Research ArticleAcetylcholinesterase Inhibitors Assay Using Colorimetric pHSensitive Strips and Image Analysis by a Smartphone

Adam Kostelnik12 Alexander Cegan1 and Miroslav Pohanka23

1Faculty of Chemical Technology University of Pardubice Studentska 95 53210 Pardubice Czech Republic2Faculty of Military Health Sciences University of Defense Trebesska 1575 50001 Hradec Kralove Czech Republic3Department of Geology and Pedology Mendel University in Brno Brno Czech Republic

Correspondence should be addressed to Miroslav Pohanka miroslavpohankagmailcom

Received 29 September 2016 Accepted 17 January 2017 Published 13 February 2017

Academic Editor Neil D Danielson

Copyright copy 2017 Adam Kostelnik et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Smartphones are widely spread and their usage does not require any trained personnel Recently smartphones were successfullyused in analytical chemistry as a simple detection tool in some applications This paper focuses on immobilization ofacetylcholinesterase (AChE) onto commercially available pH strips with stabilization in the gelatin membrane AChE degradesacetylcholine into choline and acetic acid which causes color change of acid-base indicator Smartphone served as a tool formeasurement of indicator color change from red to orange while inhibitors blocked this process AChE inhibitors were measuredwith limits of detection 149 nM and 223 nM for galanthamine and donepezil respectively Organic solvents were measured formethod interferences Measurement procedure was performed on 3D printed holder and digital photography was evaluated usingred-green-blue (RGB) channels The invented assay was validated to the standard Ellmanrsquos test and verified on murine plasmasamples spiked with inhibitors We consider that the assay is fully suitable for practical performance

1 Introduction

AChE is an enzyme splitting the neurotransmitter acetyl-choline in cholinergic synapsis into choline and acetic acid[1] Sensitivity of AChE to neurotoxic compounds (anti-Alzheimerrsquos drugs pesticides and nerve agents) can be usein their measurement [2] Commonly used method is basedon reaction of thiocholine formed from acetylthiocholineduring enzymatic hydrolysis with Ellmanrsquos reagent produc-ing yellow 5-thio-2-nitrobenzoatemeasurable by spectropho-tometry in 412 nm [3] Despite broad use this methodhas some drawbacks like instability of Ellmanrsquos reagent andhemoglobin interference [2 4 5] Furthermore there is pos-sibility of measuring enzyme activity electrochemically [6 7]Colorimetric detection can be also based on pH measure-ment Many acid-base indicators are known for this purposewhen phenol red was used in our work previously [8 9]Different techniques for enzyme immobilization and matrixlike gelatin were described as well [10ndash13] Simple physicalimmobilization of enzyme onto cellulose and stabilization

into gelatin matrix was successfully used for preparation ofbiosensor in an application where AChE was embedded intogelatin membrane on a paper matrix and performed for theassay of neurotoxic compounds [14 15] In the cited papersthere was no however made evaluation of enzyme activityby a camera because a naked eye assay and another type ofsubstrate were preferred Gelatin provides good propertiesfor enzyme immobilization combined with biocompatibilityand zero toxicity [16] Modern mobile phones dispose highresolution cameras which gives them ability to serve as atool for diagnostics [17 18] Previously mobile phones havebeen used for some applications in analytical chemistry[19 20] This paper deals with preparation of biosensorbased on commercial pH strips with immobilized AChE andstabilized in gelatinmembrane Performance of the biosensorwas verified on neurotoxic compounds This approach offerseasy way for AChE inhibitors determination especially ifwe considered well-established spectrophotometric assayfor the presented method here no special equipment ortrained personnel are required There is also innovation in

Hindawi Publishing CorporationInternational Journal of Analytical ChemistryVolume 2017 Article ID 3712384 8 pageshttpdxdoiorg10115520173712384

2 International Journal of Analytical Chemistry

evaluation of color based reaction compared to previouslydescribed methods The major advantage of our assay isbased on the opportunity to link it to a smartphone which isconsidered as the detector device providing wide availabilityto less equipped laboratories and for field tests without anyexpensive gear

2 Material and Methods

21 Materials and Devices Acetylcholinesterase from elec-tric eel (ge1000 unitsmg protein) acetylcholine chloride(AChCl) acetylthiocholine chloride (ATChCl) 551015840-dithio-bis(2-nitrobenzoic acid) (DTNB) donepezil hydrochloridemonohydrate galanthamine hydrobromide tetraisopropylpyrophosphoramide (iso-OMPA) phosphate buffer saline(PBS) pH 74 dimethyl sulfoxide (DMSO) and isopropylalcohol were purchased from Sigma-Aldrich (St LouisMO USA) denatured ethanol and gelatin were suppliedby PENTA (Prague Czech Republic) Indicator strips pH-Fix 60ndash77 were obtained from Macherey-Nagel (DurenGermany) Color change was detected by Sony XperiaMT27iwith 5Mpx camera and LED light using operation systemAndroid 237 device version number 60B3184 (TokyoJapan) For 3D print 3D printer Prusa i3 fromPrusa Research(Prague Czech Republic) was used Murine plasma sampleswere obtained from 20 female BALBc mice which werepurchased from Velaz (Unetice Czech Republic) The micewere kept under standard ambient temperature and humidity50 plusmn 10 Light and dark periods lasted equally for 12 hourseach The mice were sacrificed in the age of 8 weeks bycutting of carotid under carbon dioxide narcosis and theblood was taken into tubes pretreated with lithium heparin(Dialab Prague Czech Republic) and centrifuged at 1000timesgfor 5 minutes Fresh plasma was kept at minus80∘C until use inthe assay The whole experiment was both permitted andsupervised by ethical committee Faculty of Military HealthSciences (Hradec Kralove Czech Republic)

22 Solutions Preparation AChCl solutions were preparedin concentration range from 031 to 10mM and placedin microtubes Galanthamine solutions were prepared inconcentration range from 16 to 2500120583M Donepezil solu-tions were prepared in concentration range from 031 to500120583M Each solution was prepared in PBS 74 and finalconcentration in microtube was 10 times lower Gelatin wasprepared in 1 concentration by stirring of 10mg of gelatinin 1ml of water for 20min All solutions for Ellmanrsquos assaywere prepared in PBS 74 DTNB solution was prepared inconcentration 1mM and ATChCl in 10mM Concentrationrange of galanthamine was from 625 to 100 120583M and 13 to20120583M in case of donepezil Final concentrations in cuvettewere 10-fold less concentration of ATChCl and 40-fold less incase of galanthamine or donepezil Iso-OMPA was preparedin PBS 74 in concentration 1mM final concentration inplasma samples was 01mM

