Castellino Micaela
Graduate School in Physics
and Astrophysics - XX Cycle
Towards Biosensors:
Characterisation and
Functionalisation of
Diamond Surface
1
Outline


Introduction
Why Diamond?
•
•
•
General properties (“the Biggest & the Best”)
Production methods
Surface:
•
•
•

Properties (Electrochemical model)
Characterisation (XPS, AFM, electrical measurements)
Functionalisation (Proteins attachment)
Conclusions
M. Castellino - NIS & DFS Torino
2
Research Line 5:
Molecular interactions
in complex and nanostructured biointerphases:
solid-protein-cell
Research Line 2:
Nano-structured thin
films for coatings and
functional applications
My PhD thesis is a MIUR scholarship
(D.M. 198 – 23/10/03)
M. Castellino - NIS & DFS Torino
3
Aim of the work
Diamond Surface study to develop a device for
simultaneous recording of electrical and optical signals
from living cells, as sensor elements (Biosensors)
Variation of the physiochemical
(temperature, pH, ion
concentration….) and physiological
(growth factor, hormones….)
environments can greatly influence
cellular health
M. Castellino - NIS & DFS Torino
4
Aim of the work
What is the physical observable ?
Electrochemical response
The membrane potential changes which
occur during nerve impulse
propagation are collectively called
Action Potential (AP)
Potential use of cell based sensors:

Environmental monitoring
(chemical/biological warfare
agents, groundwater
contamination…..)

Pharmaceutical screening

Drug discovery
M. Castellino - NIS & DFS Torino
5
State of the art
Intracellular recording: patch clamp
ADVANTAGES
•Well established technique
•Best signal-to-noise ratio
DRAWBACKS
•Fragile interfaces that limit the duration of the
recordings
•Difficult simultaneous recording from various cells
Extracellular recording with metallic multi electrode array
ADVANTAGES
•Capability to record and stimulate multiple cells simultaneously
•Feasible to conduct long term and repeated measurements on
the same culture.
DRAWBACKS
•Signal amplitude is too smaller
•No optical transmission trough metallic surfaces
6
Why Diamond?
“… silicon has been used in most of the past and current developments. However,
its poor biocompatibility and chemical instability prevent silicon from becoming
the ideal material for biosensors applications. In contrast, diamond is known as a
biocompatible material in itself, consisting of just carbon atoms”
(A. Hartl et al, Nature Materials, 3, 736- 742, 2004)
“Diamond possesses unique properties (biocompatibility, optical
transparency, possibility of modifying the electronic and
hydrophilic/hydrophobic properties at the nanoscale)”
(P. Ariano et al. Diam. Relat. Mater, 14 - 669, 2005)
“Diamond has some of the most extreme physical properties of any
material, yet its practical use in science or engineering has been
limited due its scarsity and expense ”
(P. May. Phil. Trans. R. Soc. Lond. A, 358, 473 - 495, 2000)
M. Castellino - NIS & DFS Torino
7
Diamond
Natural diamond:
too much expensive, with structure and
morphology not suitable for technical
applications
Artificial diamond:
1953 – Stockholm (Quintus-ASEA project):
first synthetic diamond (8.4 GPa, 1500°C)
(the discovery was kept secret )
1954 – USA (T. Hall, General Electric):
first published result
Primary Tecniques:

HPHT (High Pressure High Temperature)
works in P and T ranges where diamond is the
stable form of carbon (A,B,C)

