L
LL
Description and Drawings of a Direct
Quantification Portable Salivary Melatonin
Biosensor for Determining Circadian
Rhythms
Ryan J. Love
DRDC Toronto
Michel A. Paul
DRDC Toronto
Heinz-Bernhard Kraatz
University of Toronto
Defence R&D Canada
Technical Report
DRDC Toronto TR 2013-108
March 2014
Limited D
Description and Drawings of a Direct
Quantification Portable Salivary Melatonin
Biosensor for Determining Circadian
Rhythms
Ryan J. Love
DRDC Toronto
Michel A. Paul
DRDC Toronto
Heinz-Bernhard Kraatz
University of Toronto
Defence Research and Development Canada – Toronto
Technical Report
DRDC Toronto TR 2013-108
March 2014
IMPORTANT INFORMATIVE STATEMENTS
The work described here has been protected by the following US provisional patent:
Love RJ, Kraatz HB, Paul MA. Method and Device for Melatonin Detection. U.S. Provisional Patent
Application 61867270, filed August 19, 2013.
Funding for this work was provided by WBE 04KD07.
© Her Majesty the Queen in Right of Canada, as represented by the Minister of National Defence, 2014
© Sa Majesté la Reine (en droit du Canada), telle que représentée par le ministre de la Défense nationale,
2014
Abstract ……..
An individual’s circadian rhythm governs cycles of fatigue and alertness, which permits nighttime
sleep and optimal performance of mental and physical tasks during the day. Circadian
misalignment, caused by shift work or eastward/westward travel, therefore impairs our ability to
sleep and remain mentally alert. This is particularly problematic in military and civilian
occupations in which human error may result in death; such is the case for transport vehicle
operators and medical professionals. As a result, knowing and understanding an individual’s
baseline circadian rhythm may be critical in fatigue-related accident prevention strategies.
Currently, accurate identification of a person’s circadian rhythm requires salivary melatonin
assessment by a plate-based immunoassay, which takes 24 hours and must be performed in a
laboratory. Therefore, the goal of this project is to develop and test a user-friendly device that
performs nearly instantaneous salivary melatonin assessment. Direct electrochemical
quantification of melatonin was performed in water and saliva on a disposable ceramic screenprinted electrode with a chemically modified surface. The peak current of melatonin oxidation in
2
both mediums (water and saliva) was highly correlated (R > 0.95) to the concentration of
melatonin in the samples. In conclusion we have shown proof-of-concept that melatonin can be
directly quantified on a gold electrode.
Résumé ….....
Le rythme circadien d'une personne régit ses cycles de fatigue et de vigilance, ce qui permet le
sommeil la nuit et un rendement optimal des tâches mentales et physiques pendant la journée. Le
désordre circadien, causé par le travail par postes ou les déplacements vers l’Est ou vers l’Ouest,
affecte donc notre capacité à dormir et à rester alerte. Cela est particulièrement problématique
dans les occupations militaires et civiles où l'erreur humaine peut entraîner la mort, comme dans
le cas des conducteurs de véhicules et des professionnels médicaux. Par conséquent, la
connaissance et la compréhension du rythme circadien de base d'un individu peuvent être
essentielles dans les stratégies de prévention des accidents liés à la fatigue. Actuellement,
l'identification précise du rythme circadien d'une personne nécessite une évaluation de la teneur
en mélatonine de la salive qui est effectuée au moyen d’un immunodosage sur plaque, qui prend
24 heures et doit être effectué en laboratoire. Par conséquent, l'objectif de ce projet est de
développer et de tester un appareil convivial qui effectuerait une évaluation salivaire presque
instantanée de la teneur en mélatonine. La quantification électrochimique directe de la mélatonine
a été réalisée dans de l'eau et de la salive sur une électrode en céramique sérigraphiée jetable
ayant une surface chimiquement modifiée. Le courant de crête d'oxydation de la mélatonine dans
les deux milieux (eau et salive) est étroitement corrélé (R2 > 0,95) avec la concentration de la
mélatonine dans les échantillons. En conclusion, nous avons prouvé le principe selon lequel la
mélatonine peut être directement quantifiée sur une électrode d'or.
DRDC Toronto TR 2013-108
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DRDC Toronto TR 2013-108
Executive summary
Description and Drawings of a Direct Quantification Portable
Salivary Melatonin Biosensor for Determining Circadian
Rhythms
Ryan J. Love; Michel A. Paul; Heinz-Bernhard Kraatz; DRDC Toronto TR 2013108; Defence Research and Development Canada – Toronto; March 2014.