23 Preparation of pH Strips with AChE To pH strips 10 120583lof AChE (activity for acetylthiocholine 173 times 10minus8mols120583l)was added and let to dry in laboratory temperature Then

pH strips were covered by 10 120583l of 1 gelatin After dryingin laboratory temperature pH strips were stored in 4∘C untilused in the assay

24 Preparation of 3D Printed Holder Holder was created inAutodesk 123D Design (open source software) 3D printersetupwas as follows acrylonitrile butadiene styrene shaped in3mm filaments was used as material nozzle temperature wasat 285∘C and bottom temperature at 100∘C and individualdeposited layers were 01mm thick Size of holder was 80mmin height and 105mm in length and inner diameter of tubewas set to 40mm (Figure 1)

25 Smartphone Assay The smartphone assay was made inthe following way 450120583l of PBS pH 74 and 50 120583l of 10mMAChCl solution were added to microtubes and strip was putinto it After incubation of 15min excess of reaction mediumwas drained and picture of the still wet pH strip was takenDuring photographing the camera was placed on the 3Dprinted holder and the strip inside hence no outer lightinfluenced the photography and integrated LED light was theonly one source Distance between the strip and camera wasalso constant just due to the holder

26 Ellmanrsquos Assay To standard cuvette 400120583l of DNTBsolution 25 120583l of AChE 475 120583l of PBS and 100 120583l of ATChClwere added Absorbance was measured in 412 nm immedi-ately and after incubation of 2min

27 Data Processing RGB color values were obtained byprocessing of photography in GIMP 2816 (open sourcesoftware) using Color Picker function ΔColor intensity wasobtained as follows intensity of strip before reaction ndash inten-sity of strip after the reaction This difference correspondsto AChE activity in different concentration of used substrateor inhibitor 119870

119872value for AChE and AChCl substrate was

calculated using nonlinear curve fitting by Hill function withcoefficient of cooperativity 119899 = 1 Limit of detection wascalculated as signal to noise ratio equal to three For thesepurposes Origin software 8 PRO (OriginLab NorthamptonMA USA) was used

3 Results and Discussion

AChE splitting acetylcholine into choline and acetic acidresulted in decreasing pH of medium Our method is basedon color change of indicator in pH sensitive zone of pHstrip from red to orange (Figure 2) During photographyprocessing color change was observed in green channel whilered and blue ones were without change

31 Gelatin Optimization Amount of gelatin was tested in0 0001 001 01 1 and 10 concentration Thestrips were covered with 10 120583l of gelatin in tested concentra-tion and dipped into PBS 74 and color change of strip intored was observed after 5min Gelatin in 10 concentrationdid not allow us to enter buffer to pH sensitive zone and colordid not change into red one while it stayed orange which wasrepresented by decreasing of color intensity (Figure 3) Color

International Journal of Analytical Chemistry 3

(a) (b)

Figure 1 Tube shaped holder printed by 3D print technology (a) and the holder with a smartphone adjusted on the hole to providephotographs by an integrated camera

Acetylcholine + water choline + acetic acid

AChE

Inhibitor

Figure 2 pH sensitive method using AChE principle

RcGcBc

minus3 minus1minus2 10log c gelatin ()

0

50

100

150

ΔC

olor

inte

nsity

Figure 3 Gelatin optimization Drop in ΔColor intensity indicatesthat pH strip did not change into red color Error bars representstandard error of the mean for 119899 = 3 Rc = red channel Gc = greenchannel and Bc = blue channel

change into red was held while using gelatin in 1 and lessconcentration but for further measurement 1 gelatin waschosen just for the better stabilization effect for AChE

30

35

40

45

50

ΔC

olor

inte

nsity

5 10 15 20 25 300Time (min)

Figure 4 Optimization of pH strip incubation time with ATChClas a substrate Error bars represent standard error of the mean for 119899= 3

32 Time Optimization Strip with AChE and 1 gelatin wasincubated with 1mM AChCl Incubation time with AChClwas observed in 5min intervals from 0 to 30min Thebiggest color change was held up to 15min Over this timecolor change was not significant for longer incubation time(Figure 4)

4 International Journal of Analytical Chemistry

05 1000c ACh (mM)

2

4

6

8

10

ΔC

olor

inte

nsity

Figure 5 Saturation curve for AChE and AChCl as a substrateError bars represent standard error of the mean for 119899 = 3

33 Substrate Measurement Saturation curve for AChE andAChCl as a substrate was measured in concentration rangefrom 0031 to 10mM and 119870

119872value was calculated as

described above to 5426 120583M (Figure 5) while 739 120583M wasreported by Xu and coworkers [24] 119870

119872value however

depends on type of used buffer as proved byWille et al usinghuman AChE isolated from erythrocytes and achieved 119870

119872

equal to 714 120583M in MOPS buffer 982 120583M in PBS buffer01mM in Tyrode buffer and 0122mM in Tris buffer whenpH was set to 74 [25] There are also big differences betweenorganisms as shownby Shaonan et al whoworkedwithAChEisolated from fish species and found out values above 01mM[26] and by Jiang et al who reported119870

119872to be 6385 120583Mwhen

measured with mosquito AChE [27] Recombinant enzymesexhibit slightly higher 119870

119872values compared to wild types as

showed in experiments with mice AChE carried out by Boydet al who found out119870

119872to be 46 120583M in wild type and 58120583M

for recombinant AChE [28]

34 Inhibitors Measurement AChE is sensitive to neurotoxiccompounds like drugs nerve agents or pesticides Some ofthese compounds are widely used in treatment of Alzheimerdisease [29] Galantamine and donepezil can be examples forthe currently available drugs [30] We performed calibrationcurve of galanthamine in concentration range from 0156to 25 120583M with limit of detection calculated to 149 nMand quantification limit of 05 120583M was achieved (Figure 6)Linearity of the assay is limited to 25 120583M when higherconcentrations appeared to be indistinguishable Comparingto the standard Ellmanrsquos assay it is only 10 times higherdetection limit when 183 nM of galanthamine was achievedin our experiment From previously published methodsfor galanthamine measurement based on AChE inhibitionwe can conclude that our method is competitive [21 22]Although detection limits are similar there is advantage infabrication time of pH strip with immobilized AChE whichis not time consuming

R = minus09975

1015 middot log c + 1292

minus10 minus05 05 1000log c galantamine (120583M)

10

15

20

ΔC

olor

inte

nsity

ΔColor intensity =

Figure 6 Calibration curve of galanthamine concentration is givenin logarithm Error bars represent standard error of the mean for 119899= 3