CVD (Chemical Vapour Deposition)
works in ranges where graphite is the carbon
stable form (D)
A - shockwave synthesis (sound pulses of
t  ms)
B – with catalyst (es. Ni)
C – without catalyst
D - low P (mbar) and low T (700 – 900 °C)
M. Castellino - NIS & DFS Torino
8
Diamond
The crystal structure of diamond is
equivalent to a face-centred cubic (FCC)
lattice.
The conventional unit cell is cubic with a
side length a0 approximately equal to
3.567 Å at room temperature.
The C – C bond length d is equal to
1.54 Å. The atomic density is
1.76×1023 atoms/cm3.
Its covalent bonds between hybrid sp3
orbitals make it the hardest material in
nature (from the Greek “adamas ” =
indestructible)
M. Castellino - NIS & DFS Torino
9
Diamond Bulk
Property
Diamond
Best
Alternative
Mechanical Hardness
(g/mm2)
5700 - 10400
4500 (cubic BN)
Thermal Conductivity
(W cm-1 K-1)
20
6 (BeO)
Electrical Resistivity
(W cm)
1015
1015 (Al2O3)
Band Gap (eV)
5.45
1.12 (Si)
Lattice Constant (Å)
3.56
5.43 (Si)
Optical Transmission
220 nm < l
< 2500 nm
l > 6000 nm
Sapphire
150 nm < l
< 5000 nm
M. Castellino - NIS & DFS Torino
10
Diamond Surface
H - Termination
Face (100) properties
a)
Without adsorbates: C
symmetrical dimers,
linked together with
double bond (s+p).
b)
With H (single): C atoms
arranges as dimers, but
only with s bonds, while
the left bond is
attached to the H atom.
S. J. Sque et al. - Physical Rev B 73, 2006
M. Castellino - NIS & DFS Torino
11
Diamond Surface
O - Termination
Face (100) properties
a)
“Ketone” arrangement:
the O single atom is
double-bonded to a
single surface C atom.
b)
“Ether”: the O atom
bridges two surface C
atom and makes a
single bond to each.
S. J. Sque et al. - Physical Rev B 73, 2006
The last one is a more stable configuration, due to the fact that the
highest occupied level in the ether system is significantly lower in
energy than the same level in the ketone system.
M. Castellino - NIS & DFS Torino
12
Diamond Surface
“Origin of Surface Conductivity in
Diamond”
(Maier et al., Phys. Rev. Lett. 85, 16 (2000) )
Different diamond samples show surface
conductivity (C=10-6-10-5A/V) if
hydrogenated and then exposed to air.
If the sample is hydrogenated but left in
UHV enviroment, the surface
conductivity reaches at least 10-10 A/V.
Chemisorbed hydrogen is a
necessary but not a sufficient
prerequisite for Surface
Conductivity (SC)
De-hydrogenation made with
1keV e- for 90min, with a flux of
0.2 mA/cm2
T= 300°C – aqueous layer desorption
T= 700°C – H desorption
M. Castellino - NIS & DFS Torino
13
Diamond Surface
Negative Electron Affinity - NEA (c)
When the vacuum level is below the
Conduction Band Minimum (CBM): in
this case the surface layer acts as an
“electrons well”, which can escape
from the material with an E=|c|.
c can be modified by the presence of
adsorbates, which create a “dipolar
layer”, varying the vacuum level :
a)
Without adsorbates: c =+0.38 eV
b)
With H- termination: c =-1.27 eV
c)
With O - termination: c =+1.77 eV
Element Electron Affinity (eV):
C (2.55), O (3.44), H (2.20)
(J. Ristein, Appl. Phys. A 82, 377-384, 2006)
c0 = clean surface electron affinity
e= elementary charge
e0= vacuum dielectric constant
n = dipoles density
pz = dipole moment
Diamond Surface
Surface Conductivity (SC):
(Ravi et al. ,Appl. Phys. Lett. 55, 1989))
p-type with r=1016 to 105 W cm
Holes density= 1012 – 1013 cm-2
Holes accumulation layer Xa = (0.4-1.2) nm.
Thomas - Fermi equation
e = diamond dielectric constant
Ne = carriers density (1020 cm-3)
