dopamine biosensor
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Dopamine SensorTRANSCRIPT
ANALYTICAL SCIENCES JANUARY 2A07, V OL. 23
2AA7 @ The Japan Society for Analytical Chemistry
81
Real-Time Detection of Dopamine Released from a Nerve ModelCell by an Enz,yme -Catalyied Lumine§cence Method and ltsApplication to Drug Assessment
Iliroaki §m¡os¿n¿'t and Feifei WaNc
Field. of Life, I$ormation and System Scierues, Graútate Scttool of Scimce and Engineering
IJnivercity of Toywta 3 DA Gofuha Toyuna 9 j O85 5 5' Japan
A real-time observatioa of neurctraasmiüer release from a nerve cell is a useful method for not only- neuroscieoce
researctr, but also .rroriog of the influence of chemicals, including drugs, on *te human nervou§ sy§tem' In this stud¡ a
more simple and sensitive-method for real-time monñoring of do,pamine release from a nerve modsl ce¡l was developed"
Higtüy sensitive detection of dopamine was performed by using tyr¿mine oxidase for dopamine oxida{9n whilh'was
rolowe¿ by a luminol luminesc-ence reactiou. rhis enryme-catalyzed luminescence method was applied to observe
dopamine ,elease from the pC12 cell as a nerve model cell upon stimulation with acetylcholine and an acetylcholine
rpcepúor agonist The resuln demonstrated that the real-time moniodng of the activation of rhe PC12 cell was e4sily
perfórmeO-Uy ttris method, This method possessed many advantage§, such as high sensitivity, rapid measurement and no
prerrr.rn*ifo, cells. It might be applied to drug scr;ning and the as§es§ment of harmful influences of food addiüves
and pesticides on the nerves.
(Received october 3{), 20ffi; Accepd November 6 2&K; Published January 1g'?Ñl\
Introduction
Easier and more sensitive methods for the biological assessment
of chemical compounds, such as drugs, food additives and
environmental pollutants, to the human nervous system have
been increasingly required. The real-time monioring of nerve-
cell acüvation, or the release of neurotra¡smitters, is very
effectivs for these purposes. Real-time mea§urement§ of nerve-
cell activation have been conventionally performed by using
fluorescence microscopy with a calcium indicatol.r'z However,
in this meüoü the indicator reagent should be loaded into the
cell bodies, and any excess of the reagent should be removed by
washing each time. Although HPLC analysis is also the usual
method to investigate neurotransmitter release by nerve-cell
activation,3l ÍIPLC analysis is complicated, and takes much
tirne. Therefore, the development of easier, real-time
observation methods is further requested.
In üis study, we aimed to develop a novel method for the
real-time detection of dopamine, a typical neuroransmitter,
released from the rat pheochromocytoma (PC12) cell as a
dopaminergic neuron model.!' Acetylcholine (ACh) and 1,1-
dimettryt-2}-phenylpiperazinium iodide (DMPP) were used as
stimulation agents for the PCl2 cell. It was well-known that
these compounds induce an increase of cytosolic Ca2n through
depolarization-induced activation of voltage-sensitive Ca?"
channels, and the release of Caa from the cytosolic vesicles'
and following neurotransmitter release ftom PCl2 cells.Ho
In this paper, a modified enzyme-luminescence detection
method for doPaminc is int¡oduccd as a novcl real-time
r To whom correspondence should be addressed.
E-mail: [email protected] jp
observation method to observe dopamine release from PCl2' In
this method, dopamine is firsüy oxidized to produce IIrO, by
tyramine oúdase (TOD). The produced H:Oz reacts ,wiÚ¡
tuminol to gerierate chemiluminescence in the presence ofhorseradish percxidase (IIRP)'
- The appücationr of
lactoperoxidase (trJOD)tt or tyr<xinaset2 for dopamine
oxidation was previously reported, however these euz¡nneg had
insufficient activity to detect dopamine in the examination
experiments" Therefore, in ttris study, ryramineoxidase (TOD'gC t.¿.¡.¿ t¡om Ar-throbacter sp.) was fir§t te§ted to oxidize
dopamine iastead of LFOD or tyrosinasé, because üe structure-
of its original substrate, tyramine is similar to thal ofdopamine.ri Furthenr¡ore, the ToD-catalyzed luminescence
¿eLction of dopamine was applied to detect dopamine deased
from PC12 cells upon ACh stimulhtion. Finally, we tried to
apply this method for drug assessment.