R = 09922ΔColor intensity = 3172 middot absorbance + 198

015625 120583M

03125 120583M

0625 120583M

125 120583M

25 120583M10

15

20

ΔC

olor

inte

nsity

(sm

artp

hone

assa

y)

03 04 05 0602Absorbance (Ellmanrsquos assay)

Figure 7 Validation of galanthamine assay compared to standardEllmanrsquos assay Error bars for smartphone assay represent standarderror of the mean and for Ellmanrsquos assay standard deviation for 119899 =3

Validation of method for galanthamine measurementwas done using standard Ellmanrsquos assay with correlationcoefficient of 09922 (Figure 7)

Method was verified using murine plasma Plasma sam-ples were pretreated by iso-OMPA selective inhibitor ofbutyrylcholinesterase [31] which naturally occurs in plasmaand has ability to split acetylcholine Then galanthamine wasspiked into plasma samples in appropriate concentrationsand smartphone assay has been performed (Figure 8) Formeasurement with plasma PBS buffer had to be replaced bywater because plasma strongly buffered itself then PBS bufferwas not required

In literature there are plenty of references about determi-nation of donepezil by chromatography techniques [32 33]Likewise there are electrochemical methods for donepezilmeasurementwith similar detection limits as ourmethod [23

International Journal of Analytical Chemistry 5

015625 120583M

03125 120583M

0625 120583M

125 120583M

25 120583M

R = 09673ΔColor intensity (plasma) = 136 middot ΔColor intensity (standard) + 030

15 2010ΔColor intensity (standard galanthamine solution)

15

20

25

30

35

ΔC

olor

inte

nsity

(spi

ked

plas

ma s

ampl

es)

Figure 8 Verifying of galanthamine assay in plasma samplescompared to standard galanthamine solution Error bars representstandard error of the mean for 119899 = 3

R = minus09844minus844 middot log c + 438

minus10 minus05minus15 00log c donepezil (120583M)

10

15

20

ΔC

olor

inte

nsity

ΔColor intensity =

Figure 9 Calibration curve of donepezil concentration is given inlogarithm Error bars represent standard error of the mean for 119899 =3

34] and some spectrophotometricmethods for quantificationof donepezil in pharmaceuticals or human plasma [35 36]However no evidence about determination of donepezil viainhibition of AChE was found Donepezil calibration curvewas done in concentration range from 0031 to 050 120583M andlimit of detection equal to 223 nMwas achievedwhile limit ofquantification was found to be 02120583M (Figure 9) Also abovecalibration range there is limited linearity For comparisondetection limit achieved by Ellmanrsquos assay in this experimentwas equal to 382 nM

Validation of method for donepezil measurement wasperformed using standard Ellmanrsquos assay with correlationcoefficient equal to 09895 (Figure 10)

Verifying of donepezil in plasma samples was done aswell Donepezil was spiked in appropriate concentrationsinto pretreated plasma samples and smartphone assay was

R = 098954372 middot absorbance + 682ΔColor intensity =

003125 120583M

00625 120583M

0125 120583M

025 120583M

05 120583M

01 02 0300Absorbance (Ellmanrsquos assay)

10

15

20

ΔC

olor

inte

nsity

(sm

artp

hone

assa

y)

Figure 10 Validation of donepezil assay compared to standardEllmanrsquos assay Error bars for smartphone assay represent standarderror of the mean and for Ellmanrsquos assay standard deviation for 119899 =3

003125 120583M

00625 120583M

0125 120583M

05 120583M

R = 09924ΔColor intensity (plasma) = 143 middot ΔColor intensity (standard) minus 044

025 120583M

10

15

20

25

30

ΔC

olor

inte

nsity

(spi

ked

plas

ma s

ampl

es)

15 2010ΔColor intensity (standard donepezil solution)

Figure 11 Verifying of donepezil assay in plasma samples comparedto standard donepezil solution Error bars represent standard errorof the mean for 119899 = 3

performed (Figure 11) PBS buffer was also replaced by waterfor reason mentioned in the principle based on pH changewhere a strong buffering could interfere It appears that bothmeasurements are in a good correlation

35 Organic Solvents Because AChE activity can be reducedby organic solvents in small concentration [37] we decidedto consider them as possible interferents in the AChEbased assay For this purpose ethanol isopropyl alcoholand dimethyl sulfoxide were tested all in 5 concentrationInfluence of DMSO to AChE activity has been investigatedbefore and results showed that DMSO slightly decreasesenzyme activity which was confirmed in our work [38]

6 International Journal of Analytical Chemistry

Table 1 Comparison of presented assay with Ellmanrsquos assay and literature

Detection limit Fabrication time Assay time Necessary equipmentPresented smartphoneassay

Galanthamine 1491 nMDonepezil 223 nM Aprox 1 hour 15min Smartphone

Standard Ellmanrsquos assay Galanthamine 183 nMDonepezil 382 nM NA 10min Spectrophotometer

Spectrophotometricassay [21] Galanthamine 005 nM Aprox 35 hours Aprox 20mn Spectrophotometer

Potentiometric assay[22] Galanthamine 54 nM Aprox 10 hours Aprox 10min Electrodes

potentiometerSquare-wavevoltammetry [23] Donepezil 151 nM NA Aprox 20min Electrodes

electrochemical analyser

G 25120583MD 05120583MNo inhibition EtOH IsoPr DMSO0

5

10

15

ΔC

olor

inte

nsity

Figure 12 Method interferences by organic solvents EtOH =ethanol IsoPr = isopropyl alcohol DMSO = dimethyl sulfoxideD 05 120583M = donepezil in 05120583M concentration and G 25 120583M= galanthamine in 25 120583M concentration Error bars representstandard error of the mean for 119899 = 3

Ethanol has been reported to decrease AChE activity [37 39]however there is evidence that at low concentration it canenhance enzyme activity [38 40] No activity change againstuninhibited enzyme was observed in our work Isopropylalcohol as well as ethanol can drop AChE activity [41]which is in agreement with our results To compare usedinhibitors donepezil and galanthamine are included in graph(Figure 12)

4 Conclusion

Here the presented method proved its ability for deter-mination of neurotoxic compounds with promising limitsof detection for galanthamine and donepezil 149 nM and223 nM respectively Assay was successfully validated tothe standard Ellmanrsquos spectrophotometric test and showedfeasibility of measurement in plasma samples Additionallyno specialized equipment and trained personnel are requiredcombined with low cost portability easy preparation andminiaturization are considered as big advantages of the here

invented method Comparison of presented method withstandard Ellmanrsquos assay and literature is given in Table 1