(H. Kawarada, “The Physics of diamond: proceedings of the international school of physics Enrico Fermi”)
Diamond Surface
Electrochemical Model
cad=Egap+ cC-H= 5.5 - 1.3 = 4.2 eV
Electron affinity of molecular atmospheric
species lie in the range of 2.5<c<3.7 eV:
direct e- transfer from the diamond into an
atmospheric adsorbate seems to be
impossible.
A thin water layer provides a system which
can act as a surface acceptor for diamond.
me is the chemical potential of e- in the liquid
phase: when is below the diamond Fermi
level, e- are transferred from diamond to
water (left to right) until the band bending
equals the me to the EF (H2O me= -4.26 eV )
Maier et al., Phys. Rev. Lett. 85, 16 (2000) 3472
Electrons exchange by redox reaction:
2H3O++2e-
H2+2H2O
M. Castellino - NIS & DFS Torino
16
So far …
HTD
Control (Plastic)
M. Castellino - NIS & DFS Torino
Neurons grown on
HTD adhere, survive
and emit long neuritic
processes; no
differences with
control cultures
17
Hydrogenation
Hot Filament CVD tecnique
Gas Inlet
Bias
electrode
Filament
electrodes
Diamond sample
Sample
Atomic hydrogen
H• H•
2200 °C
Hot
Filament
(Ta, W, Re)
Gas outlet
H2
electrodes
ElettroRava, Savonera (TO) (Dott. P. Bonino)
Molecular hydrogen
Just to resume
Diamond surface can be modified trough H –
termination, becoming a conductor material, which
can acts as an electrode of a Biosensor : this has
also the diamond bulk properties such as Hardness,
Biocompatibility and Optical Transparency.
•
•My PhD research purpose is the diamond surface
characterisation, which can be subjected to several
functionalisations to create biosensors.
M. Castellino - NIS & DFS Torino
19
Characterisation
XPS (X-ray Photoelectron Spectroscopy)
VSW Scientific Instruments Ltd.
Experimental Physics Dep.
X-Ray source: Mg Ka (1253.6 eV), Al Ka (1486.6 eV)
Analyser: Concentric Hemispherical Analyser (CHA)
Energy resolution: (1.0 ± 0.1) eV (calculated on Au 4f7/2, Cu 2p3/2 , Cu L3VV)
Copyright by ASTM (AMERICAN SOCIETY FOR TESTING AND MATERIALS INTERNATIONAL)
Our Samples
Homoepitaxial (deposited on diamond substrates)
• Ulm 1 & 2 (supplied by Ulm University): 300 nm IIa (100) oriented diamond deposited
by plasma CVD on Ib HPHT diamond, with two different type of Boron doping surface
layer (1 nm) (3.7x3.7 mm2).
• Roma 1 & 2 (supplied by Tor Vergata University): 300 nm IIa (100) oriented diamond
deposited by HF CVD on HPHT Ib diamond substrate supplied by Sumitomo Electric Co.
Ltd. (3x3 mm2).
Heteroepitaxial (deposited on other materials)
• NCD (1 to 5) (supplied by Rho-best coating - Innsbruck): 100 nm IIa nano crystalline
diamond deposited by HF CVD on optical quartz (1x1 cm2)
• Ulm film on Si (supplied by Ulm University): 300 nm IIa oriented diamond deposited
by plasma CVD on Si wafer (5x8 mm2)
Other two samples will be presented later on (Article section)
M. Castellino - NIS & DFS Torino
21
XPS Analysis
How to distinguish diamond from other Carbon allotropes?
I (17.7 eV)
Counts (a.u.)
8.5
2s orbitals
II (13.4 eV)
8.0
sp3 orbitals
III (8.7 eV)
7.5
2p orbitals
Valence Band (Ulm 1 )
7.0
30
1
25
20
15
10
5
EB (eV)
0
s(2s)/s(2p) = 12
Structure
E (CVD)
eV
E (natural) 1
eV
E (peak) 2
eV
Peak I
17.7
17.9
21 - 16
Peak II
13.4
13.2
15 - 10
Peak III
~ 8.7
-
10 - 0
Cavell et al. Phys. Rev. B, 7, (1972) 5313
2
McFeely et al. Phys. Rev. B, 9 (1973) 5268
NCD #5 (hydrogenated)
EK = (250-1500) eV
E = 1 eV
Dwell = 500 ms
10 scans
FAT = 22 eV
Counts (a.u.)
16000
C1s
Sample NCD 5
C: 76.9%
O: 17.1%
Ta: 6.0%
Hydrogenated
8000
C: 93.2%
NotHydrogenated
O 1s
O KL23L23
O: 6.5%
Ta: 0.4%
Ta 4f5/2,7/2
0
1200
1000
800
600
400
