Fabrication of a Diamond-Based Cellular Biosensor with Ion...
Transcript of Fabrication of a Diamond-Based Cellular Biosensor with Ion...
Fabrication of a Diamond-Based Cellular Biosensor with Ion Beam Lithography
F. Picollo1,2,3,4, V. Carabelli2,4,5, E. Carbone2,4,5, D. Gatto Monticone1,2,3,4, S. Gosso2,4,5, P. Olivero1,2,3,4, A. Pasquarelli6, E. Vittone1,2,3,4.
1Physics Department, University of Torino, Torino, Italy. 2NIS Centre of Excellence, University of Torino, Torino, Italy.3INFN Sezione di Torino, Torino, Italy.
4Consorzio Nazionale Interuniversitario per le Scienze Fisiche della Materia (CNISM), Sezione di Torino, Italy. 5Department of Neuroscience, University of Torino, Torino, Italy.
6Institute of Electron Devices and Circuits, Ulm University, Ulm, Germany.
LNL Annual ReportNuclear Physics 1
After ion implantation, the sample was annealed in
vacuum at a temperature of 1100 °C for 2 hours. As
reported in previous works [1-6], the process resulted in
the conversion of the amorphized layer to a graphitic
phase, while the surrounding regions which were damaged
below a critical threshold reconverted to the pristine
diamond form. One of the electrically conductive graphitic
microchannels was subsequently connected to the
acquisition electronic chain by means of a standard metal
contact, while the other emerging end defined the location
at the sample surface where biochemical sensing was
performed, as schematically shown in figure 2.
It is worth underlying that the micro-electrode is
employed to monitor the activity of a living cell, which
during the in vitro measurements is immersed in a
physiological solution: for this reason buried connections
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1[skrowsuoiver - corpeht,]6
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ivilafoytivitcaehtrotinom
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dradnats latem
noitacolehtden
la sawgnisnes
e 2.
or - siedortcele
hcihw,llecgni
anidesremm
de snoitcennoc
Fig. 1. Schematics of the variable-thickness process allowing the
definition of the buried amorphous layer at variable depth.
.1.giF amehcS
htfonoitiniffied
elbairavehtfoscita - corpssenkciht
bairavtareyalsuohprrpomadeirube
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htpedelb .
As reported in previous works [1-6], high-fluency MeV
ion implantation determines the formation of a sub-surface
amorphized layer in correspondence of the ion end-of-
range. In the present work, ion implantation led to the
formation of a �300 nm thick amorphous layer at a depth
of 3.2 μm below the sample surface. The employment of
variable-thickness masks defined on the sample surface by
means of non-contact metal evaporation allowed the
gradual modulation of the penetration depth of the MeV
ions, thus ensuring the emergence of the buried layers at
the sample surface at specific locations, as schematically
shown in figure 1.
detropersA
itatnalpminoi
dezihprrpoma l
d ni skrowsuoiverp [1-6 hgih,]
noi onoitamrrmoffoehtsenimreted
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VeMehtfoht
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INTRODUCTION
Following our previous studies on the formation of
conductive graphitic microchannels in single-crystal
diamond by means of direct lithography with a scanning
ion microbeam [1-6], we report on the fabrication and
preliminary testing of a cellular biosensor with the above-
mentioned technique.
The so-called “Lab-on-chip” devices are highly
demanded in modern biotechnology to cultivate living cells
for long periods while inducing and revealing a broad
range of electrical biosignals, such as nerve excitations and
synaptic transmission. An integrated diamond-based
device can effectively address many open issues in the
existing cell-sensing research which to different extents are
not met by conventional bio materials (silicon, metals and
metals oxides and polymers), such as robustness and
reproducibility in performance over repeated biosensing
cycles, biocompatibility and long term stability for in vivo
measurements and high transparency for optical
interfacing.
Here we report on the MeV ion-beam fabrication process
of a diamond cellular substrate with graphitic electrodes,
and on the results of its preliminary characterization for the
detection of exocytosis activity from chromaffin cells.
DEVICE MICROFABRICATION
In the present work an artificial single-crystal diamond
sample produced by Sumitomo Electric by means of “high
pressure high temperature” (HPHT) technique was
employed. Sample size is 3�3�1.5 mm3 and the crystal is
classified as type Ib, i.e. its substitutional nitrogen
concentration is comprised between 10 and 100 ppm.