Competing Interests

The authors declare that there is no conflict of interests withregard to the publication of this paper

Acknowledgments

A Long-Term Organization Development Plan 1011 (Facultyof Military Health Sciences University of Defense CzechRepublic) is gratefully acknowledged

References

[1] M Pohanka ldquoCholinesterases a target of pharmacology andtoxicologyrdquo Biomedical Papers vol 155 no 3 pp 219ndash230 2011

[2] M Pohanka ldquoBiosensors containing acetylcholinesterase andbutyrylcholinesterase as recognition tools for detection ofvarious compoundsrdquo Chemical Papers vol 69 no 1 pp 4ndash162015

[3] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961

[4] P M George and M H Abernethy ldquoImproved Ellman proce-dure for erythrocyte cholinesteraserdquo Clinical Chemistry vol 29no 2 pp 365ndash368 1983

[5] V Gorun I Proinov V Baltescu G Balaban and O BarzuldquoModified Ellman procedure for assay of cholinesterases incrude enzymatic preparationsrdquoAnalytical Biochemistry vol 86no 1 pp 324ndash326 1978

[6] M Pohanka ldquoVoltammetric assay of butyrylcholinesterase inplasma samples and its comparison to the standard spectropho-tometric testrdquo Talanta vol 119 pp 412ndash416 2014

[7] A Kostelnik A Cegan and M Pohanka ldquoElectrochemicaldetermination of activity of acetylcholinesterase immobilizedon magnetic particlesrdquo International Journal of ElectrochemicalScience vol 11 no 6 pp 4840ndash4849 2016

[8] F Morıs-Varas A Shah J Aikens N P Nadkarni J D Rozzelland D C Demirjian ldquoVisualization of enzyme-catalyzed reac-tions using pH indicators rapid screening of hydrolase librariesand estimation of the enantioselectivityrdquo Bioorganic andMedic-inal Chemistry vol 7 no 10 pp 2183ndash2188 1999

International Journal of Analytical Chemistry 7

[9] A Kostelnik A Cegan and M Pohanka ldquoColor change ofphenol red by integrated smart phone camera as a tool for thedetermination of neurotoxic compoundsrdquo Sensors vol 16 no9 article 1212 2016

[10] S Timur and A Telefoncu ldquoAcetylcholinesterase (AChE) elec-trodes based on gelatin and chitosan matrices for the pesticidedetectionrdquo Artificial Cells Blood Substitutes and Biotechnologyvol 32 no 3 pp 427ndash442 2004

[11] J Zhang J Zhang F Zhang et al ldquoGraphene oxide as a matrixfor enzyme immobilizationrdquo Langmuir vol 26 no 9 pp 6083ndash6085 2010

[12] M Mogharabi N Nassiri-Koopaei M Bozorgi-KoushalshahiN Nafissi-Varcheh G Bagherzadeh and M A FaramarzildquoImmobilization of laccase in alginate-gelatin mixed gel anddecolorization of synthetic dyesrdquo Bioinorganic Chemistry andApplications vol 2012 Article ID 823830 6 pages 2012

[13] R A Sheldon ldquoEnzyme immobilization the quest for optimumperformancerdquo Advanced Synthesis and Catalysis vol 349 no 8-9 pp 1289ndash1307 2007

[14] M Pohanka ldquoCholinesterases in biorecognition and biosensorsconstruction a reviewrdquo Analytical Letters vol 46 no 12 pp1849ndash1868 2013

[15] M Pohanka ldquoAcetylcholinesterase based dipsticks with indoxy-lacetate as a substrate for assay of organophosphates andcarbamatesrdquo Analytical Letters vol 45 no 4 pp 367ndash374 2012

[16] Y Zheng Z Liu Y Jing J Li and H Zhan ldquoAn acetyl-cholinesterase biosensor based on ionic liquid functionalizedgraphene-gelatin-modified electrode for sensitive detection ofpesticidesrdquo Sensors and Actuators B Chemical vol 210 pp389ndash397 2015

[17] K Grudpan S D Kolev S Lapanantnopakhun I D McKelvieand W Wongwilai ldquoApplications of everyday IT and commu-nications devices in modern analytical chemistry a reviewrdquoTalanta vol 136 pp 84ndash94 2015

[18] A Garcıa M M Erenas E D Marinetto et al ldquoMobile phoneplatform as portable chemical analyzerrdquo Sensors and ActuatorsB Chemical vol 156 no 1 pp 350ndash359 2011

[19] Y Lu W Shi J Qin and B Lin ldquoLow cost portable detectionof gold nanoparticle-labeled microfluidic immunoassay withcamera cell phonerdquo Electrophoresis vol 30 no 4 pp 579ndash5822009

[20] M Pohanka ldquoPhotography by cameras integrated in smart-phones as a tool for analytical chemistry represented by anbutyrylcholinesterase activity assayrdquo Sensors vol 15 no 6 pp13752ndash13762 2015

[21] B J White J A Legako and H J Harmon ldquoSpectrophoto-metric detection of cholinesterase inhibitors with an integratedacetyl-butyrylcholinesterase surfacerdquo Sensors and Actuators BChemical vol 89 no 1-2 pp 107ndash111 2003

[22] M Cuartero M S Garcıa F Garcıa-Canovas and J AOrtuno ldquoNew approach for the potentiometric-enzymatic assayof reversible- competitive enzyme inhibitors Application toacetylcholinesterase inhibitor galantamine and its determina-tion in pharmaceuticals and human urinerdquo Talanta vol 110 pp8ndash14 2013

[23] A Golcu and S A Ozkan ldquoElectroanalytical determination ofdonepezil HCl in tablets and human serum by differential pulseand osteryoung square wave voltammetry at a glassy carbonelectroderdquo Pharmazie vol 61 no 9 pp 760ndash765 2006

[24] Z Xu S Yao Y Wei et al ldquoMonitoring enzyme reaction andscreening of inhibitors of acetylcholinesterase by quantitative

matrix-assisted laser desorptionionization fourier transformmass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 19 no 12 pp 1849ndash1855 2008

[25] TWilleHThiermann and FWorek ldquoEffect of different bufferson kinetic properties of human acetylcholinesterase and theinteraction with organophosphates and oximesrdquo Archives ofToxicology vol 85 no 3 pp 193ndash198 2011

[26] L Shaonan X Xianchuan Z Guonian and T Yajun ldquoKineticcharacters and resistance to inhibition of crude and puri-fied brain acetylcholinesterase of three freshwater fishes byorganophosphatesrdquo Aquatic Toxicology vol 68 no 4 pp 293ndash299 2004