200
0
EB (eV)
C: 38.3%
• Atoms Identification
O: 45.5%
• Quantification (%)
Ta: 12.5%
Ca: 3.7%
NCD 5 was hydrogenated in our groups,
while NCD 1 was hydrogenated by Rhobest coating (Innsbruck)
NCD #1 (hydrogenated)
EK = (250-1500) eV
E = 1 eV
Dwell = 500 ms
6 scans
FAT = 22 eV
16
Counts (a.u.)
Elemental Analysis:
12
O KL23L23
O 1s
Ca 2p1/2,3/2
8
Ta 4p3/2
Ta 4d3/2,5/2
Ta 4p1/2
4
Ta 4f5/2,7/2
C1s
O 2s
Ca 3s
0
1200
1050
900
750
600
EB (eV)
450
300
150
23
0
NIST – National Institute of Standars & Technology
O 1s (530.9 eV)
Counts (a.u.)
105
C: 15%
90
O: 60.4%
NCD 4 (oxygenated)
75
Ta: 24.6%
60
O 1s: 531 eV (standard reference)
45
• if bonded with Ta (Ta2O5): 530.6 eV
30
528
529
530
531
532
533
534
535
536
537
• if bonded with C (CO): 532 eV
EB (eV)
This is due to the peaks
O 1s (532.8 eV)
35
“Chemical Shift”:
Counts (a.u.)
30
NCD 3 (hydrogenated)
25
20
we can understand which kind of bonding
are involved in our sample surface
15
C: 63.6%
10
O: 33.7%
5
528
529
530
531
532
533
534
535
536
537
Ta: 2.7%
EB (eV)
Measurements made at ITC-irst Institute (Trento)
24
New Method
We have noticed that our hydrogenation device
introduces Ta contamination (due to the Ta hot
filament sublimation); so we decided to try
another way to hydrogenate our sample.
We used what is called
“Thermal hydrogenation”:
the sample is heated by a resistive heating
element and its surface interacts with H2
molecular gas, instead of an atomic gas.
M. Castellino - NIS & DFS Torino
25
Characterisation
Hydrogenation process
ElettroRava, Savonera (TO) (Dott. P. Bonino)
M. Castellino - NIS & DFS Torino
26
Characterisation
Sheet Resistance:
Electrical measurements
- Collinear four-probe head with
tungsten carbide tips from Jandel
Engineering Ltd.
ρ
π V
V
=
 = 4.532 
t ln2 I
I
- The distance between the tips was
0.635 mm and their radii was 0.04 mm.
[
Ω
]
t = sample thickness
- Calibrated constant current source
was used in order to supply 0.01 mA
through the two outer tips of the 4point probe, while measuring the
voltage across the two inner probe tips
Experimental Physics Dep.
M. Castellino - NIS & DFS Torino
27
Characterisation
Sample A is a IIa (110) oriented natural diamond supplied by
Drukker International (3x3x1 mm3).
Sample B is a IIa (100) oriented 5 mm thick homoepitaxial diamond
film deposited on highly resistive (boron free) HPHT Ib diamond
substrate supplied by Sumitomo Electric Co. Ltd. (3x3x0.5 mm3).
Sheet Resistance:
“Virgin State” *
After Thermal Hydrogenation
Sample A
>109 W/
Sample B
>109 W/
(2.8±0.8) 104 W/
>109 W/
*samples
were initially oxidised using a sulfochromic acid solution at 120 °C for 4
hours, rinsed in DI water, then heated at 70 °C in a H2O2 (36 vol.) : NH4OH (30%)
1:1 mixture, rinsed in DI water and after in acetone and finally dried in Ar flow.
After that they were annealed for 1 hour at 900 °C in high vacuum conditions (10-5
Pa) to induce hydrogen and/or oxygen desorption and to start with a clean surface.
28
Characterisation
Sheet Resistance of Natural IIa (110) oriented diamond
ohmic character
M. Castellino - NIS & DFS Torino
29
Characterisation
Dependence of Sheet Resistance
from sample Temperature
M. Castellino - NIS & DFS Torino
30
Characterisation
Sensitivity to environmental conditions
M. Castellino - NIS & DFS Torino
31
Characterisation
Sample A
In order to estimate the
average roughness of the
surfaces, several noncontact AFM maps were
acquired over hydrogenated
surfaces of both the samples
using an AFM PSIA XE-100.
Sample B
Sample A showed a
maximum vertical excursion
(Rpv) of 120 nm , whereas
