CONSTRUCTIA SI

ASAMBLAREA
SENSORILOR ELECTROCHIMICI
PENTRU DETECTIA
NEUROTRANSMITATORILOR

Catecholamines
Catecholamines originate from a wide range of neural pathways by employing
biogenic amines as neurotransmitters (Stoica et al., 2004).
The neurotransmitter metabolites released into the cerebrospinal fluid can be a
sensitive indicator of neuronal functioning in nearby diencephalon structures
(Wightman et al., 1988).
Therefore, it is of great clinical importance to measure neurotransmitters and their
metabolites level in the extracelluar fluid in order to monitor neurotransmission
process (Peters et al., 2000).
Dopamine (DA) is an important neurotransmitter because it involved in motor and
cognitive functions; deficits in brain may cause Parkinsons disease in human
beings. DA has also been associated with the reward system, the circuitry in
the brain is responsible for the motivation to seek out stimuli as well as the
emotions for feeling satisfied and satiated in ones environment (Vinton and
Wightman, 2003).
From the view of point of physiological importance, it is a challenge to monitor DA
and its metabolite of 3,4-dihydroxyphenylacetic acid (DOPAC), because DA
level control is vital in the treatment of Parkinsons disease.
Stoica, L., Lindgren-Sjlander, A., Ruzgas, T., Gorton, L., 2004. Biosensor based on cellobiose dehydrogenase for detection of
catecholamines. Anal. Chem. 76, 46904696.
Wightman, R.M., May, L.L., Michael, A.C., 1988. Detection of dopamine dynamics in the brain. Anal. Chem. 60, 769A779A.
Peters, J.L., Yang, H., Michael, A.C., 2000. Quantitative aspects of brain microdialysis. Anal. Chim. Acta 412, 112.
Vinton, B.J., Wightman, R.M., 2003. Psychoanalytical electrochemistry: dopamine and behavior. Anal. Chem. 75, 414A421A.

Neurotransmitters

Neurotransmitters

Catecholamines

Catecholamines

Metabolism of L-DOPA
Entacapone

COMT
COMT = Catechol Omethyl transferase

Carbidopa

AAD

AAD = Aromatic L-amino

acid decarboxylase
MAO = Monoamine
Oxidase

COMT

Selegiline

MAO
AD

NE

COMT

MAO
AD

AD = Aldehyde
dehydrogenase
NE = Norepinephrine

Biological Sensor - Dopamine

DA is electrochemically active the development of an electrochemical method
that is capable of sensing DA is important.
Electrochemcial detection suffers from interference (from Ascorbic Acid (AA)
and Uric Acid (UA). Typical concentrations of AA is 10-4 to 10-3 mol dm-3 while DA
(10-7 to 10-5 mol dm-3).

OBIECTIVUL GENERAL
IL REPREZINTA PROIECTAREA SI REALIZAREA UNOR ELECTROZI
care sa permita detectia simultana a mai multor neurotransmitatori
eliminindu-se totdata si efectele interferentilor posibili (ex. acidul ascorbic)

SCOPUL
Realizarea si validarea unor instrumente analitice care sa poata determina in timp real
neurotransmitatorii

O POSIBILA CALE DE ATINGERE A ACESTUI SCOP

O reprezinta constructia unor sensori amperometrici enzimatici

the Neurotransmitter sensors based on the SWCNT-W

modified microelectrodes;
towards the neurotransmitter biosensors using the Lglutamate oxidase enzyme but starting from the model
enzyme (using for GOx the glucose biosensor assembly);
conclusions and perspectives.

In the planned work

1. The applicability of CNT modified surfaces with
selected enzymes (e.g. glucose oxidase or
cellobise dehydrogenase) and/or
electrocatalists (like metallo phtalocyanines)
will be investigated and used for developing
different kind of microbiosensors for
neurotransmitters detection;
2. The stability and morphological characteristics
of these modified surfaces will be studied by
different surface analytical methods.

ASPECTE DE STUDIAT
Proiectarea si realizarea unor microbiosensori
electrochimici bazati pe NTC pentru evaluarea
neurotransmitatorilor;
Elaborarea unei metodologii adcevate de lucru cu
microbiosensorii pentru detectia neurotransmitatorilor in
probe reale;
Validarea datelor obtinute prin utilizarea metodelor clasice
(standard)

Catecolaminele fac parte din clasa NEUROTRANSMITATORIILOR

fiind sintetizate la nivelul sistemului nervos central
+
NH3

+
NH3

HO
-

Structurile chimice ale catecolaminelor

HO

OH

+
NH2
-

X
OH

OH

OH

OH

DOPAMINA

NOREPINEFRINA

OH
EPINEFRINA

Variatii ale concentratiilor de catecolamine:

serotonina,
norepinefrina
si dopamina
conduc la instalarea unor boli neuropsihice
Concentratii mari de catecolamine conduc la manie.
Concentratii mici de catecolamine conduc la depresie

Neurotransmitatorii sunt molecule chimice care exista pe o perioada

mica de timp in fluidul extra-celular, ele fiin eliberate in mediu in spatiul
sinaptic pe perioada transmiterii unui mesaj neurochimic

Realizarea unei sinapse intre 2 celule nervoase (neuroni)

Neurotransmitatorii sunt molecule chimice care exista pe o perioada

mica de timp in fluidul extra-celular, ele fiind eliberate in mediu in
spatiul sinaptic pe perioada transmiterii unui mesaj neurochimic

Realizarea unei sinapse intre 2 celule nervoase (neuroni)

CATECOLAMINELE sunt SINTETIZATE

la nivelul sistemului nervos central in cadrul unei
secvente de reactii catalizate enzimatic

Cooper JR, Bloom FE, and Roth RH (1991) Dopamine, in The Biochemical Basis of
Pharmacology, pp 285337, Oxford University Press, New York.