[27] H Jiang S Liu P Zhao and C Pope ldquoRecombinant expres-sion and biochemical characterization of the catalytic domainof acetylcholinesterase-1 from the African malaria mosquitoAnopheles gambiaerdquo Insect Biochemistry andMolecular Biologyvol 39 no 9 pp 646ndash653 2009

[28] A E Boyd A B Marnett L Wong and P Taylor ldquoProbingthe active center gorge of acetylcholinesterase by fluorophoreslinked to substituted cysteinesrdquoThe Journal of Biological Chem-istry vol 275 no 29 pp 22401ndash22408 2000

[29] M Pohanka M Hrabinova J Fusek et al ldquoElectrochemicalbiosensor based on acetylcholinesterase and indoxylacetatefor assay of neurotoxic compounds represented by paraoxonrdquoInternational Journal of Electrochemical Science vol 7 no 1 pp50ndash57 2012

[30] J I da Silva M C de Moraes L C C Vieira A G CorreaQ B Cass and C L Cardoso ldquoAcetylcholinesterase capillaryenzyme reactor for screening and characterization of selectiveinhibitorsrdquo Journal of Pharmaceutical and Biomedical Analysisvol 73 pp 44ndash52 2013

[31] D R Liston J A Nielsen A Villalobos et al ldquoPharmacologyof selective acetylcholinesterase inhibitors implications for usein Alzheimerrsquos diseaserdquo European Journal of Pharmacology vol486 no 1 pp 9ndash17 2004

[32] N Yasui-Furukori R Furuya T Takahata and T TateishildquoDetermination of donepezil an acetylcholinesterase inhibitorin human plasma by high-performance liquid chromatographywith ultraviolet absorbance detectionrdquo Journal of Chromatog-raphy B Analytical Technologies in the Biomedical and LifeSciences vol 768 no 2 pp 261ndash265 2002

[33] C Apostolou Y Dotsikas C Kousoulos and Y L LoukasldquoQuantitative determination of donepezil in human plasma byliquid chromatographytandem mass spectrometry employingan automated liquid-liquid extraction based on 96-well formatplates Application to a bioequivalence studyrdquo Journal of Chro-matography B vol 848 no 2 pp 239ndash244 2007

[34] E M Ghoneim M A El-Attar and M M Ghoneim ldquoDeter-mination of donepezil hydrochloride in pharmaceutical formu-lation and human serum by square-wave adsorptive cathodicstripping voltammetryrdquo Chemia Analityczna vol 54 no 3 pp389ndash402 2009

[35] M Yasir and U Sara ldquoDevelopment of UV spectrophotometricmethod for the analysis of acetylcholinesterase inhibitorrdquo Inter-national Journal of Pharmacy and Pharmaceutical Sciences vol6 no 9 pp 128ndash131 2014

[36] C Patel N Patel and C Kothari ldquoQuantitative determina-tion of donepezil hydrochloride by a simple and accuratesynchronous spectrofluorimetric method in human plasmardquoJournal of Young Pharmacists vol 6 no 4 pp 47ndash50 2014

[37] M Pohanka J Fusek V Adam and R Kizek ldquoCarbofuranassay using gelatin based biosensor with acetylcholinesterase as

8 International Journal of Analytical Chemistry

a recognition elementrdquo International Journal of ElectrochemicalScience vol 8 no 1 pp 71ndash79 2013

[38] T Montesinos S Perez-Munguia F Valdez and J-L MartyldquoDisposable cholinesterase biosensor for the detection of pes-ticides in water-miscible organic solventsrdquo Analytica ChimicaActa vol 431 no 2 pp 231ndash237 2001

[39] S Andreescu T Noguer V Magearu and J-L Marty ldquoScreen-printed electrode based on ache for the detection of pesticidesin presence of organic solventsrdquo Talanta vol 57 no 1 pp 169ndash176 2002

[40] S SookW Portia and C Chang-Hwei ldquoBiochemical studies ofthe actions of ethanol on acetylcholinesterase activity ethnol-enzyme-solvent interactionrdquo International Journal of Biochem-istry vol 23 no 2 pp 169ndash174 1991

[41] A D C Obregon M R C Schetinger M M Correa etal ldquoEffects per se of organic solvents in the cerebral acetyl-cholinesterase of ratsrdquo Neurochemical Research vol 30 no 3pp 379ndash384 2005

International Journal of Analytical Chemistry 3

(a) (b)

Figure 1 Tube shaped holder printed by 3D print technology (a) and the holder with a smartphone adjusted on the hole to providephotographs by an integrated camera

Acetylcholine + water choline + acetic acid

AChE

Inhibitor

Figure 2 pH sensitive method using AChE principle

RcGcBc

minus3 minus1minus2 10log c gelatin ()

0

50

100

150

ΔC

olor

inte

nsity

Figure 3 Gelatin optimization Drop in ΔColor intensity indicatesthat pH strip did not change into red color Error bars representstandard error of the mean for 119899 = 3 Rc = red channel Gc = greenchannel and Bc = blue channel

change into red was held while using gelatin in 1 and lessconcentration but for further measurement 1 gelatin waschosen just for the better stabilization effect for AChE

30

35

40

45

50

ΔC

olor

inte

nsity

5 10 15 20 25 300Time (min)

Figure 4 Optimization of pH strip incubation time with ATChClas a substrate Error bars represent standard error of the mean for 119899= 3

32 Time Optimization Strip with AChE and 1 gelatin wasincubated with 1mM AChCl Incubation time with AChClwas observed in 5min intervals from 0 to 30min Thebiggest color change was held up to 15min Over this timecolor change was not significant for longer incubation time(Figure 4)

4 International Journal of Analytical Chemistry

05 1000c ACh (mM)

2

4

6

8

10

ΔC

olor

inte

nsity

Figure 5 Saturation curve for AChE and AChCl as a substrateError bars represent standard error of the mean for 119899 = 3

33 Substrate Measurement Saturation curve for AChE andAChCl as a substrate was measured in concentration rangefrom 0031 to 10mM and 119870

119872value was calculated as

described above to 5426 120583M (Figure 5) while 739 120583M wasreported by Xu and coworkers [24] 119870

119872value however

depends on type of used buffer as proved byWille et al usinghuman AChE isolated from erythrocytes and achieved 119870

119872

equal to 714 120583M in MOPS buffer 982 120583M in PBS buffer01mM in Tyrode buffer and 0122mM in Tris buffer whenpH was set to 74 [25] There are also big differences betweenorganisms as shownby Shaonan et al whoworkedwithAChEisolated from fish species and found out values above 01mM[26] and by Jiang et al who reported119870