The sample was implanted at the ion microbeam line of
the AN2000 accelerator of the Legnaro National
Laboratories with a scanning beam of 1.8 MeV He+ ions at
typical fluences of �5�1017
cm-2
. Ion beam size was
10 μm, while beam currents were comprised between 5 nA
and 8 nA, thus ensuring typical implantation times of 50
minutes. The sample was metal-coated to avoid surface
charging and fluence was accurately monitored with an
electrometer connected to the sample chamber, which is
electrically insulated from the rest of the beam line, thus
acting effectively as a Faraday cup.
ruogniwolloF
NOITCUDORTNI
offoehtnoseidutssuoiverpr
fonoitamro
g
argevitcudnoc
aemybdnomaid
maeborcimnoi
pr itsetyrryanimile
nhcetdenoitnem
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doirepgnolroffo
acirtcelefoegnar
msnartcitpanys
id ffff
p
isnislennahcorcimcitihp
htiwyhpargohtiltceridfosna
[1-6] rbaffaehtnotroperew,
htiwrosnesoibralullecafogn
.euqi
baL“d -on- secived”pihc
etavitlucotygolonhcetoibnred
laeverdnagnicudnielihws i
al oib slangis sahcus, cxeevren
noissim aiddetargetninA.
iddlit
elgni - latsyrc
gninnacsah
dnanoitacir
evobaehth -
ylhgihera
sllecgnivile
daorbagni
snoitatic dna
dnom - desab
hti
ecived can ceffefffe
llecgnitsixe - snes
vnocybtemton
sedixoslatem a
iytilibicudorper
selcyc , oib apmoc
stnemerusaem
.gnicaffaretni
ereH ew trrtoper
ecdnomaidafo
tluserehtnodna
sinepoynamsserddaylevitc
nereffefffidothcihwhcraesergnis
cilis(slairetamoiblanoitnev ,no
dna sahcus,)sremylop ubor
i detaeperrevoecnamrofforepn
ytilibatsmretgnoldnaytilibita
dna fycnerapsnarthgih
VeMehtnot noi - acirbaffamaeb
citihparghtiwetartsbusralulle
ziretcarahcyranimilerpstifost
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yp
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TACIRBAFORCIMECIV NOI
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LNL Annual Report Nuclear Physics2
[1] P. Olivero et al., LNL Annual Report 2008 (2009) 118.[2] P. Olivero et al., Diamond Relat. Mater. 18 (2009) 870.[3] P. Olivero et al., Eur. Phys. J. 75 (2010) 127.[4] F. Picollo et al., Diamond Relat. Mater. 19 (2010) 466.[5] P. Olivero et al., Nucl. Instr. and Meth. B 269 (2011) 2340.[6] F. Picollo et al., Fabrication and characterization of three-
dimensional graphitic microchannels in single crystal diamond,accepted for publication in New J. Phys. (2012).
As shown in figure 3, when immersed in a saline
solution containing 10 mM adrenaline, the device can
detect the oxidation of the adrenaline molecules at the
electrode when the voltage reaches +0.8 V, as opposed to
the case when a standard saline solution (in mM:
128 NaCl, 2 MgCl2, 10 glucose, 10 HEPES, 10 CaCl2,
4 KCl) is employed.
Following these preliminary tests, the device was tested
by positioning an isolated chromaffin cell (i.e. a specific
type of excitable neuroendocrine cell releasing
catecholamines) in correspondence of the active region,
while keeping the whole system in physiological solution.