Brain Disorders
Neurotransmitters, Addiction and
Depression

Burden of disease in Europe

Original data: Projected Years Lived with Disability (YLD) by selected disorders for the EU (Olesen & Leonardi, 2003)

The Need
Brain disorders e.g.
Depression
Anxiety
Pain
Epilepsy
Schizophrenia
Dementias
Addiction
Account for about HALF of all the
burden of disease in Europe and
growing

Brain disorders and their neurotransmitter targets

Depression - Serotonine
Antidepressants designed to increase 5HT
-but we still do not know
1. if 5HT is low in depression
2. if antidepressants increase this
3 and if so where?
Microbiosensors could be a solution to
answer these and other important
questions by using them for quantitative
determination of neurotransmitters

Schizophrenia Dopamine
Released by stimulant drugs [cocaine,
amphetamine]
More dopamine more pleasure
addiction (dependenta)
Where in brain?
Increased release in schizophrenia
Reduced in Parkinsons disease
can be restored by brain implants
May be reduced in depression

Dregarile ale sistemului nervos si

neurotransmitatorii implicati

Depresia: serotonine (5HT), noradrenaline

Anxietatea: GABA, noradrenaline, 5HT
Pain: endorphins, noradrenaline
Epilepsy: GABA, glutamate
Schizophrenia: dopamina, 5HT, noradrenaline
Dementia; Acetylcholine, glutamate
Addiction: GABA, glutamate, endorphins

Dopamina - schizofrenie
Eliberata de droguri drugs stimulatorii
(cocaina, amfetamina)
Mai multa dopamina
mai multa
placere dependenta
Unde in creer?
Cresterea secretiei - in schizophrenie
Scaderea secretiei - in Parkinson
Poate fi redusa in depresie

Serotonina - depresie
Antidepressants designed to increase 5HT
-but we still do not know
1. if 5HT is low in depression
2. if antidepressants increase this
3 and if so where?
Microbiosensors could be a solution to
answer these and other important
questions by using them for quantitative
determination of neurotransmitters

PROBLEME legate de DETECTIA

neurotransmitatorilor:
in situ
Limitari (constringeri) temporale si spatiale impuse de insasi mediul de determinare
Timpul de existenta a neurotransmitatorilor in fluidul extracelular este limitat (citeva
sute de milisecunde
proba de analizat se afla intr-un volum extrem de mic (tpic citiva picolitrii)

in fluidul extra-celular
Acesta este un mediu extrem de complex:
toate moleculele care intra si ies dintr-o celula trebie sa treaca prin acest spatiu
(ex. metaboliti, hormoni, neurotransmitatori)
Matrice ostila
Contine surfactanti (ex. lipide), proteins, electrocatalizatori (ex. glutation si ascorbat)

In literatura de specialitate sunt descrise un numar relativ restrins de tehnici care permit
monitorizarea in vivo a neurotransmitatoriolr
voltametria ciclica cu baleere rapida bazata pe microelectrozi
care prezinta caracteristicile legate de
sensibilitate,
selectivitate,
dimensiune a probei
si mai important rezolutie temporala
si care conduce la realizarea unor masuratori relevante ale dinamicii
neurotransmitatorilor in vivo

Problema interferentele datorate acidului ascorbic in raspunsul voltametric

R. M. Wightman et. al., Nature 262 (1976) 145.

METODE ANALITICE
folosite pentru determinarea neurotransmitatorilor

ELECTROFOREZA CAPILARA
SI
CROMATOGRAFIA DE LICHIDE
CU
DETECTIE ELECTROCHIMICA

Exista un interes major in realizarea electrozilor pentru determinarea

electrochimica a neurotransmitatorilor: dopamina, norepinefrina,
acetilcolina si serotonina (5-HT).
SCOPUL
Dezvoltatrea si validarea sensorilor (bio)chimici pentru determinarea in timp real a
neurotransmitatorilor

Neurotransmitatori pot fi oxidati

usor si deci,

Au fost realizate o serie de TEHNICI ELECTROCHIMICE pentru

detectia lor avind la baza diferite materiale de electrod

LISTA MATERIALELOR DE ELECTROD

FOLOSITE IN DETECTIA NEUROTRANSMITATORILOR in vivo si in vitro(selectiv)

microelectrozi de Pt, Au
Carbon sticlos modificat
Pasta de carbon
Microfibre de carbon
Nanotuburi de carbon

F. Valentini, S.Orlanducci, E.Tamburri, M.Terranova, A. Curulli, G. Palleschi, Electroana

In domeniul medical, monitorizarea, neurotransmitatorilor la pacientii

depresivi reprezinta o necesitate stringenta care genereaza informatii
legate de starea de sanatate a pacientilor depresivi, aceasta avind
implicatii sociale si economice majore.

In ultimii ani, in literatura de specialitate au fost

realizati si caracterizati un numar mare de
biosensori pentru detectia neurotransmitatorilor.
Electrozii pe baza de nanotuburi de carbon
beneficiaza de o serie de avantaje (ex. dimensiuni
reduse, etc) si de aceea sunt utilizati in realizarea
microbiosensorilor.

Compatibilitate lor cu tehnologia de microfabricatie precum si pretul lo

acesti electrozi sa reprezinte un instrument analitic promitator pentru
neurotransmitatorilor in vivo.