119872to be 6385 120583Mwhen

measured with mosquito AChE [27] Recombinant enzymesexhibit slightly higher 119870

119872values compared to wild types as

showed in experiments with mice AChE carried out by Boydet al who found out119870

119872to be 46 120583M in wild type and 58120583M

for recombinant AChE [28]

34 Inhibitors Measurement AChE is sensitive to neurotoxiccompounds like drugs nerve agents or pesticides Some ofthese compounds are widely used in treatment of Alzheimerdisease [29] Galantamine and donepezil can be examples forthe currently available drugs [30] We performed calibrationcurve of galanthamine in concentration range from 0156to 25 120583M with limit of detection calculated to 149 nMand quantification limit of 05 120583M was achieved (Figure 6)Linearity of the assay is limited to 25 120583M when higherconcentrations appeared to be indistinguishable Comparingto the standard Ellmanrsquos assay it is only 10 times higherdetection limit when 183 nM of galanthamine was achievedin our experiment From previously published methodsfor galanthamine measurement based on AChE inhibitionwe can conclude that our method is competitive [21 22]Although detection limits are similar there is advantage infabrication time of pH strip with immobilized AChE whichis not time consuming

R = minus09975

1015 middot log c + 1292

minus10 minus05 05 1000log c galantamine (120583M)

10

15

20

ΔC

olor

inte

nsity

ΔColor intensity =

Figure 6 Calibration curve of galanthamine concentration is givenin logarithm Error bars represent standard error of the mean for 119899= 3

R = 09922ΔColor intensity = 3172 middot absorbance + 198

015625 120583M

03125 120583M

0625 120583M

125 120583M

25 120583M10

15

20

ΔC

olor

inte

nsity

(sm

artp

hone

assa

y)

03 04 05 0602Absorbance (Ellmanrsquos assay)

Figure 7 Validation of galanthamine assay compared to standardEllmanrsquos assay Error bars for smartphone assay represent standarderror of the mean and for Ellmanrsquos assay standard deviation for 119899 =3

Validation of method for galanthamine measurementwas done using standard Ellmanrsquos assay with correlationcoefficient of 09922 (Figure 7)

Method was verified using murine plasma Plasma sam-ples were pretreated by iso-OMPA selective inhibitor ofbutyrylcholinesterase [31] which naturally occurs in plasmaand has ability to split acetylcholine Then galanthamine wasspiked into plasma samples in appropriate concentrationsand smartphone assay has been performed (Figure 8) Formeasurement with plasma PBS buffer had to be replaced bywater because plasma strongly buffered itself then PBS bufferwas not required

In literature there are plenty of references about determi-nation of donepezil by chromatography techniques [32 33]Likewise there are electrochemical methods for donepezilmeasurementwith similar detection limits as ourmethod [23

International Journal of Analytical Chemistry 5

015625 120583M

03125 120583M

0625 120583M

125 120583M

25 120583M

R = 09673ΔColor intensity (plasma) = 136 middot ΔColor intensity (standard) + 030

15 2010ΔColor intensity (standard galanthamine solution)

15

20

25

30

35

ΔC

olor

inte

nsity

(spi

ked

plas

ma s

ampl

es)

Figure 8 Verifying of galanthamine assay in plasma samplescompared to standard galanthamine solution Error bars representstandard error of the mean for 119899 = 3

R = minus09844minus844 middot log c + 438

minus10 minus05minus15 00log c donepezil (120583M)

10

15

20

ΔC

olor

inte

nsity

ΔColor intensity =

Figure 9 Calibration curve of donepezil concentration is given inlogarithm Error bars represent standard error of the mean for 119899 =3

34] and some spectrophotometricmethods for quantificationof donepezil in pharmaceuticals or human plasma [35 36]However no evidence about determination of donepezil viainhibition of AChE was found Donepezil calibration curvewas done in concentration range from 0031 to 050 120583M andlimit of detection equal to 223 nMwas achievedwhile limit ofquantification was found to be 02120583M (Figure 9) Also abovecalibration range there is limited linearity For comparisondetection limit achieved by Ellmanrsquos assay in this experimentwas equal to 382 nM

Validation of method for donepezil measurement wasperformed using standard Ellmanrsquos assay with correlationcoefficient equal to 09895 (Figure 10)

Verifying of donepezil in plasma samples was done aswell Donepezil was spiked in appropriate concentrationsinto pretreated plasma samples and smartphone assay was

R = 098954372 middot absorbance + 682ΔColor intensity =

003125 120583M

00625 120583M

0125 120583M

025 120583M

05 120583M

01 02 0300Absorbance (Ellmanrsquos assay)

10

15

20

ΔC

olor

inte

nsity

(sm

artp

hone

assa

y)

Figure 10 Validation of donepezil assay compared to standardEllmanrsquos assay Error bars for smartphone assay represent standarderror of the mean and for Ellmanrsquos assay standard deviation for 119899 =3

003125 120583M

00625 120583M

0125 120583M

05 120583M

R = 09924ΔColor intensity (plasma) = 143 middot ΔColor intensity (standard) minus 044

025 120583M

10

15

20

25

30

ΔC

olor

inte

nsity

(spi

ked

plas

ma s

ampl

es)

15 2010ΔColor intensity (standard donepezil solution)

Figure 11 Verifying of donepezil assay in plasma samples comparedto standard donepezil solution Error bars represent standard errorof the mean for 119899 = 3

performed (Figure 11) PBS buffer was also replaced by waterfor reason mentioned in the principle based on pH changewhere a strong buffering could interfere It appears that bothmeasurements are in a good correlation

35 Organic Solvents Because AChE activity can be reducedby organic solvents in small concentration [37] we decidedto consider them as possible interferents in the AChEbased assay For this purpose ethanol isopropyl alcoholand dimethyl sulfoxide were tested all in 5 concentrationInfluence of DMSO to AChE activity has been investigatedbefore and results showed that DMSO slightly decreasesenzyme activity which was confirmed in our work [38]

6 International Journal of Analytical Chemistry

Table 1 Comparison of presented assay with Ellmanrsquos assay and literature

Detection limit Fabrication time Assay time Necessary equipmentPresented smartphoneassay

Galanthamine 1491 nMDonepezil 223 nM Aprox 1 hour 15min Smartphone

Standard Ellmanrsquos assay Galanthamine 183 nMDonepezil 382 nM NA 10min Spectrophotometer

Spectrophotometricassay [21] Galanthamine 005 nM Aprox 35 hours Aprox 20mn Spectrophotometer