Experimental
Reagents and clwmicalsLuminol (5-amino2'3'ttihydro-l$'phthalazinedigne)'
tyrosinase from mushroom, hydrogen peroxide, and acetylcholine
b¡omide were obtained from Wako Pure Chemical Industries,
Ltd. Dopamine hydrochloride and lactoperoxidase (LPOD)
from bovine milk was obtained from Sigma' Peroxidase (POD)
wa.§ obtained from TOYOBO Co., Ltd. Tyrarnineoxidase
(fOD) from Anhrobacter sp' was obtained from ASAHI
KASEI PI{ARMA. DMPP (1,1 -dimethyl4-phenylpiperazihiumiodide) was obtained from ICN Biomedicals' Inc'
Cell culturePC12 cells (RC80009) were purchased from the cell bank of
Doparnine rele*edñom FC12
PCf8cdls
Fig. 1 Schematic illusnation of the principle of real-time detectionof dopamine released from PCl2 cells by drug stimulation with aluminescence detectable piate reader.
RIKEN BioResouce Center. Cells were grown in Dulbecco'smodified Eagle medium (DMEM) supplemented with 10% (v/v)horse serum (GIBCO), 57o (vlv) fetal bovine serum (ICNBiomedicals, Inc.), and 1% penicillin-streptomycin (GIBCO) at37'C in a humidified atmosphere containing 5%, COz. On theday before an experiment, cells were harvested with trypsin-EDTA and re-plated in a 9Gwell plate at a density of 0.5 x IffcellVml, and incubated for Z h to allow aüachment for a cellassay.
Characterizption of tle enzynes for doparnine oidationA comparison of three enzJme (TOD, tyrosinase, POD)
activities for dopamine oxidation was performed by a
colorimetric assay. The assay mixture contained 1 mM phenol,
0.5 mM 4-aminoanüpyrine, 0.5 mg/ml horseradish peroxidase,
10 pM - 2 mM dopamine, 0.1 M phosphate buffer (pH 7.4) at a
final volume of 3 ml. The reaction was started by adding üeenzyme in a final concentration of 0.1 mgiml. The absorbance
of üe solution was measured at 500 nm for 5 min with the v-560 spectrophotometer (JASCO Co.). The enzyme activity was
expressed as üe amount of dopamine oxidized per min and mg
of protein.
Deuction of dopamine with the e¡tzyme-luminescetrce method
The detection of dopamine was performed with a
iuminescence detectable plate reade¡ FLUOstar OIrIIMA(BMG LABTECH). Before a measurement, the medium was
replaced with [,ocke's solution Gü{ 7.4, 200 pl), then 0.25
mg/ml POD (30 td), 1.0 mg/mi TOD (30 ¡rl) and I mM luminol(30 pl) were added to each well. A dopamine solution of 10 plwas injected into the wells of the 96-well plate using an
automatic reagent injector to start the enzymatic reaction.
Lurninescence was detected by an equipped photomultiplier.
Enzync-catalyzed hnnine scence detection of dopunine released
fronPC|Z cellsPCl2 cells were plated in the 96-well plate at a density of
0.5 x ltr cellVml, aod incubarcd ovemight to Promotc ccll
adhesion. Just before a measurement, the Srowth medium was
removed from each well and cells were washed twice withLocke's solution. After washing, Locke's solution (200 U$,
ANALYTICAL SCIENCES JANUARY 2Ñ7, Y OL. 23
Table I Characterization of three enzymes for dopamineoxidation
Activityfu mgt KJ¡uM.
Tyr4mineoxidase CfOD)Tyrosinaselactoperoxidase &POD)
0.25 mg/¡ril POD (30 pl), l'0 mg/ml TOD (30 pi) and I mM
luminol (30¡tl) were added to each weil. The 96-well plate was
set in tl¡e plate reader and cell excitation was induced by the
injection of Ach or DMFP with the equipped auto-injector. The
principle of the real-time detection of dopamine released from
PCl2 cells by drug injection with the luminescence-detectable
plate reader is schematically shown in Fig. l.HPLC-ECD analysis was atso carried out to determine
dopamine released from the PCl2 cells by drug stimulation in
Locke's solution. Ion-pair chromarography with an ODS
capiltary column was used to separate dopamine after
deproteinization by a perchlorate pretreatment and
centrifugation. An LC-100 pump, ail I-C-165 on line valve, a
DA-5 chromatograph interface, and an LC-4C amperometric
detector were purchased from BAS.
Results and Discussion
Enzyme se lection for d.opamine oxidartonWe aimed firstly to find a good enzyme to detect dopamine
released from nerve model cells (PCl2) in real-time by enzyme-
catalyzed luminescence mea§urements. Table I rePresents the
three enzyme activities upon dopamine oxidation. TOD had the
highest activity (more than 6 times for tyrosinase and more than
2(tr times for LPOD) for dopamine oxidation, while there was
little difference in the K, value of three enzymes. Theretbre' itwas considered that TOD is much more efficient tbr dopamine
oxidation ar¡d HzO: production as compared with the previous
two enzymes.