Strategia actuala in domeniu este aceea de a proiecta electrozi care pe

a mai multor neurotransmitatori, eliminandu-se efectul interferent al

O CALE POSIBILA
de dctectie a neurotransmitatorilor este bazata pe folosirea

electrozilor chimic modificati bazati pe enzime

Substrate

Product

Product

Substrate

Substrate

O2

H2O2

Medox

Product

Medred

electrod

FOLOSIREA MEDIATORILOR REDOX a imbunatatit semnificativ performantele

biosensorilor electrochimici

With respect to dynamic of electroanalytical

techniques the concept of the modified
electrode is certainly one of the exciting
developments of the last three decades
Reasons for surface modification are:
Improved electrocatalysis
Freedom from surface fouling effects

By using electron transfer mediators oxidation or reduction of the desired s

occurs at a potential near to its expected thermodynamic potential
The voltammetric sensors have shown an additional interest due
to their:
high sensitivity,
versatility
simplicity,
with a choice of potential range, waveform and electrode

In acest sens s-a investigat posibilitatea utilizarii ftalocianinelor pentru detectia neurotransmitatorilor
Complecsii de metalici ai ftalocianinelor (MePhC) sunt binecunoscutii ca fiind electrocatalizatori pentru
multe reactii

Proprietatile cele mai importante ale MePhCs, sunt:

solubilitate mica in majoritatea solventilor
Prezinta un domeniu larg de potentiale care se modifica odata cu
schimbarea metalului central

Potentiale de oxidare vs. SCE

Structura generala a MePhC

Ni (II) PhC
Fe (II) PhC
Co (II) PhC

Derivati de studiat:

+1.05 V
+0.16 V
+ 0.77 V

4,4,4,4-tetraamino PhC
4,4,4,4-tetrakis-2-aminofenoxi PhC

A. Ciucu, R. P. Baldwin, Electroanlysis, 4 (1992) 515

A. Ciucu, C. Negulescu, R. P. Baldwin, Biosens. Bioelectron., 18 (2003) 293

SENSORII amperometrici

Beneficiaza de o serie de avantaje

pret de cost scazut

usor de fabricat in orice format si dimensiune

Oxidoreductazele

sunt elemente binecunoscute de recunoastere moleculara

Care impreuna fac posibila that makes possibla fabricarea la un pret scazut a unor

BIOSENSORI ELECTROCHIMICI

DETECTIA NEUROTRANSMITATORILOR BAZATA PE

ELECTROZI CHIMIC MODIFICATI BAZATI PE ENZIME
Un biosensor pentru glucoza bazat pe un electrod modificat cu Gox si pe cuplul redox Q/HQ poate fi folosit
pentru detectia compusilor dihidroxi fenolici (hidrochinona, dopamina, epinefrina, norepinefrina etc.)

Gox este o enzima ideal de folosit in constructia biosensorilor

datorita:
Specificitatii fata de glucoza
Capacitatii catalitice mari
Stabilitatii

Mecanismul de aciune al gluco-oxidazei

Bioelectrocatalytic reaction of glucose oxidation at the surface

of an amperometric carbon paste electrode modified with Gox.

Dihydroxyphenols are consumed at the electrode surface, but regenerated by Gox in presence of
glucose which generate a signal amplification.

This Gox based sensory system is applicable for detection of dihydroxyphenols

(HQ, catechol, dopamine,epinephrine and norepinephrine) with increased sensitivity
due to the response amplification.
F. Mizutani, et. al.. Biosens. Bioelectron., 6 (1991) 305
A.
Ciucu, C. Ptroescu, Anal. Lett., 17, (1984) 1417
A. Ciucu, V. Magearu, C. Luca, Anal. Lett., 18, (1985) 299

(II) Another strategy is to use the clasical glucose biosensor

A glucose sensor could be prepared by using an electrode coated
with a layer containing immobilized Gox and Q/HQ redox couple.

Bioelectrocatalytic reaction of glucose oxidation at the surface of an

amperometric carbon paste electrode modified with Gox.

Dihydroxyphenols are consumed at the electrode

surface, but regenerated by Gox in presence of glucose
which generate a signal amplification.
This Gox based sensory system is used for detection of dihydroxyphenols
(HQ, catechol, dopamine,epinephrine and norepinephrine)
with increased sensitivity due to the response amplification.

Gox is ideally suited for use in biosensors

design due to its:
high specificity for glucose
high turnover rate
high stability

F. Mizutani, et. al.. Biosens. Bioelectron., 6 (1991) 305

A. Ciucu, C. Ptroescu, Anal. Lett., 17, (1984) 1417
A. Ciucu, V. Magearu, C. Luca, Anal. Lett., 18, (1985) 299

(III) O posibila alternativa in constructtia biosensorilor pentru detectia

neurotransmitatorilor este utilizarea celobioz dehidrogenazei (CDH) o enzima
cu cofactori micsti
CDH a fost descoperita de Ulla Westermark si Karl-Erik Eriksson
in Stockholm

CDH este produsa extracelular de fungi precum:

Phanerochaete chrysosporium
Structura CDH: este o flavoenzima care
contine
un cofactor flavinic,
si un al II-lea cofactor de hem

B. A. Gregg and A Heller, Anal. Chem., 62 (1990) 258-263.

Mecanismul bioelectrocatalitic de functionare al biosensorului pe baza de CDH

Viteza de transfer de electroni intramolecular este

mica

Solutie

folosirea unui mediator in scopul cresterii

vitezei de transfer de electroni dintre enzima si
electrod

Transferul direct (sus) si mediat (jos) de electronidintre CDH si suprafata unui electrod de grafite

Folosindu-se aceasta schema de detectie s-a detectat dopamina limita de detectie fiind de
2.5 nM
T. Larsson, et al., Anal. Chim. Acta 331 (1996) 207.
A. Lindgren, L. Stoica, T. Ruzgas, A. Ciucu, L. Gorton, The Analyst, 124, (1999)527-532.
C. Nistor, J Emneus, L. Gorton, A. Ciucu, Anal. Chim. Acta, 387, (1999) 309-326.