Introduction or background: The daily cycle of fatigue and alertness that promotes nighttime
rest and mental/physical activity during the day is governed by each individual’s circadian
rhythm. Shift work and eastward/westward travel causes the individual to work and rest at times
that are misaligned with his/her circadian rhythm leading to fatigue during desired work hours
and insomnia during desired rest hours. Effective countermeasures are available, but depend on
accurate quantification of a hormone, melatonin, which is strongly linked to the circadian rhythm.
Melatonin quantification in an easy-to-obtain biological fluid (e.g. saliva) is currently performed
by a long, complicated laboratory procedure known as an immunoassay. Several military and
civilian occupations would benefit from having the capability to measure salivary melatonin
concentration quickly in the field with a disposable detection device since such a device would
permit accurate, user-initiated entrainment of his/her circadian rhythm.
Results: Direct electrochemical quantification of melatonin was performed in water and saliva on
a disposable ceramic screen-printed electrode (SPE) with a chemically modified surface.
Chemical modification of the gold working-electrode surface was performed by a self-assembled
monolayer (SAM) assembly of lipoate N-hydroxysuccinimide ester, followed by anti-melatonin
immunoglobulin (Ig) G conjugation to lipoate, followed by aqueous ethanolamine treatment. Any
residual bare gold was filled with pooled human saliva that was collected mid-day. X-ray
photoelectron spectroscopy of the gold surface following attachment of the self-assembled
monolayer showed elevated carbon and nitrogen peaks indicative of SAM attachment. Melatonin
quantification on the chemically modified gold electrode was performed by square-wave
voltammetry (SWV). The SWV peak current of melatonin oxidation in both mediums (water and
2
saliva) was found to be highly correlated (R > 0.95) to the concentration of melatonin in the
samples with a detection limit of 15.6 pg/ml.
Significance: Herein we show clear proof-of-concept that melatonin can be detected directly on
an electrode surface that has been chemically modified with a highly specific anti-melatonin
antibody. Such electrode can be utilized by the Canadian Forces to identify melatonin production
onset and circadian rhythm timing by personnel in the field, which permits optimization of
psychological alertness during critical missions or tasks.
Future plans: Further device development is required to produce a biosensor that is inexpensive,
reliable, and user friendly. Concurrently, we will be developing a second-generation electrode
that has a lower detection limit.
DRDC Toronto TR 2013-108
iii
Sommaire .....
Description and Drawings of a Direct Quantification Portable
Salivary Melatonin Biosensor for Determining Circadian
Rhythms
Ryan J. Love; Michel A. Paul; Heinz-Bernhard Kraatz Heinz-Bernhard Kraatz;
DRDC Toronto TR 2013-108; Recherche et développement pour la défense
Canada – Toronto; mars 2014.
Introduction ou contexte : Le cycle quotidien de fatigue et de vigilance qui favorise le repos
nocturne et les activités mentales et physiques pendant la journée est régi par le rythme circadien
de chaque individu. Le travail par postes et les déplacements vers l’Est et vers l’Ouest ont pour
effet que l'individu sera en capacité de travail et de repos à des moments qui ne correspondent pas
à son rythme circadien, ce qui amène la fatigue pendant les heures où l’on souhaite travailler et
l'insomnie durant les périodes souhaitées pour le repos. Des contre-mesures efficaces sont
disponibles, mais elles exigent une quantification précise d'une hormone, la mélatonine, qui est
étroitement liée au rythme circadien. La quantification de la teneur en mélatonine d’un fluide
biologique facile à obtenir (par exemple la salive) est effectuée au moyen d’une procédure de
laboratoire compliquée longtemps connue comme dosage immunologique. Plusieurs occupations
militaires et civiles auraient avantage à faire appel à une capacité de mesurer rapidement la
concentration de mélatonine salivaire sur le terrain avec un dispositif de détection jetable car un
tel dispositif permettrait à l’utilisateur d’entraîner précisément et rapidement son rythme
circadien.
Résultats : La quantification électrochimique directe de la mélatonine a été réalisée dans de l'eau
et de la salive sur une électrode en céramique sérigraphiée jetable (SPE) ayant une surface
chimiquement modifiée. La modification chimique de la surface active d’or de l'électrode a été
réalisée par une monocouche autoassemblée (SAM) de lipoate ester de nhydroxysulfosuccinimide, suivie d'une conjugaison lipoate et immunoglobuline anti-mélatonine
G (Ig), suivie d'un traitement à l'éthanolamine aqueuse. Toutes les surfaces d'or nu résiduel ont
été couvertes avec de la salive humaine ordinaire qui a été recueillie au milieu de la journée. La
spectroscopie de photoélectrons XPS de la surface de l'or après la fixation de la monocouche
autoassemblée présente des pics de carbone et d'azote élevés indicatifs de la fixation de la SAM.
