatings’ morphology was characterized by scanning electron microscopy (SEM) equipment, before and after the 30 day period. Energy dispersive X-ray spectroscopy (EDS) was applied on these surfaces to characterize their elemental composition. For the electrical measurements two copper wires, 8 mm apart, were fixed with conductive silver glue to the diamond surfaces. These contacts were then isolated from the surrounding environment using an epoxy based resin. The I-V response of each conducting surface in contact with phosphate buffer saline (PBS), SBF and SBF with BSA was obtained using a specially developed homemade equipment which incorporates a 16bit USB DAQ (National Instruments) controlled by a pc. These measurements were performed in continuous stirred solutions maintained at 37ºC. Finally, protein adsorption on these surfaces was estimated using the bicinchoninic acid (BCA) assay in conjunction with Fourier Transformed Infrared absorption spectroscopy (FT-IR). 3. Results and discussion The SEM micrographs of the six diamond surfaces presented in Fig. 1 confirm the growth of two distinctive morphologies: microcrystalline (MCD) for the A, C and E samples grown with CH4/H2=0.016, and nanocrystalline (NCD) for samples B, D and F grown with CH4/H2=0.063.
A
B
200 nm C
D
200 nm E
F
200 nm Fig.1. SEM micrographs of the six as-grown diamond surfaces obtained at: 25mbar (samples A and B), 50mbar (samples C and D), 100mbar (samples E and F), with two CH4/H2 ratios.
119
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M.A. Neto et al. / Procedia Chemistry 6 (2012) 117 – 124
It is clearly seen in the micrographs of Fig. 1 that the MCD surfaces have bigger diamond grains than the NCD coatings, resulting in rougher surfaces. Furthermore, for the MCD morphology, the deposition pressure seems to influence the surface roughness, reducing the grain size with increasing pressure. These different microstructures should incorporate boron in different ways and have different electrical behaviour. The typical I-V response of the diamond surfaces in contact with PBS, SBF and SBF with BSA is illustrated by the graphs of Fig. 2. These graphs show that the two surface morphologies behave differently in contact with the physiological media. While the more conductive MCD surfaces are essentially inert, the current passing through the NCD samples is influenced by the fluid’s ionic composition. This is explained by the differential electrical potential created along the 8mm gap on the NCD diamond surface that induces charge transfer at the surface/fluid interface. Due to the higher electrical conductivity of the MCD samples this differential potential is very small as indicated on the axis of the correspondent graph of Fig. 2. Consequently, negligible charge transfer at the surface/fluid interface is expected to occur for such surfaces. Also, the current collected by the NCD surfaces is reduced upon contact with the BSA containing SBF solution when compared to the pure SBF fluid. This could be explained by the binding capability of serum albumin molecules to both negative and positive ions in solution [6]. This, locally, reduces the amount of free ions in solution therein reducing the charge transfer at the surface/fluid interface.
Current (A)
1,0x10
-4
-4
H2O PBS SBF SBF+BSA
Current (A)
2,0x10
0,0
-1,0x10
-2,0x10
-4
MCD
-4
-0,2
-0,1
0,0
Voltage (V)
0,1
0,2
1,0x10
-4
8,0x10
-5
6,0x10
-5
4,0x10
-5
2,0x10
-5
H2O PBS SBF SBF+BSA
0,0 -2,0x10
-5
-4,0x10
-5
-6,0x10
-5
-8,0x10
-5
-1,0x10
-4
-1,2x10
-4
-12 -10
NCD -8
-6
-4
-2
0
2
4
6
8
10
12
Voltage (V)
Fig.2. Typical I-V response of the MCD and NCD diamond surfaces in contact with distilled water, PBS, SBF and SBF with BSA.
All of these surfaces were then rinsed in distilled water and placed in contact with a SBF solution containing BSA (40mg/ml) for 30 days at 37ºC. After this period, the samples were rinsed again in distilled water in an ultrasonic bath for 30 minutes. The new SEM micrographs of the diamond surfaces are presented in Fig. 3 and illustrate radical changes. The typical MCD and NCD morphologies are no longer observed. Rather, the diamond surfaces are now covered by a more or less uniform deposit with small variations between the samples. This suggests a marked interaction between the diamond surfaces and the biological fluid.
M.A. Neto et al. / Procedia Chemistry 6 (2012) 117 – 124
To identify the chemical composition of the deposit, EDS spectra were obtained on every diamond surface. The data presented by the graph of Fig. 4 for samples A and B, prove that Ca, P, Mg, O and Si are the main elements present in the deposited layer. While the detection of Si could be attributed to the silicon nitride substrate or to silicon impurities incorporated in the diamond surfaces during their growth, the other elements can only originate from the physiological fluid.
A
B
10 μm C
10 μm D
10 μm E
10 μm F
10 μm
10 μm
Fig.3. SEM micrographs of the diamond surfaces after 30 days in contact with the SBF solution containing BSA.