The micro-electrode was polarized at the fixed voltage
corresponding to the oxidation of the adrenaline (i.e. 0.8 V,
as reported in figure 2), in order to detect, via
amperometry, the quantal release occurring during
exocytosis. As reported in figure 4, the device is able to
detect with high signal-to-noise ratio the amperometric
spikes of 3-22 pA corresponding to the quantal release of
catecholamines.
nwohssA
tnocnoitulos
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ni fiigf eru esremminehw,3
gniniat 10 Mm enilanerddra , ht
omenilanerdaehtfonoitadix
.0+sehcaeregatlovehtne 8 ,V
nide enilasa
nacecivedeh
ehttaselucelo
otdesopposa,
hwesaceht
128 2,lCaN
4 )lCK pmesi
tgniwolloF
ninoitisopyb
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enimalohcetac
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gnidnopserroc
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ag detalosin niffifffamorhc llec
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noit :Mmni(
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8.0.e.i(enila ,V
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A.sisotycoxe
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enimalohcetac
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gnirruc gnirud
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emorepma cirt
foesaelerlatna
Fig. 4. Time course of amperometric signals detected from a
single chromaffin cell located in correspondence of the active
region of the device: the spikes (zoom in the inset) correspond to
single exocytotic events.
4.giF . cemiT
famorhcelgnis
dehtfonoiger
elgnis tycoxe ot
esruoc langiscirtemorepmafo s
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evitcaehtfoecn
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CONCLUSIONS
Ion beam lithography demonstrated to be useful to
microfabricate cellular biosensing devices in diamond,
which will be further investigated in future works.
maebnoI
tacirbaffaorcim
bllihih
SNOISULCNOC
otdetartsnomedyhpargohtil
oibralullece secivedgnisnes
htf tfidtiti
luffuesueb ot
dnomaidnis ,
k
eblliwhcihw
rehtruffu werutuffunidetagitsevni
.skrow
PRELIMINARY FUNCTIONAL TESTS
The responsiveness of the device towards catecho-
lamines (adrenaline, noradrenaline) was preliminarily
tested by means of cyclic voltammetry measurements.
ILERP
ehT visnopser
YRANIMI TSETLANOITCNUF
ssenev ecivedehtfo drawot
ST
sd ohcetac -
ehT visnopser
anerda(senimal
detset snaemyb
ssenev ecivedehtfo drawot
psaw)enilanerdaron,enila
fo erusaemyrrytemmatlovcilcyc
sd ohcetac
yliranimilerp
stneme .
Fig. 2. (Top) Lateral view schematics of the diamond device
incorporating the graphitic microchannel; (Bottom) top view
micrograph of the microelectrode at the endpoint of the graphitic
microchannel.
2.giF . taL)poT(
ehtgnitaroprrpocni
ehtfohpargorcim
orcim lennahc .
aidehtfoscitamehcsweivlaret
citihparg lennahcorcim mottoB(;
ehttaedortceleorcim fotniopddpne
eciveddnoma
weivpot)m
citihpargehtf
Fig. 3. Cyclic voltammetry tests on saline and adrenaline
containing solutions. The adrenaline oxidation feature at V=0.8 V
is clearly visible.
3.giF . vcilcyC
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.elbisivylraelcsi
naenilasnostsetyrrytemmatlov
ehT.sn rutaeffenoitadixoenilanerda
dn enilanerddra
8.0=Vtaer V
and the metal macro-contacts are shielded with a
biocompatible insulating mask in “sylgard” polymer.
Before proceeding with the functional tests reported in
the following section, two-points electrical characterization
was performed to check that i) the buried electrical
channels get in electrical contact with the sample surface at
their endpoints and ii) the electrical resistivity of the
microchannels is compatible with that of polycrystalline
graphite (i.e. �3�10-3
�·cm).
latemehtdna
oib nielbitapmoc
eecorperoffoeB
cesgniwolloffoeht
orcam -c dleihserastcatno
s“niksamgnitalusn y ”dragl lop
stsetlanoitcnuffuehthtiwgnid
,noitc owt - rahclacirtcelestniop
ahtiwde
remy .
nidetroper
noitaziretcar
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enitegslennahc
stniopdnerieht
slennahcorcim i
.e.i(etihparg �3�
eirubeht)itahtkcehcot
pmasehthtiwtcatnoclacirtcele
itsiserlacirtceleeht)iidna
lopfotahthtiwelbitapmocs
�10-3�·cm).
lacirtcelede
taecaffarusel
ehtfoytivi
l enillatsyrcy
embedded in the insulating diamond matrix are demanded, nideddebme xirtamdnomaidgnitalusnieht ,dednamedera nideddebme xirtamdnomaidgnitalusnieht ,dednamedera