La quantification de la mélatonine sur l'électrode d'or modifiée chimiquement a été réalisée par
voltampérométrie à onde carrée (SWV). Le courant de pic de l’oxydation de la mélatonine dans
les deux milieux (eau et la salive), mesuré en SWV, s'est révélé être étroitement corrélé (R2 >
0,95) avec la concentration de la mélatonine dans les échantillons, avec une limite de détection de
15,6 pg/ml.
Portée : Nous avons donc éprouvé avec succès le principe selon lequel la mélatonine peut être
détectée directement sur une surface d'électrode qui a été modifiée chimiquement avec un
anticorps anti-mélatonine très spécifique. Cette électrode peut être utilisée par les Forces
canadiennes pour identifier le début de la production de mélatonine et l’horaire du rythme
circadien par le personnel sur le terrain, ce qui permet d'optimiser la vigilance psychologique lors
des missions ou des tâches critiques.
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DRDC Toronto TR 2013-108
Recherches futures : La poursuite de l'élaboration de l'appareil est nécessaire pour produire un
biocapteur peu coûteux, fiable et facile à utiliser. Parallèlement, nous allons développer une
électrode de deuxième génération qui a une plus basse limite inférieure de détection.
DRDC Toronto TR 2013-108
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DRDC Toronto TR 2013-108
Table of contents
Abstract …….. ................................................................................................................................. i
Résumé …..... ................................................................................................................................... i
Executive summary ........................................................................................................................ iii
Sommaire ....................................................................................................................................... iv
Table of contents ........................................................................................................................... vii
List of figures ............................................................................................................................... viii
1 Background ............................................................................................................................... 1
2 Limitations of the Current Techniques for Analyzing Salivary Melatonin .............................. 2
3 Concept of a New Rapid Melatonin Sensor.............................................................................. 3
3.1 Background ................................................................................................................... 3
4 Materials and Methods.............................................................................................................. 4
4.1 Materials and Reagents.................................................................................................. 4
4.2 Preparation of the Antibody-Modified Electrode .......................................................... 4
4.3 Electrochemical Measurements ..................................................................................... 4
5 Results and Discussion ............................................................................................................. 6
5.1 X-Ray Photoelectron Spectroscopy (XPS) Characterization of Electrode Surface ....... 6
5.2 Electrochemical Characterization of Electrode Surface ................................................ 6
5.3 Aqueous Melatonin Detection and Measurement ......................................................... 7
5.4 Salivary Melatonin Detection and Measurement .......................................................... 8
6 Summary and Discussion........................................................................................................ 10
References ..... ............................................................................................................................... 11
List of symbols/abbreviations/acronyms/initialisms ..................................................................... 13
DRDC Toronto TR 2013-108
vii
List of figures
Figure 1 Cyclic voltammogram (4 cycles) of a 62.5ȝg/ml Melatonin solution illustrates the
terminal nature of melatonin oxidation. The inset depicts the chemical reaction
that occurs following melatonin oxidation and the three stable metabolites that
form. .............................................................................................................................. 3
Figure 2 An illustration of the self-assembled monolayer (SAM) that is formed on the gold
electrode surface to permit selective detection of melatonin. ....................................... 4
Figure 3 X-ray photoelectron spectroscopy characterization of the gold working electrode
after crticial SAM assembly steps. (A) XPS scan of the lipoic acid SAM attached
to the gold working electrode. (B) XPS scan of the antibody SAM attached to
lipoic acid on the gold working electrode. .................................................................... 6
Figure 4 Investigation of the electrochemical characteristics of the bare and surface-modified
electrodes by cyclc voltammetry (CV). (A) Formation of the SAM of lipoic acid
reduced the Faradaic redox currents that can be seen from the oxidation/reduction
of Fe[CN]63-/4- on bare gold. (B) The anti-melatonin antibody was anchored on the
surface by reacting selectively with the N-Hydroxysuccinimide on lipoic acid,
which caused a further decrease in the redox current. To block the non-specific
adsorption of proteins and other species to the surface of the electrode, the surface
was treated with pooled human saliva (melatonin-free), which reduced redox
currents even further by blocking direct access of the conducting ions to the
electrode surface............................................................................................................ 7
Figure 5 A Potential vs. Current Density graph after square-wave voltammetry (SWV)
experiments using aqueous melatonin solutions of known concentration. The plot
of melatonin concentration vs. peak current clearly shows a linear relationship
between melatonin and current produced by the SWV experiments ............................ 8
Figure 6 A Potential vs. Current graph from SWV experiments performed in redox probed
after the electrodes were incubated for 1 hr in saliva with a known concentration
of melatonin. The plot of melatonin concentration vs. peak current clearly shows a
linear relationship with a correlation coefficient of 0.9936. ......................................... 9
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DRDC Toronto TR 2013-108
1
Background
Jet lag and shift lag are major concerns in many critical military and civilian occupations,
especially in transport vehicle operation and medical professions, due to the concomitant
fatigue and associated cognitive performance decrements. Countermeasures for reducing jet
lag and shift lag exist but their application depends on knowledge of the individual’s baseline
circadian rhythm, which currently requires extensive laboratory work [1-4]. Therefore, the
goal of this project was to develop a sensor that can be used to identify endogenous
melatonin, the major biomarker of circadian rhythms. This manuscript describes the initial
studies on the direct detection of melatonin on the surface of a gold electrode following
modification with an anti-melatonin antibody to form a self-assembled monolayer (SAM).