SELECTIVE RESPONSE OF DOPAMINE IN THE PRESENCE

OF ASCORBIC ACID AT CARBON NANOTUBES PASTE MODIFIED ELECTRODE
A. Ciucu, A. Ciobanu*
University of Bucharest, Faculty of Chemistry, Romania,
*University of Medicine and Pharmacy Carol Davila, Bucharest, Romania

INTRODUCTION
There is considerable interest in developing electrodes for electrochemical
determination of neurotransmitters such as dopamine (DA) and serotonin (5-HT). Low
levels of DA have been found in patients with Parkinsons disease. Serotonin 5-HT is
widely distributed in the brain, and together with other neurotransmitters, plays an
important role in brain functions. Both DA and 5-HT are readily oxidized; hence,
electrochemical techniques have been explored for their analysis [1-4] ascorbic acid
(AA) represent a major interferent in the determination.

OBJECTIVE
Selective electrochemical detection of DA and 5-HT in the
presence of AA at multi-wall carbon nanotube paste electrode
modified with cobalt(II) phthalocyanine (MWCNT-CoPC).

Metallophthalocyanines (MPhc) complexes are coordination

compounds where a transition metal is coordinated with a
phthalocyanine ring
Properties:
acts as electrocatalysts for many reactions [4,1214],
the electrochemical properties of MPhc can be modulated by:
changing the central metal atom or
introducing substituents in the phthalocyanine ring ,
very low solubility in nearly all solvents, both aqueous and organic,

Figure 1. The structure of metallophthalocyanines MPhc

The use of MPhc complexes as electrocatalysts for the determination of
neurotransmitters has not received much attention.
In this work, voltammetric electrodes based on MPhc compounds (Fig. 1) have been used as the working electrode in
cyclic voltammetry experiments, for evaluating neurotransmitters.

The voltammetric sensors have shown an additional interest due to their:

high sensitivity,
versatility
and simplicity,
with a choice of potential range, waveform and electrode material.

ELECTRODES USED FOR THE DETECTION OF

NEUROTRANSMITTERS FROM TISSUE, IN VIVO AND IN VITRO

modified glassy carbon electrodes (GCE),

carbon nanotube electrodes,
carbon-based ring disk electrodes,
carbon paste electrodes (CPE),
Pt microelectrodes,
carbon micro fiber electrodes,
single wall carbon nanotubes

The strategy now is to design electrodes that can allow for simultaneous detection
of several neurotransmitters, while eliminating the interfering effects of ascorbic acid.
APPROACHES FOR THE DETERMINATION OF NEUROTRANSMITTERS:
capillary electrophoresis with electrochemical detection
liquid chromatography with electrochemical detection
fast-scan cyclic voltammetry (FSCV) using microelectrodes (for in vivo studies)

METHOD
Electrochemical approach
Cyclic voltammetry
Diferential pulse voltammetry
Results

W.E. =

MWCNTP-CoPC

AUX.E. = Pt
REF.E. =Ag|AgCl (3M)

Cyclic voltammograms for a 10 mM K3[Fe(CN)6] solution at a unmodified

MWCNTP electrode ( ) and at a MWCNTP-CoPC electrode (----).
Conditions: 1,0 M KNO3, v= 50 mV/sec

Electrocatalytic mechanism:
On the basis of these observations, it would appear that the most reasonable mechanism for the
dopamine oxidation is then a catalytic CE sequence:
Co(III)PC + DA
Co(II)PC - e-

Co(II)Phc + DA oxidation product

Co(III)PC

A similar mechanism would be expected for 5-HT.

Interference of ascorbic acid in the MWCNTP modified electrodes response

Ascorbic acid (M)

Dopamine (M)

Ip,a A

1.0 x 10-6

5.2 x 10-6

36.0

1.0 x 10-6

2.10 x 10-6

33.0

1.0 x 10-6

1.1 x 10-6

30.4

1.0 x 10-6

5.2 x 10-6

32.3

1.0 x 10-6

2.0 x 10-6

29.7
mean 32.3

observed at + 0.12 V vs Ag/AgCl

Conclusion
Electrochemical approach is used to detect dopamine at low
applied potential.
The present study proposes an easy-to-make and low-cost
sensor construction for the selective determination of
dopamine.
The chemically-modified multi walled carbon nanotube paste
electrode is capable of enhanced electrochemical monitoring
of dopamine due to the properties of the electrode material
and metalo-phthalocyanines used as mediators.
Interference of ascorbic acid in the multi walled carbon
nanotube paste modified electrodes response was eliminated.

Baza Biologica a
Depresiei

Bolile mentale reprezinta o arie enorma si complexa de cercetare in care se simte

nevoia dezvoltarii unor noi directii de abordare a problematicii din acest domeniu.

Bolile mentale disturba

gandirea, afectivitatea, starea psihica si capacitatea de integrare a persoanelor
suferind de aceste maladii.

Depresia
este o boala a mintii si corpului;
are cea mai mare incidenta in lume afectand milioane de oameni;
depresia apare de doua ori mai frecvent la femei decat la barbati, din
motive care nu sunt total intelese;
netratata, depresia conduce la suicid;
Rata de sinucidere in tarile est europene este de 4 pana la 6 ori mai
mare comparativ cu cea din SUA.

"Depresia este cancerul

emotiilor" (Lewis Wolpert)

Depression is diagnosed on the

basis of a patient's behaviour, not
on the basis of laboratory test.

Depresia: Dimensiunea
Problemei

5 - 12 % of men and 10 - 20 % of women in the U.S. will

suffer from a major depressive episode at some time in
their life.
15% of depressives commit suicide each year
In 1996 the Centers for Disease Control and Prevention
listed suicide as the ninth leading cause of death in
the U.S. (slightly behind infection with the AIDS virus),
taking the lives of 30.862 people
In 1992 the estimated costs of depression totalled $43
billion, mostly from reduced or lost worker productivity.
(Source: Nemeroff, 1998)

Depresia
Depresie primar
unipolar
bipolar
mixt

Depresie secundar
boal Parkinson
depresia din schizofrenie
depresia din bolile cardiovasculare
afeciuni neurologice degenerative

Unipolar depressive disorders

Unipolar depressive disorders are

characterized by depressive symptoms
only, without any history of a manic,
mixed or hypomanic episode.
This criterion distinguishes them from the
group of bipolar (affective) disorders.