Detection of doparuine with the enzyme-lwninescence mcthodTOD and POD were combined to detect dopamine by rhe
luminol luminescence method. When dopamine was injected
into the measurement soiution, luminescence was quickly
geúerated and reached the peak within a few seconds, as shown
in Fig. 2(A). The luminescence peak intensity increased withincreasing dopamine concenration within I UM. The detection
timit of dopamine was l0 nM, and its seositivity was
comparable to that of the ÍIPLC method. Figure 2(B) shows the
calibration cun¡es for dopamine with our TOD + POD method
and the LPOD + POD method. The comparison indicates that
the TOD + POD method is more sensitive to detect dopamine'
Real-time detection of dopamine rcleased from PCl2 cells by
the eniyme- catalyzed lurnine scenc e rnetho dPrior to the detection of dopamine released from PCl2 cells
by the developed enzyme-catalyzed lumine§cence method'
HPLC detection of catecholamine release<I from PCl2 cells was
performed. The HPLC anaJysis demonsuated that PCl2 cells
release only dopamine by ACh stimulation after one day ofculturing, though norepinephrine was also released after more
than n¡vo days. Next, the real-time observation of dopamine
release from PC12 cells after one day of culturing was
examined by the deveiopcd enzyme-cataiyzed luminescence
oñtr-:-'ry 5l3994
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ANALYTICAL SCIENCES JANUARY 2N7, YOL, 23
DM
510Time / sec,
0 t00 200 3m 4fl) 500
[DopamineJ /dr{
Fig. 2 Detection of dopamine by the enzyrre-luminescencerrrthod. (A) Tine course of luminescence after dopamine injection.(B) Dependence of the luminescence intensity on the dopamine
concenrratio¡. (a) TOD + POD method; (¡)) LPOD + POD ñpthod.
method. As shown in Fig. 3(A), the luminescence peak was
observed quickly within a few seconds after the injection ofACb. There was good reproducibility for three wells. The
dependence of the luminescence peak intensity on the
co¡centration of ACh was safurated at 100 pM with a detection
li¡nit of 5 !rM, as shorvn in Fig. 3(B). The EGo for ACh was 6ó
¡rM by this method and it was almost the sarne as the ECso (70
pM) by our HPLC analysis.Mo¡eover, we tried to apply this method to drug as§essment
for FCl2 cells. Luminescence geoer¿¡tion was quickly observed
by the injecüon of DMPP (a typical ACh receplor agonist) as
similar as ACt stimulation. The luminescence peak intensity
depended on the concentration of DMPP, a§ §hown in Fig. 4'
The EC5qby this meüod forDMPP was 20 pM and the ECsoby
our ÍIPIf analysis was 30 ¡tM. This result demonstrated that
this method can be used for dn¡g assessment.
Conclusions
It was demonstrated that our modified enzymeluminescencemethod is very useful for rcal-time observations of dopamine
release from PCl2 ceüs, as a doperminergic neuron model, by
acetylcholine and DMPP stimulation. The dependence of the
cell response on the drug concentration by this method
coresponded well to the dependence investigated by an HPLCanalysis. These results support that this method can be applied
to drug assessment for dopaminergic neuron§ or their model
10 20 30
Time/ sec.
0 100 2a0 300 400 500
lAchl I FtM
Fig. 3 Real-time dercdiou of dopamine ¡eleased ftom PCl2 cells.(A) Luminescence observation indicating dopamine ¡etrease fm¡nPCl2 cells by ACh stimulation The responses of th¡ee wells are
overlaid. The ACh concentration was 100 t¿M. (B) Dependence ofttre luminesence intessity on the ACh concenfation.
0 100 200 300 400 500
IDMPPI / l¡I,t
Fig. 4 Dependence of the luminescence intensity on the DMPP
conentration.
cells. This method has many advantages, such as highsensitivily for dopamine, rapid measurement (high througi¡pu$,and no pretreatment for cells (simple procedure). In addition,
the meüod mighr be usefui tbr the screening of chemical
compounds that have a harmful influence on the human nerve
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system. As our next direcüon, we would like o develop ttrismethod for the real-time imaging of dopamine rclease f¡omdopaminergic neurons, or rheir model cells. This fuure studymay help to find dopaminergic neurons in brain slice andprimary culturcd cells without any staini¡g. AIso it may beusefi¡l for monitoring the activity of focused dopaminergicneurons.
Acknowledgements
This work was supported by a grant of Toyama-Medical BioCluster ProjecL MEXT in Japan.
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