For these studies, aqueous melatonin solutions of decreasing concentration were utilized.
Experimental work with saliva spiked with known concentrations of melatonin was also
performed to show that saliva components have no effect on the electrochemical
quantification of melatonin
A biosensor for salivary melatonin has been desired by the chronobiology research
community for over the past two decades; however the necessary specifications of such a
sensor are such that many challenges had to be overcome. First, the sensor must be able to
detect dim light melatonin onset (DLMO), which means that the minimum detection
sensitivity of the sensor should be approximately 1.0 pg/ml. Second, with regards to the
specificity, the sensor must be able to detect melatonin without interference from tryptophan,
or tryptophan-derived endocrines such as serotonin. Therefore, melatonin binding at the
surface must be achieved with a melatonin receptor or antibody with a binding pocket that is
specific for melatonin. For this research we chose to work with an anti-melatonin antibody
that has been shown by the manufacturer to bind only melatonin and not related tryptophanlike molecules.
The chemistry developed for this sensor can be easily applied towards the development of
sensors for other small-molecule endocrines. For example, cortisol, androgens and estrogens
can be detected in saliva and other biological fluids using electrodes with similar chemistry.
As a result, we expect to use the technology platform described here for the invention of
endocrine sensors capable of quantifying physical/psychological stress, androgenic potential
and overtraining, and detection of a range of endocrine-related pathologies (e.g. adrenal
insufficiency).
DRDC Toronto TR 2013-108
1
2
Limitations of the Current Techniques for Analyzing
Salivary Melatonin
Quantification of hormone concentration in a biological fluid, including salivary melatonin, is
currently performed by a plate-based immunoassay that has been developed specifically for the
hormone of interest. An immunoassay requires well-trained personnel to perform the test, and
generally requires 24 hours (± 1h) to complete. A hormone conjugate probe may be labeled with
either a radiological isotope, for increased sensitivity, or biotin, which is subsequently reacted
with a streptavidin-peroxidase for a colorimic change of 3,3ƍ,5,5ƍ-tetramethylbenzidine. When a
radiological isotope is used, the immunoassay is referred to as a RadioImmunoAssay (RIA), and
the quantification of the analyte is performed indirectly by a gamma counter. Immunoassays that
quantify the analyte by a colorimic change in the test well are known as Enzyme-Linked
ImmunoSorbent Assays (ELISA), and require a spectrophotometer to quantify the analyte
concentration.
The primary limitations of the current technology for salivary melatonin quantification are 1) the
time required to perform the assay, § 24 hrs; and 2) the bulky and expensive equipment that is
required. Furthermore, radiologically active probes are required for increased sensitivity of the
immunoassay, which poses a health risk to the technician that is performing the procedure. The
financial expense of the current technology is also a limitation as a kit for the analysis of 40
samples costs approximately $600.
Ultimately, the current technology does not permit self-assessment of endogenous melatonin
concentration, which is required for circadian rhythm and fatigue analysis in the field. The
technology presented below permits self-assessment of endogenous melatonin concentration
rapidly, and in any environment. The financial expense associated with individual sample
analysis of this portable biosensor is also expected to be much lower than the current technology.
2
DRDC Toronto TR 2013-108
3
Conce
ept of a New
N
Rapid Mela
atonin S
Sensor
3.1
Backg
ground
Well known as a powerful an
ntioxidant, melatonin is kknown to be oxidized at approximatelly
700m
mV, which peermits electro
ochemical deetection of thhe molecule at an electrrode interface.