Criteria of major depressive disorder

A. Over the last 2 weeks, 5 of the following
features should be present most of the day,
or nearly every day (must include 1 or 2) :

1. depressed mood
2. loss of interest or pleasure in almost all
activities
3. significant weight loss or gain (more than
5% change in 1 month) or increase or
decrease in appetite nearly every day
4. insomnia or hypersomnia
5. psychomotor agitation or retardation

 6. fatigue or loss of energy

7. feelings of worthlessness or
excessive or inappropriate guilt
8. diminished ability to think or
concentrate, or indecisiveness
9. recurrent thoughts of death , or
recurrent suicidal ideation, or a
suicide attempt, or a specific plan for
committing suicide.

 B. The symptoms cause clinically significant

distress or impairment in social, occupational,
or other important areas of functioning.
C. The symptoms are not due to a
physical/organic factor or illness (e.g., a drug
abuse, a medication, a general medical
condition).
D. The symptoms are not better explained by
bereavement (although this can be
complicated by major depression).
4th Revision of the American Psychiatric Associations Diagnostic and
Statistics Manual (American Psychiatric Association 1994)

PRINCIPALII NEUROTRANSMITORI
IMPLICAI IN TULBURAREA DEPRESIV
Norepinefrina

Serotonina
Anxietate
Iritabilitate

Energie
Interes

Impulsivitate
Stare psihica, emotii,
functia cognitiva

Apetit
sexual
Agresivitate

Motivatie

Motilitate
Dopamina

S.M. Stahl Essential Psychopharmacology, in Neuroscientific Basis and Practical

Applications. 2nd ed. Cambridge, UK, Cambridge University Press, 2000, p 152.

MAIN NEUROTRANSMITTERS IMPLIED IN

DEPRESION TULBURAREA
Norepinephrine

Serotonin

Energy
Interest

Anxiety
Irritability
Impulsivity
Mood, emotion,
cognitive function

Motivation

Sex
Appetite
Aggression

Drive
Dopamine

S.M. Stahl Essential Psychopharmacology, in Neuroscientific Basis and Practical

Applications. 2nd ed. Cambridge, UK, Cambridge University Press, 2000, p 152.

There are several reasons to search for

biological markers for depression.
Depression is diagnosed on the basis of a
patient's behaviour, not on the basis of
laboratory test.
The biological markers for depression could
be used for :

a better diagnostic tool

to track improvement under drug treatment
to better understand the biological basis of depression
to develop more effective drug treatments

Monoamine hypothesis of
depression
has been the theory explaining biological
basis of depression (Stahl, 1998).
This theory states that depression is
essentially due to a deficiency in one of
three catecholamines : serotonin,
norepinephrine, or dopamine ( notably
norepinephrine - NE and serotonin 5
hydroxytryptamine - 5HT ).

Permissive Biogenic Amine

Theory
If catecholamine activity is increased then the
patient will exhibit mania .
If catecholamine activity is decreased then the
patient will exhibit depression .
Consequently depression could be treated by
either treating the underlying serotonin
abnormality, or by restoring NA activity to
normal.

NA metabolism and depression

1. depression is associated with a
functional deficiency of noradrenalin
(NA) and 3-4 MHPG (specially in
urine )
2. mania is associated with an excess of NA
and 3-4 MHPG.
(Schildkrant J. J. and all. 1973)

 MHPG (3-methoxy-4-hydroxy-phenylglycol) is produced when NA is

broken down by enzymes.
MHPG (3-methoxy
4hydroxyphenylglycol) is the principal
NA metabolite found in the human
cerebrospinal fluid (CSF) which
suggests that it may provide a good
estimate of NA activity.

Depresia i serotonina (5-HT)

Deficitul transmisiei serotoninergice este anomalia de
neurotransmisie cel mai frecvent implicat n etiopatogenia
depresiei (Asberg i Van Praag 1984):
numr crescut de receptori 5-HT2 n cortexul frontal la
sinucigai (Stanley - 1990);
curb 5-HIAA mult sczut la sinucigai (Asberg - 1984);
curb sczut de 5-HIAA n LCR la cei cu tentative de
suicid violent (Cocarro - 1989);
valori sczute ale triptofanului liber la depresivi.

Din punct de vedere clinic, depresia cu deficit de serotonin se

caracterizeaz prin:
prezena anxietii
comportament de tip ostil
risc suicidar crescut

Evaluarea markerilor de tip indolic submparte depresia

prin deficit de serotonin n dou categorii
cu nivel sczut de indoli
potenial suicidar ridicat ( Oreland 1985), pentru care
activitatea MAO plachetar este un marker biologic controlat
genetic, nivelele sczute ale activitii MAO plachetare sunt
corelate cu hiperactivitatea MAO neuronal i deficiena
serotoninic.

cu nivel normal sau crescut al indolilor

sugereaz o alt subform biochimic de depresie i riscul
instalrii sindromului serotoninergic n situaia utilizrii medicaiei
antidepresive de tip inhibitori de recaptare a serotoninei

5-HT metabolism and depression

Serotonin (5-HT), and a possible

interaction between 5-HT and
NAdrenaline, may play an important
role in mood, such as interaction
between 5-HT and hypothalamus
pituitary adrenal system .