Potentiometric assay[22] Galanthamine 54 nM Aprox 10 hours Aprox 10min Electrodes

potentiometerSquare-wavevoltammetry [23] Donepezil 151 nM NA Aprox 20min Electrodes

electrochemical analyser

G 25120583MD 05120583MNo inhibition EtOH IsoPr DMSO0

5

10

15

ΔC

olor

inte

nsity

Figure 12 Method interferences by organic solvents EtOH =ethanol IsoPr = isopropyl alcohol DMSO = dimethyl sulfoxideD 05 120583M = donepezil in 05120583M concentration and G 25 120583M= galanthamine in 25 120583M concentration Error bars representstandard error of the mean for 119899 = 3

Ethanol has been reported to decrease AChE activity [37 39]however there is evidence that at low concentration it canenhance enzyme activity [38 40] No activity change againstuninhibited enzyme was observed in our work Isopropylalcohol as well as ethanol can drop AChE activity [41]which is in agreement with our results To compare usedinhibitors donepezil and galanthamine are included in graph(Figure 12)

4 Conclusion

Here the presented method proved its ability for deter-mination of neurotoxic compounds with promising limitsof detection for galanthamine and donepezil 149 nM and223 nM respectively Assay was successfully validated tothe standard Ellmanrsquos spectrophotometric test and showedfeasibility of measurement in plasma samples Additionallyno specialized equipment and trained personnel are requiredcombined with low cost portability easy preparation andminiaturization are considered as big advantages of the here

invented method Comparison of presented method withstandard Ellmanrsquos assay and literature is given in Table 1

Competing Interests

The authors declare that there is no conflict of interests withregard to the publication of this paper

Acknowledgments

A Long-Term Organization Development Plan 1011 (Facultyof Military Health Sciences University of Defense CzechRepublic) is gratefully acknowledged

References

[1] M Pohanka ldquoCholinesterases a target of pharmacology andtoxicologyrdquo Biomedical Papers vol 155 no 3 pp 219ndash230 2011

[2] M Pohanka ldquoBiosensors containing acetylcholinesterase andbutyrylcholinesterase as recognition tools for detection ofvarious compoundsrdquo Chemical Papers vol 69 no 1 pp 4ndash162015

[3] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961

[4] P M George and M H Abernethy ldquoImproved Ellman proce-dure for erythrocyte cholinesteraserdquo Clinical Chemistry vol 29no 2 pp 365ndash368 1983

[5] V Gorun I Proinov V Baltescu G Balaban and O BarzuldquoModified Ellman procedure for assay of cholinesterases incrude enzymatic preparationsrdquoAnalytical Biochemistry vol 86no 1 pp 324ndash326 1978

[6] M Pohanka ldquoVoltammetric assay of butyrylcholinesterase inplasma samples and its comparison to the standard spectropho-tometric testrdquo Talanta vol 119 pp 412ndash416 2014

[7] A Kostelnik A Cegan and M Pohanka ldquoElectrochemicaldetermination of activity of acetylcholinesterase immobilizedon magnetic particlesrdquo International Journal of ElectrochemicalScience vol 11 no 6 pp 4840ndash4849 2016

[8] F Morıs-Varas A Shah J Aikens N P Nadkarni J D Rozzelland D C Demirjian ldquoVisualization of enzyme-catalyzed reac-tions using pH indicators rapid screening of hydrolase librariesand estimation of the enantioselectivityrdquo Bioorganic andMedic-inal Chemistry vol 7 no 10 pp 2183ndash2188 1999

International Journal of Analytical Chemistry 7

[9] A Kostelnik A Cegan and M Pohanka ldquoColor change ofphenol red by integrated smart phone camera as a tool for thedetermination of neurotoxic compoundsrdquo Sensors vol 16 no9 article 1212 2016

[10] S Timur and A Telefoncu ldquoAcetylcholinesterase (AChE) elec-trodes based on gelatin and chitosan matrices for the pesticidedetectionrdquo Artificial Cells Blood Substitutes and Biotechnologyvol 32 no 3 pp 427ndash442 2004

[11] J Zhang J Zhang F Zhang et al ldquoGraphene oxide as a matrixfor enzyme immobilizationrdquo Langmuir vol 26 no 9 pp 6083ndash6085 2010

[12] M Mogharabi N Nassiri-Koopaei M Bozorgi-KoushalshahiN Nafissi-Varcheh G Bagherzadeh and M A FaramarzildquoImmobilization of laccase in alginate-gelatin mixed gel anddecolorization of synthetic dyesrdquo Bioinorganic Chemistry andApplications vol 2012 Article ID 823830 6 pages 2012

[13] R A Sheldon ldquoEnzyme immobilization the quest for optimumperformancerdquo Advanced Synthesis and Catalysis vol 349 no 8-9 pp 1289ndash1307 2007

[14] M Pohanka ldquoCholinesterases in biorecognition and biosensorsconstruction a reviewrdquo Analytical Letters vol 46 no 12 pp1849ndash1868 2013

[15] M Pohanka ldquoAcetylcholinesterase based dipsticks with indoxy-lacetate as a substrate for assay of organophosphates andcarbamatesrdquo Analytical Letters vol 45 no 4 pp 367ndash374 2012

[16] Y Zheng Z Liu Y Jing J Li and H Zhan ldquoAn acetyl-cholinesterase biosensor based on ionic liquid functionalizedgraphene-gelatin-modified electrode for sensitive detection ofpesticidesrdquo Sensors and Actuators B Chemical vol 210 pp389ndash397 2015

[17] K Grudpan S D Kolev S Lapanantnopakhun I D McKelvieand W Wongwilai ldquoApplications of everyday IT and commu-nications devices in modern analytical chemistry a reviewrdquoTalanta vol 136 pp 84ndash94 2015

[18] A Garcıa M M Erenas E D Marinetto et al ldquoMobile phoneplatform as portable chemical analyzerrdquo Sensors and ActuatorsB Chemical vol 156 no 1 pp 350ndash359 2011

[19] Y Lu W Shi J Qin and B Lin ldquoLow cost portable detectionof gold nanoparticle-labeled microfluidic immunoassay withcamera cell phonerdquo Electrophoresis vol 30 no 4 pp 579ndash5822009

[20] M Pohanka ldquoPhotography by cameras integrated in smart-phones as a tool for analytical chemistry represented by anbutyrylcholinesterase activity assayrdquo Sensors vol 15 no 6 pp13752ndash13762 2015

[21] B J White J A Legako and H J Harmon ldquoSpectrophoto-metric detection of cholinesterase inhibitors with an integratedacetyl-butyrylcholinesterase surfacerdquo Sensors and Actuators BChemical vol 89 no 1-2 pp 107ndash111 2003