Howeever, unlike other
o
antioxid
dants, melaton
nin does not undergo redoox cycling; uupon oxidationn,
melatonin becomees highly reacctive and freequently bindds to a seconnd melatonin molecule (seee
figuree 1). The disaadvantage of this
t is that a melatonin
m
sollution is imm
mediately com
mpromised afteer
one ro
ound of oxid
dation and eleectrochemicall detection m
must be achievved on the fi
first round off a
potential sweep of
o the electro
ode surface. However,
H
if sufficient m
melatonin is ppresent on thhe
electrode surface, the electric current
c
that reesults from m
melatonin oxiidation correllates extremelly
well with
w the conceentration of th
he original so
olution as we show in our rresults.
Fiigure 1 Cyclicc voltammogrram (4 cycles)) of a 62.5ȝg//ml Melatoninn solution illuustrates the
terrminal naturee of melatonin
n oxidation. The
T inset depiicts the chemiical reaction that occurs
follow
wing melatonin
n oxidation and the three sstable metaboolites that form
m.
DRDC
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2
3
4
Materiials and Method
ds
4.1
Materiials and Reagents
R
s
The electrodes,
e
pu
urchased from
m Dropsens (Oviedo,
(
Spaain), are screeen-printed onn ceramic annd
contaiined a gold working
w
electrrode with a 4.0
4 mm diameeter, a platinuum counter ellectrode, and a
silver reference electrode.
e
Po
olyclonal antti-melatonin antibody waas purchasedd from Piercce
Biotecchnology In
nc. (Rockforrd, IL). Meelatonin stanndard was ppurchased fr
from Biotrennd
Chem
mikalien Gmb
bH (Destin, FL).
F
Lipoic acid, N-Hyddroxysuccinim
mide (NHS), Poly(ethylenne
glycol) methyl eth
her thiol (Mn
n=2000), Eth
hanolamine, aand Tris(hydrroxymethyl) aminomethanne
(TRIS
S) was purchaased from Sig
gma-Aldrich (Canada).
(
Alll other reagennts were of annalytical gradde.
Deion
nized water fiiltered with a Millipore Milli-Q
M
system
m was used foor aqueous soolutions and aall
electrode washing steps.
4.2
Preparation of the Antib
body-Mod
dified Ele
ectrode
Beforre modificatio
on, all electro
odes were wasshed thoroughhly with deioonized water and dried witth
N2. Th
he electrode was
w then bath
hed in 2mM dihydrolipoic
d
acid NHS esster dissolvedd in basic wateer
for 72
2 hours at 4°°C. Subsequeently, the surfface was rinssed and then incubated w
with 1mM anttimelatonin pAb in phosphate-bu
uffered salinee (PBS) for 244 h at 4°C. A
Any active essters remaininng
were then quencheed with a 1%
% ethanolamiine solution ((pH 8) for 1 h and emptyy spots on thhe
electrode surface were
w filled by
y incubating th
he modified eelectrode withh pooled hum
man saliva for 1
h at room
r
temperaature. Figure 2 illustrates the
t sensor suurface modificcation that iss performed oon
the go
old electrode surface to maake it specificc and selectivee for melatonnin.
Figure
F
2 An illlustration of the
t self-assem
mbled monolayyer (SAM) thaat is formed oon the gold
electrode surrface to perm
mit selective deetection of meelatonin.
4.3
Electrochemica
al Measu
urements
All ellectrochemicaal studies weere performan
ned at room temperaturee in a groundded, enclodseed
Farad
day cage with a potentiostaat/galvanostat (CompactStaat, Ivium Techhnologies US
SA, Fernandinna
Beach
h, FL) conneccted to a personal computeer. The screenn-printed elecctrode was coonnected to thhe
potentiotstat with a three-electtrode contact edge connecctor (Dropsenns, Oviedo, Spain). Cyclic
voltam
mmetry (CV) and square wave
w
voltamm
metry (SWV)) were commeenced from reest potential iin
4
DRDC Toronnto TR 2013-1008
a 10mM Tris buffer (pH 7). The CV experiments were performed with a scan rate of 100mVs-1 in
the potential range from -100 to 350 mV with a step potential of 5 mV, amplitude of 25 mV, and
a frequency of 10 Hz. Electrochemical impedance spectroscopy (EIS) was performed in a 10mM
Tris buffer (pH 7) containing 5mM sodium perchlorate and 1.0 mM ferro-/ferricyanide
[Fe(CN)6]3-/4- as a redox probe. The EIS measurements were conducted in the frequency range of
100 hHz to 0.1 Hz, at a formal potential of 100 mV and AC amplitude of 5 mV. The
aforementioned electrochemical measurements were all repeated at least three times with separate
electrodes to ensure reproducibility.