 Studies suggest that levels of serotonin (5HT)

and serotonin metabolites ( 5HIAA 5hydroxy indole acetic acid ) are reduced in brain tissue
and the levels of serotonin metabolites are
decreased in cerebrospinal fluid (CSF) of
depressed patients.
Patients with low CSF 5HIAA tend to be the
patients with impulsive behaviors, such as suicide.
Low levels 5-HIAA may be a biological marker for
suicidal behaviour
Reference:
Asberg ( Science , 191, 478- 480, 1976)

Depresia i dopamina
Din punct de vedere clinic, depresia prin deficit de
dopamin se caracterizeaz prin:

inhibiie psihomotorie
tensiune intrapsihic marcat
tendine abulice
risc suicidar

Markeri biochimici:
prolactina
acidul homovanilmandelic (HVA)
nivele sczute depresie inhibat cu risc suicidar crescut
nivele crescute tendin la depresie delirant sau viraj
dispoziional.

DA metabolism and depression

Clinical data has clearly shown a
correlation between dopamine deficiency
and depression.
The strongest evidence implicating
dopaminergic involvement in depression
has come from studies of the dopamine
metabolite, homovanillic acid (HVA) in
cerebrospinal fluid.

 Depressed patients who had attempted

suicide had significantly lower urinary
concentrations of HVA and lower total body
concentrations of dopamine than patients
with depression who did not attempt
suicide. (Bowden et al. 1997).

Depresia i noradrenalina
Bunney - 1965 consider deficitul noradrenergic la
nivel limbic ca mecanismul de baz n depresie.
Din punct de vedere clinic, deficitul de
noradrenalin determin o form particular a
depresiei, caracterizat prin:
inhibiie psihomotorie
tendine apato-abulice
pseudo-deficit cognitiv

Tot noradrenalina

NA metabolism and depression

1. depression is associated with a
functional deficiency of noradrenalin
(NA) and 3-4 MHPG (specially in
urine )
2. mania is associated with an excess of NA
and 3-4 MHPG.
(Schildkrant J. J. and all. 1973)

 MHPG (3-methoxy-4-hydroxy-phenylglycol) is produced when NA is

broken down by enzymes.
MHPG (3-methoxy
4hydroxyphenylglycol) is the principal
NA metabolite found in the human
cerebrospinal fluid (CSF) which
suggests that it may provide a good
estimate of NA activity.

Valorile indicatorilor biochimici ai deficitului NA

MHPG (Metilhidroxifenilglicol) plasmatic sau
urinar
valori normale indic depresie cu activitate
predominent serotoninergic sau dopaminergic.
nivelul crescut constituie un argument pentru ipoteza
hipercolinergic a depresiei (Leong - 1987)
valori sczute indic depresie prin deficit NA.

DOPEG (Dihidroxifeniletilenglicol) plasmatic

marker fidel al metabolismului NA, la depresivi
DOPEG avnd un nivel sczut (Loo - 1983, Scatton 1986).

Indicatorii enzimatici ai depresiei NA

scderea activitii dopaminbetahidroxilazei
(DBH) nu are valoare de marker la depresivii
bipolari (Eisemman - 1983, Rihmer - 1989),
la unipolari atribuindu-i-se rolul unui index de
risc pentru depresie major (Meltzer - 1995);
MAO are valori variabile, nivelul sczut al
acestei enzime fiind expresia unui stigmat
genetic al depresiei bipolare.

In ciuda identificarii markerilor neurobiologici implicati in bolile neuropsihice

(ex. depresia majora si dereglarile bipolare) diagnosticarea acestor boli se face
pe baza simptomelor caracteristice asociate dereglarilor starilor psihice care
pot include episoade alternante de cresteri extreme ale starii de spirit (mania)
si depresie severa. In mod evident, in domeniul neurostiintelor se impune
dezvoltarea unor metode de laborator pentru detectia si monitorizarea
neurotransmitatorilor in scopul diagnosticarii clinice a dereglarilor nervoase.

Una dintre solutiile de perspectiva este reprezentata de posibilitatea utilizarii

micro-(bio)sensorilor pentru evaluarea neurotransmitatorilor, a caror proiectare
presupune un efort interdisciplinar bazat pe dezvoltarea si proiectarea unor
nanotehnologii aferente micro-nanosistemelor integrate. Potentialul utilizarii
micro-(bio)sensorilor rezulta din capacitatea acestora de a masura interactiile
dintre sistemele nanosensorile si neurotransmitatori. Micro-(bio)sensorii au
capacitatea de a detecta intr-un mod simplu, rapid, selectiv si specific
substantele de interes avand posibilitatea de a genera o informatie continua.

Proiectarea micro(bio)sensorilor este unul din exemplele elocvente din domeniul

cercetarilor interdisciplinare. Abordarea stiintifica propusa pentru determinarea
neurotransmitatorilor este una inovativa care pleaca de la metodele clasice de analiza
chimica. Proiectarea unor instumente miniaturizate care retin sensibilitatea ridicata si
selectivitatea metodelor instrumentale de laborator sofisticate reprezinta o provocare
analitica majora. In cadrul tehnologiei propuse pentru detectia neurotransmitatorilor au
fost folositi (bio)sensori relevanti din punt de vedere clinic care au condus la
identificarea si detectia neurotransmitatorilor intr-un timp mult redus comparativ cu
orice alte metode clasice.

Rezultatele obtinute au permis evaluarea unor neurotransmitatori din probe

reale si prin aceasta fac posibila corelarea acestora cu diagnosticarea unor boli
neuropsihice (precum dereglari uni- si bipolare). Acestea au condus la o mai
buna intelegere a noilor cerinte analitice, incluzand noi parametri (biomarkerii)
care descriu si conduc la diagnosticare tulburarilor neuropsihice; ele pot
conduce si la o dezvoltare clinica in domeniul diagnosticarii bolilor neuropsihice
prin stabilirea corelatiilor clinice dintre concentratiile de neurotransmitatori din
probe reale cu bolile neuropsihice. Astfel, se pot identifica dezechilibrele in
concentratiile de neurotransmitatori, optimiza dozarea medicamentelor si
identifica rapid pacientii care raspund la medicamentatia prescrisa.