The amount of albumin adsorbed to the surface of diamonds after 30 days incubation period with SBF solution containing BSA was estimated using the BCA assay, that measure the concentration of the protein in fluid based on a calibration curve. Knowing the initial amount of BSA in the solution it was possible to estimate the concentration of adsorbed protein on the samples by subtracting the amount of albumin left in the fluid (Fig. 5a). It is clear that the concentration of adsorbed BSA is influenced by the surface morphology. For MCD, the data shows a concentration increase of adhered protein with decreasing grain size. This observation though, well perceptible for the MCD surfaces it is less pronounced for the NCD morphology. To confirm the presence of BSA proteins on the deposit layer, FT-IR spectra of the diamond surfaces, before and after the 30 day test, were obtained (Fig. 5b). The Amide I (~1649cm-1) and Amide II
121
122
M.A. Neto et al. / Procedia Chemistry 6 (2012) 117 – 124
(~1539cm-1) IR bands are clearly detected, which arise from the amide bonds that link the amino acids of the protein: while the Amide I band is due to carbonyl stretching vibrations, the Amide II is due primarily to NH bending vibrations. This result effectively proves that the BSA protein adheres to both MCD and NCD diamond surfaces. However the data presented in this work do not allow a definitive clarification on the mechanism that explains the adhesion of BSA to the diamond surfaces. In fact, the albumin protein is a very reactive molecule and can easily bind to small ions present in solution (i.e. Ca2+, Mg2+) [6, 7]. Consequently the precipitation of ionic compounds (i.e. hydroxyapatite) on the surface is affected by the presence of BSA. 2000
MCD NCD
1800 1600 1400
Counts
SBF+BSA 30 days
O
1200
P
1000
Ca
800
Si
600
Mg
400 200 0 0
2
4
6
Energy (keV) Fig.4. Typical EDS spectra of the MCD (sample E) and NCD (sample D) surfaces after 30 days in contact with the physiological fluid (SBF+BSA). 0,6
200
Absorbance
100 mbar
50 mbar
0,3
Amide I
0,2 0,1 0,0
Absorbance
160
NCD
1500
2000
3000
3500
4000
MCD as grown MCD SBF+BSA 30 days
0,15 0,10
Amide II
Amide I
0,05
1000
MCD
NCD as grown NCD SBF+BSA 30 days
Amide II
0,4
1000
25 mbar
50 mbar
180
25 mbar
100 mbar
-2
Adsorbed proteinμ(g.m m )
0,5
1500
2000
3000
3500
4000
-1
Wavenumber (cm )
Fig.5. a) Comparative graph for the estimated adsorbed BSA protein concentration on the six diamond surfaces; b) Typical FT-IR spectra of the MCD (sample E) and NCD (sample D) diamond surfaces with the highest BSA adsorption.
M.A. Neto et al. / Procedia Chemistry 6 (2012) 117 – 124
Previously published work has shown that NCD surfaces are essentially bioinert when in contact with SBF [8]. Such study did not report any deposition of Ca, P, and Mg on the diamond surfaces. Another work also showed that such diamond surfaces are not prone to unspecific interaction with BSA protein leading to negligible adsorption [9]. Nevertheless, these works were carried out on non doped diamond surfaces and for a very short period time (10 days). Consequently, their main results cannot be compared with the results of the present study. 4. Conclusions This work clearly demonstrates that the amperometric response of low boron doped NCD surfaces is much more influenced by the physiological fluids than the heavy boron doped MCD surfaces where no change is detected. This could be explained by a charge transfer between the ionic media and the diamond surface when a higher electrical potential is build up on the NCD surfaces. The contact of both boron doped MCD and NCD surfaces, for 30 days, with the SBF containing BSA solution, demonstrates the bioactivity of these surfaces. A deposit layer is formed on every NCD and MCD surface, and it is mainly composed of Ca, P, Mg, O and BSA. Acknowledgements This work was supported by project PTDC/CTM-MET/113645/2009, funded by FEDER through COMPETE programmme- Operacional Factors for Competitivity and by national funds through FCT Portuguese Science and Technology Foundation. M.A. Neto and E.L. Silva would like to acknowledge, respectively, the grants SFRH/BPD/45610/2008 and SFRH/BD/61675/2009 from FCT - Fundação para a Ciência e a Tecnologia. References [1] Tang L, Tsai C, Gerberich WW, Kruckebeu L, Kania DR. Biocompatibility of chemical-vapour-deposited diamond. Biomaterials 1995;16:483-8. [2] Härtl A, Schmich E, Garrido JA, Hernando J, Catharino SCR, Walter S, Feulner P, Kromka A, Steinmüller D, Stutzmann M. Protein-modified nanocrystalline diamond thin films for biosensor applications. Nature Mater 2004;3:736-42. [3] Yang W et al. DNA-modified nanocrystalline diamond thin-films as stable, biologically active substrates. Nature Mater 2002;1:253-7. [4] Neto MA, Silva EL, Ghumman CA, Teodoro OM, Fernandes AJS, Oliveira FJ, Silva RF. Composition profiles and adhesion evaluation of conductive CVD diamond coatings on dielectric ceramics. Thin Solid Films 2012; Sumited to publication. [5] Oyane A, Kim H-M, Furuya T, Kokubo T, Miyazaki, Nakamura Tl. Preparation and assessmet of revised simulated body fluids. J Biomed Mater Res A. 2003 May 1;65(2):188-95 [6] Deland EC, Heirschfeldt R. Protein binding of small ions – A mathematical model of serum albumin. Memorandum 1967; RM-5254-PR. [7] Fogh-Andersen N, Bjerrum PJ, Siggaard-Andersen O. Ionic Binding, Net Charge, and Donnan Effect of Human Serum Albumin as a Function of pH. Clin. Chem. 1993;39/1:48-52. [8] Popov C, Kulisch W, Jelinek M, Bock A, Strnad J. Nanocrystalline diamond/amorphous carbon composite films for
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M.A. Neto et al. / Procedia Chemistry 6 (2012) 117 – 124 applications in tribology, optics and biomedicine. Thin Solid Films 2006;494:92–7. [9] Kulisch W, Popov C, Gilliland D, Ceccone G, Reithmaier JP, Rossi F. UNCD/a-C nanocomposite films for biotechnological applications. Surface Coat Tech 2011;206:667-76.