DRDC Toronto TR 2013-108
5
5
5.1
Resultts and Discussi
D
on
X-Ray
y Photoele
ectron Sp
pectrosco
opy (XPS
S) Charac
cterization
n
of Elec
ctrode Su
urface
The XPS
X scans off the gold wo
orking electro
ode after lipoic acid (Figuure 3A) and pprotein (Figurre
3B) attachment. In
ncreased inten
nsity of adven
ntitious carbonn (C1s) electr
trons after attaachment of thhe
anti-m
melatonin antiibody indicatees that the SA
AM contains a highly dense layer of prootein.
Figu
ure 3 X-ray ph
hotoelectron spectroscopy
s
characterizaation of the goold working electrode afterr
crticcial SAM asseembly steps. (A)
( XPS scan of the lipoic aacid SAM atttached to the ggold workingg
electrode.
e
(B) XPS scan off the antibody SAM attacheed to lipoic accid on the golld working
electrode.
e
5.2
Electrochemica
al Charac
cterizatio
on of Elec
ctrode Su
urface
To eleectrochemicaally characteriize the gold electrode
e
surfface modificattion, we perfo
formed CV annd
3-/4-EIS in
i the presen
nce of the reedox probe, (Fe[CN]
(
. The CV sccans of the bare electrodde
6)
displaayed large red
dox currents corresponding
c
g to oxidationn and reductioon of potentiaal Fe[CN]6)3-/44-.
The laarge redox cu
urrents shown
n in the voltam
mmogram off the bare elecctrode surfacee indicates thhat
the ex
xpected redox
x reactions occcurred easily
y and quasi-rreversibly on the bare gold (Figure 4A
A).
After each electrod
de modification step, the CV
C scan dispplayed charginng current buut did not show
Farad
daic signal (F
Figure 4A,B). Furthermoree, the charginng current was reduced aafter each stepp.
The Nyquist
N
plots of the EIS corroborate th
he CV data (F
Figure 4). Imppedance is prresented as thhe
sum of
o the real an
nd imaginary
y Z componeents, Zre- andd Zim-, which mainly origiinate from thhe
resistaance and cap
pacitance of th
he cell respecctively. An eequivalent cirrcuit was seleected to reflect
the electrochemicaal process in order
o
to fit acccurate values.
6
DRDC Toronnto TR 2013-1008
Fig
gure 4 Investig
gation of the electrochemic
e
cal characterristics of the bbare and surfa
face-modified
eleectrodes by cyyclc voltamm
metry (CV). (A)) Formation oof the SAM off lipoic acid rreduced the
Farradaic redox currents
c
that can be seen from
f
the oxidaation/reductioon of Fe[CN]]63-/4- on bare
gold
d. (B) The antti-melatonin antibody
a
was anchored on the surface bby reacting seelectively withh
the N-Hydroxysuc
N
ccinimide on lipoic acid, which
w
caused a further deccrease in the rredox currentt.
To bllock the non-sspecific adsorrption of proteins and otheer species to tthe surface off the electrodee,
the su
urface was treeated with pooled human saliva
s
(melatoonin-free), whhich reduced rredox currents
even further by blocking direct
d
access of the conduccting ions to tthe electrode surface.
5.3
Aqueo
ous Melattonin Dettection an
nd Measu
urement
A sto
ock solution of melatonin
n standard (6
62.5 —g/ml) was prepareed in Tris buuffer (pH 7.00)
contaiining 100mM
M NaCl. The
T
stock so
olution was then seriallly diluted for melatoniin
measu
urements. Fo
or melatonin detection an
nd measurem
ment experim
ments, an 80uul drop of thhe
melatonin solution
n was depositeed on the elecctrode for meeasurement suuch that the m
meniscus of thhe
drop covered thee entire surrface of each electrodee (working, reference, and counterr).
Electrrochemical measurements
m
s were perfo
ormed immeddiately. Squaare-wave volttammetry waas
perforrmed on sep
parate electro
odes for each
h experimentt (Figure 5).. The resultiing curve waas
baseliine corrected
d using Ivium
mSoft (Ivium Technologiees, Eindhoveen, Netherlannds). The peaak
curren
nt between th
he potential raange of 0.6 V to 0.7 V wass then obtaineed from the ttable of currennt
vs. po
otential. The correlation coefficient
c
(R
R2) of melatoonin concentrration vs. peaak current waas
0.9676.