Further work applications on real samples are under study

The Value
Increases our understanding of the disease process
Identifies transmitter deficits and malfunctions
Provides new leads for drug discovery
Facilitates clinical development
Optimizes dose selection
Identifies drug responders
Rapid identification of efficacy in small groups

SWCNTs diameter:
1020

A TEM image of
SWCNTs

A theoretical model of
SWCNTs

Biliografie

FE-SEM: Field Emission

Gun Scanning
Electron Microscopy:
The Microscope Instrument:
(S4000)
Having an accelerating voltage:
20 kV

The FE-SEM investigation

showed a homogeneous and
uniform SWCNTs coating
on the W microwire
surface. In addition, the
SWCNTs deposit appears
perpendiculalry
alligned
and oriented on the W
microelectrode surfaces.

STM:
Scanning Tunneling
Microscopy:
Instrument
( Jeol JSPM-4210 )

shows
that
the
SWCNTs are organized
in very well oriented
and alligned bundles.

I: 0.100- 0.300 nA;

E (V): 0.100 - 0.300 V

F. Valentini, A. Curulli, S. Orlanducci, M. L. Terranova, and G. Palleschi, Electroanalysis 2005,

17, No. 1, 28.

SWCNT-W modified microelectrodes for the

neurotransmitter detection.
After the various deposition processes, the central portion (about 2-cm length) of each W wire was
coated with insulating varnish, whereas the two tips (about 0.5-cm length) remaining uncoated
served, respectively, as the working electrode surface and the electrical contact with the
electrochemical cell system. The modified electrode tips were washed carefully with double distilled
water prior to use for measurements. The electrochemical cell was assembled as a conventional
three-electrode system: a working electrode made of W wires 300-m in diameter coated by
SWNTs; an Ag/AgCl reference electrode (Model 805/CPG/6. from Amel Milan, Italy), and a Pt
counter electrode. All experiments were carried out at room temperature. Initial cyclic voltammetry
experiments were carried out over the range 5 1000 mV/s, while a scan rate of 100 mV/s was
eventually chosen to survey the behavior of the various electrodes being evaluated.

SWCNT-stainless steel modified microelectrodes for

the neurotransmitter detection.
All experiments were carried out at room temperature, working in a phosphate buffer solution
(0.1M, pH 7.0). Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV)
experiments were performed under quiescent conditions, respectively. Initial cyclic voltammetry
experiments were carried out over the range 5-100 mV/s, while a scan rate of 100 mV/s was
eventually chosen to survey the behavior of the various electrodes being evaluated. DPV
measurements were carried out with a pulse amplitude of 50 mV, a pulse width of 60 ms, a scan rate
of 10 mV/s, a pulse interval of 200 ms, and a sampling time of 20 ms; the E i of -0.3 V and Ef of
+0.5 V (vs. Ag/AgCl reference electrode) for epinephrine calibration curves.

A signal/noise ratio per unit area of

510.10 cm-2 for nanotube-modified W
microelectrodes, vs. 164.33 cm-2 for
Glassy Carbon bare electrodes, was
calculated.
The sensitivity of the SWCNT-W
modified microwires was significantly
better than that of the conventional GCE
(197.45 mA mol-1L cm-2).

F. Valentini, G. Palleschi, et al.; Electroanalysis 2005, 17, No. 1, 28.

The lower LODs were observed

only in the presence of Carbon
Nanotubes and Carbon Nanofiber
based sensors.

This is probably related to the higher

nominal surface area of SWCNTs
( 300 m2/g);

F. Valentini, G. Palleschi, et al.;

Electroanalysis 2005, 17, No. 1,
28.

and also to their allignement, orientation,

and nanometer-scale dimensions able to
garantee
a direct and intime contact
with the active centre of the enzymes,
biological molecules and the biocatalysts.

Figure 1 (a) shows a FE-SEM micrograph of

SWCNTs coating obtained on the SSE surfaces by
EPD method. Mechanically induced small scratches
revealed the inner nanostructure of the deposit,
containing bundles of aligned nanotubes. The
SWCNTs deposit coated entirely the microelectrode
surface, in form of a dense packed layer with a high
roughness.

The structural characterization was performed

by Raman spectroscopy, and in Figure 2a, a
typical Raman spectrum was also reported for
the CNTSSE by EPD, that showed the typical
radial breathing mode (RBM) signals, which
indicate the presence of SWCNTs, having an
average diameter of about 1.4 nm [1]. The
tangential region of Raman spectra and IR
spectroscopy showed the insertion of OH groups
on the functionalised SWCNTs, similar to those
reported in our previous work [1], via KOH pretreatment.
F. Valentini, G. Palleschi, E. Lopez Morales, S. Orlanducci, E. Tamburri, and M. L. Terranova, Electroanalysis 2006, accepted.

