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

noiretfftA

atamuucav

pnidetroper

oisrevnoceht

telihw,esahp

tircawoleb

,noitatnalpmi elpmaseht aw

foerutarepmeta 1100 offoC°

1[skrowsuoiver - corpeht,]6

reyaldezihpromaehtfono

hcihwsnoigergnidnuorruseht

tdetrrtevnocerdlohserhtlacit

nidelaennasa

ro 2 sA.sruoh

nidetluserssec

citihpargaot

degamaderew

enitsirpehtot

offodnomaid mr

lennahcorcim

lenoitisiuqca

tcatnoc elihw,

lpmasehtta

rofforep sa,dem

trowsitI

otdeyolpme

ehtgnirud i

lacigoloisyhp

.m tfoenO udnocyllacirtceleeh

wsl as yltneuqesbus nnoc

niahccinortcel afosnaemyb

niffieddnegnigremerehtoehte

oiberehwecaffarusel acimehc

fininwohsyllacitamehcss igf eru

ht gniylrednu rcimehttaht

ivilafoytivitcaehtrotinom

ni ortiv misistnemerusaem

nosaersihtroffo:noitulos irub

citihpargevitcu

ehtotdetce

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

ehtgniwollassec

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

htfoecnednopserrocnireyal

h- ycneulffl VeM

afo bus -su ecaffar

dnenoieh - f--of

dezihprrpoma l

ehtnI.egnar

fonoitamrrmoffo

fo 2.3 ebmμ

elbairav - kciht

onfosnaem

laudarg udduom

snesuht,snoi

uselpmaseht

ninwohs fiigf u

htfoecnednopserrocnireyal

tatnalpminoi,krowtneserpe

a �300 alsuohpromakcihtmn

eehT.ecaffaruselpmasehtwol

sksamssenk masehtnodeniffied

no - noitaropavelatemtcatnoc

noitalu tpednoitartenepehtfo

behtfoecnegremeehtgnirus

fiicepstaecaffaru sa,snoitacolcif

eru 1.

dnenoieh of

ehtotdelnoit

htpedatareya

emyolpme fotn

ybecaffaruselpm

n ehtdewolla

VeMehtfoht

tasreyaldeirub

yllacitamehcss

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

ehT so- dellac

omnidednamed

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

ehtniseuss

erastnetxetn

atem, dnasl

dnassentsu

oibd gnisnes

ovivniroffoy

lacitporoffo

ssecorpnoita

,sedortcelec

ehtroffonoita

coxefonoitceted

VED

tneserpehtnI

elpmas decudorp

hgiherusserp

pmaS.deyolpme

ytsadeiffiissalc

noitartnecnoc si

lhT

yp

c niffifffamorhcmorffrytivitcasisoty

TACIRBAFORCIMECIV NOI

krow na aiciffiitra elgnisl - syrc

aemybcirtcelEomotimuSybd

hcet)THPH(”erutarepmet

5.1�3�3sieziselp mm3

htdna

.e.i,bIepy its utitsbus noit

001dna01neewtebdesirpmoc

biihdli

n .sllec

dnomaidlat

hgih“fosna

euqinh saw

latsyrryceh is

negortinlan

0 .mpp

filb

elpmasehT w

0002NAeht

htiwseirotarobaL

secneulffllacipyt

10 μm aebelihw,

dna 8 esuht,An

.setunim asehT

ulffldnagnigrahc

nocretemortcele

lusniyllacirtcele

ylevitceffefffegnitca

sa borcimnoiehttadetnalpmi

orangeLehtforotarelecca

fomaebgninnacsah 8.1 VeM

fos �5�1017

cm-2

maebnoI.

erewstnerrrrucma ebdesirpmoc

gnirusne lacipyt noitatnalpmi

elpma saw latem -coa det vaot

ecneu rotinomyletaruccasaw

ebmahcelpmasehtotdetcenn

maebehtfotserehtmorffrdetal

.pucyadaraFasay

foenilmaeb

lanoitaNo

eHV+

snoi ta

sawezism

neewte 5 An

semit fo 50

ecaffarusdiov

nahtiwder

sihcihw,re

m suht,enil

yevceegc

.pucydsy

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

xoehttceted

ehwedortcele

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

efoepyt

enimalohcetac

gnipeekelihw

orcimehT - le

gnidnopserroc

d

tulosenilasdradnatsaneh

lCgM 2 01, 01,esoculg PEH

.deyolp

eht se yranimilerp vedeht,stset

ag detalosin niffifffamorhc llec

cenircodneoruenelbaticxe

se ehtfoecnednopserrocni)

oloisyhpnimetsyselohwehtg

sawedortcel deziralop ehtta

otg anerddraehtfonoitadixoeht

iffii )2 di

noit :Mmni(

01,SE lCaC 2,

detsetsaweciv

ciffiicepsa.e.i(

llec gnisaeler

,noigerevitca

cigo al .noitulos

egatlovdexiffie

8.0.e.i(enila ,V

d i

detropersa

yrtemorepma ,

A.sisotycoxe

hhtiwtceted

sekips 3fo -2

enimalohcetac

erugiffini ,)2 tredroni

, esaelerlatnauqeht cco

nidetropersA fiigf eru edeht,4

langishgih -to- ehtoitaresion

2 Ap auqehtotgnidnopserroc

se .

,tcetedo aiv

gnirruc gnirud

otelbasiecive

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

niffifff nednopserrocnidetacolllec

esniehtnimooz(sekipseht:ecive

t ci .stneve

morffrdetceted a

evitcaehtfoecn

otdnopserroc)te

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

noitulosgniniatnoc

.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

demrofforepsaw

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