DRDC
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2
7
Figure 5 A Potential
P
vs. Current
C
Densiity graph afteer square-wavve voltammetrry (SWV)
expeeriments usin
ng aqueous meelatonin soluttions of know
wn concentratiion. The plot of melatonin
conceentration vs. peak
p
current clearly
c
showss a linear relaationship betw
ween melatonnin and currennt
produced by the SWV expperiments
5.4
Saliva
ary Melato
onin Dete
ection and Measurement
A stock solution of solution of melatonin staandard (1 ng//ml) was preppared in Millii-Q water. Thhe
stock solution wass then serially
y diluted for with
w pooled hhuman saliva to obtain meelatonin-spikeed
salivaa. For melato
onin detectio
on and meassurement expperiments, an 80ul dropp of a know
wn
conceentration of melatonin-spi
m
iked saliva solution
s
wass deposited oon the gold electrode annd
incubated for 1 hour in the dark
k at room tem
mperature. Aft
fter incubationn, the electrodde surface waas
rinsed
d in Milli-Q water and dried.
d
Squaree-wave voltam
mmetry meassurements w
were performeed
immeediately with Tris buffer containing the
t redox proobe. The ressulting curvee was baselinne
correccted using Iv
viumSoft (Iv
vium Technollogies, Eindhhoven, Netheerlands). Thee peak currennt
betweeen the poten
ntial range off 0.5 V to 0.6
6 V was thenn obtained froom the table of current vvs.
2
potential. The correlation coeffiicient (R ) of melatonin cooncentration vvs. peak curreent was 0.99366.
8
DRDC Toronnto TR 2013-1008
Figu
ure 6 A Potential vs. Curreent graph from
m SWV experiiments perforrmed in redoxx probed afterr
the electrodes weere incubated
d for 1 hr in sa
aliva with a kknown concenntration of meelatonin. The
pllot of melaton
nin concentration vs. peak current clearrly shows a linnear relationsship with a
correlation coefficient
c
off 0.9936.
DRDC
C Toronto TR 2013-108
2
9
6
Summary and Discussion
Herein we show that salivary melatonin can be quantified directly on an electrode by first
entrapping the analyte on the electrode by chemically modifying the electrode surface with a
SAM containing a unit that specifically binds melatonin, and then varying the potential of the
electrode with requisite current input. A series of such experiments were performed to detect
melatonin on a screen-printed electrode surface. Overall, the results show that the developed
biosensors are capable of melatonin quantification with a limit of detection of 15pg/ml.
Conceptually several other analytes, such as hormones, pathogens or chemicals, could be directly
detected in a similar fashion if the oxidation or reduction potential of the analyte does not overlap
with other species on the electrode. The quantity of analyte on the surface of the electrode must
also be great enough such that oxidation/reduction of the analyte results in an appreciable change
of current input through the potential scan.
In summary, the first stage of developing an electrode that permits portable, rapid, and highly
sensitive quantification of endogenous salivary melatonin has been completed. Improvements to
the electrode as well as testing a range of parameters of the square wave step sequence that are
used to vary the electric potential is necessary to achieve greater sensitivity. Other
electrochemical assay designs will also be tested to achieve a maximal signal to noise ratio at the
lowest detection limit. Finally, engineering and design of an inexpensive handheld unit that will
house the electrode set is required for production of a prototype that can be manufactured and
utilized by the Canadian Forces.
10
DRDC Toronto TR 2013-108
References .....
[1] Paul, M.A., et al., Phase advance with separate and combined melatonin and light treatment.
Psychopharmacology (Berl), 2011. 214(2): p. 515-523.
[2] Paul, M.A., et al., Melatonin treatment for eastward and westward travel preparation.
Psychopharmacology (Berl), 2010. 208(3): p. 377-387.
[3] Paul, M.A., et al., Timing Light Treatment for Eastward and Westward Travel Preparation.
Chronobiology International, 2009. 26(5): p. 867-890.
[4] Paul, M.A., et al., Phototherapy for Circadian Phase Delay: a comparison of 4
phototherapeutic devices. Aviat. Space Environ. Med, 2007. 78: p. 645-652.
DRDC Toronto TR 2013-108
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DRDC Toronto TR 2013-108
List of symbols/abbreviations/acronyms/initialisms
DLMO
Dim Light Melatonin Onset
DND
Department of National Defence
CV
Cyclic Voltammetry
DND
Department of National Defence
DRDC
Defence Research and Development Canada
DSTKIM
Director Science and Technology Knowledge and Information Management
R&D
Research & Development
SAM
Self-assembled monolayer
SWV
Square-wave voltammetry
DRDC Toronto TR 2013-108
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DRDC Toronto TR 2013-108
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Defence Research and Development Canada – Toronto
1133 Sheppard Avenue West
P.O. Box 2000
Toronto, Ontario M3M 3B9
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REVIEW: GCEC APRIL 2011
3.