10
-15
-40
0.2 0.3 0.4 0.5 0.6 0.7 0.8

30
25
20
15
10
5
-5
-10
-20
-0.25 0

0.50

0.75

1.00

E, V

E/V
Au microelectrode: = 250 m

GC bare electrode: = 2.0 mm

80

60
50
40
30
20
10
0
-10
-20 0.2 0.3 0.4 0.5 0.6 0.7 0.8

I /A

E/V

Au microelectrode: = 250 m

25.0
GC bare electrode: = 2.0 mm
22.5
20.0
17.5
15.0
12.5
10.0
7.5
5.0
2.5
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

55
30
5
20
0.2 0.3 0.4 0.5 0.6 0.7 0.8

E/
V
12.5
10.0
7.5
5.0
2.5
0
-2.5
-5.0
-7.5
-10.0
-0.25

I /A

I /A

0.25

I/A

I/A

35

I / A

60

E/V

F. Valentini, G. Palleschi, et al.; Electroanalysis 2006, accepted.

0.25

E/V

0.50

0.75

1.0

1
SWCNT KOH 25C
SWCNT KOH 50C

0,8

SWCNT KOH 100C

SWCNT Carbolex

Reflectance

The FT-IR study revealed the

presence of the OH- chemical
groups (due to the KOH pretreatment) [1]. In the experimental
working
conditions,
they
were
negatively charged, acting as an
electrostatic barrier toward the AA
and UA (i.e.; as ascorbate and
urate at pH 7.0 in 0.1M PB)
oxidation
reaction,
at
the
microelectrode surface.

0,6

OH

0,4

0,2

0
400

800

1200

1600

2000

2400

2800

3200

3600

4000

-1

Wavenumber cm

-OH
-OH
-OH
-OH

OHOH-

The Acetaminophen molecule (a neutral molecule)

OHOHOH-

The Epinephrine molecule (a positively charged molecule)

The AA and UA molecule (the negatively charged molecules)
[1] F. Valentini, G. Palleschi, et al.; Electroanalysis 2006, accepted.

55
50

I / A

45 0.05mM E+5mMAA
40

0.05 mM E

35
30
25
20
15
10

5mM AA

5
-0.30 -0.20 -0.10

buffer

0.10

0.20

0.30

0.40

0.50

performed at SWCNT-modified
stainless steel microelectrodes,
by DPV.

E/V

Linear Regression Equation:y/A =1.02+1.32 x/M; (R2 = 0.998)

Linear range of concentration: 2.0 x 10-6 1.0 x 10-4 (M)
[Epinephrine], M

L.O. D.= (3 * )= 2.0 x 10-6 (M)

Sensitivity (A M-1 cm-2) = 28.1
RSD % (n = 4) = 7.0
Response Time = 6 (s)

F. Valentini, G. Palleschi, E. Lopez Morales, S. Orlanducci, E. Tamburri, and M. L. Terranova, Electroanalysis 2006, accepted

Functionalised
SWCNTs

Enzymes: the L-Glutamate Oxidase

1. The SWCNT deposition by EPD

2
1

WE: SWCNT/Au microwires,

RE: Ag/AgCl;
CE: Pt;
[pyrrole]= 0.5M;
GOx = 0.5 mg/mL

PPy+GO
x
PPy+GO
x
PPy+GO
x
PPy+GO
x
PPy+GO
x

2. One-Step electrochemical
co-deposition of PPy and
GOx
(the biocatalyst).

The overoxidised PPy film

= 250 m;
acts as diffusion barrier
toward the interferents
(higher selectivity) and the
overoxidation
of
the
glucose
(the
extendedAn
polypyrrole films was carried out
linearity effect).
by CV, working at the potential

window ranging from

The selected amount of GOx was then added to the pyrrole -0.20 to + 1.30 V, at scan rate
solution in double-distilled water, without Cl-, as dopant agent. of 0.1 V/s, for 50 consecutive
as
described
in
Electrochemical deposition: the electropolymerization proceeded cycles,
reference [1].
by chronoamperometry (working at + 0.70V for 10 min).
[1]J. Wang, M. Musameh, Anal. Chim. Acta 2005, 539, 209.

This
Figure
shows
a
homogeneous, uniform, and
dense packed coating on the
entire
Au
microelectrode
surfaces.

A higher magnification, the

polymeric layer appears as
large and compact globular
structures, ranging from 15
to 60 m.

120

I (A)

100
80
60

Au(SWCN)/PPy/GOx

Au(bare)/PPy/GOx

40
20

The
Hydrodynamic
Voltammetry
(HDV) study
was carried out to
select the best working potential for
the
amperometric
detection
of
glucose.

0
-600

-300 -200

300

600

900

1200

The best working potential: +0.90V (vs. Ag/AgCl)

E(mV)

Linear Range of Concentration:4100 mM

0.1 mM Acetaminophen

I / A

L.O.D.=100M (1.8 mg/dl) (L.O.D. = (3x y0)/a);

5
4

y /A = (y0 y0) + (a a)*x/mM

y = (0.1990.003)+(0.0790.006)*x; R2=0.999;

Sensitivity: 2.0 A mM

-1

Response Time: 10 s

R2 = 0.999

(70-1800 mg/dl);

RSD (n = 3): 5%

y / A = 0.199 + 0.079 x / mM

cm ;
-2

2
0
0

10

20

30

40

50

60

70

[Glucose], mM

80

90 100 110

These nanostructured modified microsensors

are suitable for a sensitive (using alligned and
oriented
SWCNTs)
and
selective
(using
functionalised
SWCNTs)
neurotransmitters
detection.

will be carried out to immobilize the L-Glutamate

Oxidase
enzyme
on
the
SWCNT-modified
microelectrodes
to
assemble
sensitive
neurotransmitter
biosensors
for
the
early
detection of catecholamines involved in some
typical neurological disorders.

Typical LOD values (ng/L) of various techniques for

determination of neurotransmitters
Technique

LC+Fluor
microdial

HPLC+EC

Serotonin

5
(30 pM)

12000

Norepinephrine

4000

Dopamine

2
(20 pM)

2000

Epinephrine

Laccase
electrode

CE+Fluor
microdial

8 M

2
10 M

Glutamate,
Aspartate

0.1 M

Schematic Presentation of
Molecular Imprinting
+ Monomers

Synthesis:

Polymer
Target
molecule
Washing
Binding

Binding:

Elution

Molecularly
imprinted
polymer