sample B showed a much
smoother surface with Rpv
smaller than 20 nm.
Experimental Physics Dep. (Dott.ssa C. Manfredotti)
M. Castellino - NIS & DFS Torino
32
Characterisation
• (110) oriented natural diamond sample showes an ohmic character with a
sheet resistance of 2104 W/, while the B sample remains non-conductive.
• This different behaviour is due to a different reactivity of the two surfaces
which have different natures and orientations.
• The presence of a high surface area could have played an important role
in the hydrogen chemisorption (the few experiments on thermal
hydrogenation of diamond regard powders with high specific surface area:
Ando et al, J. Chem. Soc. Faraday. Trans., 89, (1993), 1783).
(Maier et al, Surface Science, 443, 177-185, 1999)
33
Functionalisation
(Nature Materials, 1, 253-257, 2002)
34
Functionalisation
(Nature Materials, 3, 736-742, 2004)
35
Functionalisation
Experimental Physics Dep
N 1s (402 eV)
peak is
covered by Ta
4p3/2 (404 eV)
12 h in N2 flux
Counts (a.u.)
3.0
NCD #2 (Hydrogenated)
EK = (750-1250) eV
E = 0.5 eV
Dwell = 500 ms
19 scans
FAT = 22 eV
O 1s
2.5
Deprotection mixture:
• methanol:water=2:5
• 7% K2CO3 (base)
2.0
F 1s
C 1s
1.5
boiling for 3.5 h
Ta 4p3/2
Ta 4p1/2
1.0
Ca 4p1/2,3/2
0.5
700
650
600
550
500
450
EB (eV)
400
350
300
250
General & Applied Organic Chemistry Dep.
Our results
NCD5 sample functionalised
with Cyanine CY3 dye
Excitation & Emission spectra
Chemical Structure
Animal & Human Biology Dep, (To) (Dott. P. Ariano)
Active Site
M. Castellino - NIS & DFS Torino
37
Further aims
• Reproduce the functionalisation on other types of diamond
(Ulm 1 & 2, Roma 1 & 2 );
• Control each step of the functionalisation with XPS measurement;
• Make analysis on Ulm 1 & 2 samples to understand
the C – B bond for a poster/presentation in collaboration with
Dott. Hayssam El-Hajj & Prof. E. Kohn (Ulm University) at the
“New Diamond & Nano Carbons” Conference
(May 28 - 31, 2007 – Osaka, Japan)
• Continue the thermal hydrogenation study to understand
the process mechanism
M. Castellino - NIS & DFS Torino
38
Poster & Pubblication
Poster presentation at Diamond 2006 Conference
(3 - 8 September 2006 - Estoril, Portugal) :
“Diamond surface conductivity after exposure to molecular
hydrogen”
(F. Fizzotti, A. Lo Giudice, Ch. Manfredotti, C. Manfredotti, M. Castellino, E. Vittone*)
(to be published in Diamond & Related Material - Elsevier)
M. Castellino - NIS & DFS Torino
39
M. Castellino - NIS & DFS Torino
40
Characterisation
Wettability
SAMPLE A (a)
Surface Exposed to Molecular
Hydrogen Flow
0° < q > 90°
Hydrophilic
90° < q > 180°
Hydrophobic
 = (79  1)
SAMPLE B (b)
After Hot Filament CVD
Hydrogenation
 = (91  1)
Physics Dep, Politecnico (To) (Dott.ssa P. Rivolo)
M. Castellino - NIS & DFS Torino
41
Diamond
Type of Diamonds:

Ia - This is the most common type of natural diamond, containing up to

Ib - Very few natural diamonds are this type (~0.1%), but nearly

IIa - This type is very rare in nature. Type IIa diamonds contain

IIb - This type is also very rare in nature. Type IIb diamonds
0.3% nitrogen.
all synthetic industrial diamonds are. Type Ib diamonds
contain up to 500 ppm nitrogen.
so little nitrogen that it isn't readily detected using infrared
or ultraviolet absorption methods.
contain so little nitrogen (even lower than type IIa) that the
crystal is a p-type semiconductor.
M. Castellino - NIS & DFS Torino
42
Ketone = CnH2nO organic compound
an oxygen atom connected to two
(substituted) alkyl groups
M. Castellino - NIS & DFS Torino
43