TITLE (The complete document title as indicated on the title page. Its classification should be indicated by the appropriate abbreviation (S, C or U)
in parentheses after the title.)
Description and Drawings of a Direct Quantification Portable Salivary Melatonin Biosensor for
Determining Circadian Rhythms
4.
AUTHORS (last name, followed by initials – ranks, titles, etc. not to be used)
Ryan J. Love; Michel A. Paul
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DATE OF PUBLICATION
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March 2014
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26
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Technical Report
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Defence Research and Development Canada – Toronto
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DRDC Toronto TR 2013-108
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13. ABSTRACT (A brief and factual summary of the document. It may also appear elsewhere in the body of the document itself. It is highly desirable
that the abstract of classified documents be unclassified. Each paragraph of the abstract shall begin with an indication of the security classification
of the information in the paragraph (unless the document itself is unclassified) represented as (S), (C), (R), or (U). It is not necessary to include
here abstracts in both official languages unless the text is bilingual.)
An individual’s circadian rhythm governs cycles of fatigue and alertness, which permits
nighttime sleep and optimal performance of mental and physical tasks during the day. Circadian
misalignment, caused by shift work or eastward/westward travel, therefore impairs our ability to
sleep and remain mentally alert. This is particularly problematic in military and civilian
occupations in which human error may result in death; such is the case for transport vehicle
operators and medical professionals. As a result, knowing and understanding an individual’s
baseline circadian rhythm may be critical in fatigue-related accident prevention strategies.
Currently, accurate identification of a person’s circadian rhythm requires salivary melatonin
assessment by a plate-based immunoassay, which takes 24 hours and must be performed in a
laboratory. Therefore, the goal of this project is to develop and test a user-friendly device that
performs nearly instantaneous salivary melatonin assessment. Direct electrochemical
quantification of melatonin was performed in water and saliva on a disposable ceramic screenprinted electrode with a chemically modified surface. The peak current of melatonin oxidation
2
in both mediums (water and saliva) was highly correlated (R > 0.95) to the concentration of
melatonin in the samples. In conclusion we have shown proof-of-concept that melatonin can be
directly quantified on a gold electrode.
--------------------------------------------------------------------------------------------------------------Le rythme circadien d'une personne régit ses cycles de fatigue et de vigilance, ce qui permet le
sommeil la nuit et un rendement optimal des tâches mentales et physiques pendant la journée.
Le désordre circadien, causé par le travail par postes ou les déplacements vers l’Est ou vers
l’Ouest, affecte donc notre capacité à dormir et à rester alerte. Cela est particulièrement
problématique dans les occupations militaires et civiles où l'erreur humaine peut entraîner la
mort, comme dans le cas des conducteurs de véhicules et des professionnels médicaux. Par
conséquent, la connaissance et la compréhension du rythme circadien de base d'un individu
peuvent être essentielles dans les stratégies de prévention des accidents liés à la fatigue.
Actuellement, l'identification précise du rythme circadien d'une personne nécessite une
évaluation de la teneur en mélatonine de la salive qui est effectuée au moyen d’un
immunodosage sur plaque, qui prend 24 heures et doit être effectué en laboratoire. Par
conséquent, l'objectif de ce projet est de développer et de tester un appareil convivial qui
effectuerait une évaluation salivaire presque instantanée de la teneur en mélatonine. La
quantification électrochimique directe de la mélatonine a été réalisée dans de l'eau et de la salive
sur une électrode en céramique sérigraphiée jetable ayant une surface chimiquement modifiée.
Le courant de crête d'oxydation de la mélatonine dans les deux milieux (eau et salive) est
étroitement corrélé (R2 > 0,95) avec la concentration de la mélatonine dans les échantillons. En
conclusion, nous avons prouvé le principe selon lequel la mélatonine peut être directement
quantifiée sur une électrode d'or
14. KEYWORDS, DESCRIPTORS or IDENTIFIERS (Technically meaningful terms or short phrases that characterize a document and could be
helpful in cataloguing the document. They should be selected so that no security classification is required. Identifiers, such as equipment model
designation, trade name, military project code name, geographic location may also be included. If possible keywords should be selected from a
published thesaurus, e.g. Thesaurus of Engineering and Scientific Terms (TEST) and that thesaurus identified. If it is not possible to select
indexing terms which are Unclassified, the classification of each should be indicated as with the title.)
Rapid; Portable; Salivary Melatonin Bioassay Device