[22] M Cuartero M S Garcıa F Garcıa-Canovas and J AOrtuno ldquoNew approach for the potentiometric-enzymatic assayof reversible- competitive enzyme inhibitors Application toacetylcholinesterase inhibitor galantamine and its determina-tion in pharmaceuticals and human urinerdquo Talanta vol 110 pp8ndash14 2013

[23] A Golcu and S A Ozkan ldquoElectroanalytical determination ofdonepezil HCl in tablets and human serum by differential pulseand osteryoung square wave voltammetry at a glassy carbonelectroderdquo Pharmazie vol 61 no 9 pp 760ndash765 2006

[24] Z Xu S Yao Y Wei et al ldquoMonitoring enzyme reaction andscreening of inhibitors of acetylcholinesterase by quantitative

matrix-assisted laser desorptionionization fourier transformmass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 19 no 12 pp 1849ndash1855 2008

[25] TWilleHThiermann and FWorek ldquoEffect of different bufferson kinetic properties of human acetylcholinesterase and theinteraction with organophosphates and oximesrdquo Archives ofToxicology vol 85 no 3 pp 193ndash198 2011

[26] L Shaonan X Xianchuan Z Guonian and T Yajun ldquoKineticcharacters and resistance to inhibition of crude and puri-fied brain acetylcholinesterase of three freshwater fishes byorganophosphatesrdquo Aquatic Toxicology vol 68 no 4 pp 293ndash299 2004

[27] H Jiang S Liu P Zhao and C Pope ldquoRecombinant expres-sion and biochemical characterization of the catalytic domainof acetylcholinesterase-1 from the African malaria mosquitoAnopheles gambiaerdquo Insect Biochemistry andMolecular Biologyvol 39 no 9 pp 646ndash653 2009

[28] A E Boyd A B Marnett L Wong and P Taylor ldquoProbingthe active center gorge of acetylcholinesterase by fluorophoreslinked to substituted cysteinesrdquoThe Journal of Biological Chem-istry vol 275 no 29 pp 22401ndash22408 2000

[29] M Pohanka M Hrabinova J Fusek et al ldquoElectrochemicalbiosensor based on acetylcholinesterase and indoxylacetatefor assay of neurotoxic compounds represented by paraoxonrdquoInternational Journal of Electrochemical Science vol 7 no 1 pp50ndash57 2012

[30] J I da Silva M C de Moraes L C C Vieira A G CorreaQ B Cass and C L Cardoso ldquoAcetylcholinesterase capillaryenzyme reactor for screening and characterization of selectiveinhibitorsrdquo Journal of Pharmaceutical and Biomedical Analysisvol 73 pp 44ndash52 2013

[31] D R Liston J A Nielsen A Villalobos et al ldquoPharmacologyof selective acetylcholinesterase inhibitors implications for usein Alzheimerrsquos diseaserdquo European Journal of Pharmacology vol486 no 1 pp 9ndash17 2004

[32] N Yasui-Furukori R Furuya T Takahata and T TateishildquoDetermination of donepezil an acetylcholinesterase inhibitorin human plasma by high-performance liquid chromatographywith ultraviolet absorbance detectionrdquo Journal of Chromatog-raphy B Analytical Technologies in the Biomedical and LifeSciences vol 768 no 2 pp 261ndash265 2002

[33] C Apostolou Y Dotsikas C Kousoulos and Y L LoukasldquoQuantitative determination of donepezil in human plasma byliquid chromatographytandem mass spectrometry employingan automated liquid-liquid extraction based on 96-well formatplates Application to a bioequivalence studyrdquo Journal of Chro-matography B vol 848 no 2 pp 239ndash244 2007

[34] E M Ghoneim M A El-Attar and M M Ghoneim ldquoDeter-mination of donepezil hydrochloride in pharmaceutical formu-lation and human serum by square-wave adsorptive cathodicstripping voltammetryrdquo Chemia Analityczna vol 54 no 3 pp389ndash402 2009

[35] M Yasir and U Sara ldquoDevelopment of UV spectrophotometricmethod for the analysis of acetylcholinesterase inhibitorrdquo Inter-national Journal of Pharmacy and Pharmaceutical Sciences vol6 no 9 pp 128ndash131 2014

[36] C Patel N Patel and C Kothari ldquoQuantitative determina-tion of donepezil hydrochloride by a simple and accuratesynchronous spectrofluorimetric method in human plasmardquoJournal of Young Pharmacists vol 6 no 4 pp 47ndash50 2014

[37] M Pohanka J Fusek V Adam and R Kizek ldquoCarbofuranassay using gelatin based biosensor with acetylcholinesterase as

8 International Journal of Analytical Chemistry

a recognition elementrdquo International Journal of ElectrochemicalScience vol 8 no 1 pp 71ndash79 2013

[38] T Montesinos S Perez-Munguia F Valdez and J-L MartyldquoDisposable cholinesterase biosensor for the detection of pes-ticides in water-miscible organic solventsrdquo Analytica ChimicaActa vol 431 no 2 pp 231ndash237 2001

[39] S Andreescu T Noguer V Magearu and J-L Marty ldquoScreen-printed electrode based on ache for the detection of pesticidesin presence of organic solventsrdquo Talanta vol 57 no 1 pp 169ndash176 2002

[40] S SookW Portia and C Chang-Hwei ldquoBiochemical studies ofthe actions of ethanol on acetylcholinesterase activity ethnol-enzyme-solvent interactionrdquo International Journal of Biochem-istry vol 23 no 2 pp 169ndash174 1991

[41] A D C Obregon M R C Schetinger M M Correa etal ldquoEffects per se of organic solvents in the cerebral acetyl-cholinesterase of ratsrdquo Neurochemical Research vol 30 no 3pp 379ndash384 2005

4 International Journal of Analytical Chemistry

05 1000c ACh (mM)

2

4

6

8

10

ΔC

olor

inte

nsity

Figure 5 Saturation curve for AChE and AChCl as a substrateError bars represent standard error of the mean for 119899 = 3

33 Substrate Measurement Saturation curve for AChE andAChCl as a substrate was measured in concentration rangefrom 0031 to 10mM and 119870

119872value was calculated as

described above to 5426 120583M (Figure 5) while 739 120583M wasreported by Xu and coworkers [24] 119870

119872value however

depends on type of used buffer as proved byWille et al usinghuman AChE isolated from erythrocytes and achieved 119870

119872

equal to 714 120583M in MOPS buffer 982 120583M in PBS buffer01mM in Tyrode buffer and 0122mM in Tris buffer whenpH was set to 74 [25] There are also big differences betweenorganisms as shownby Shaonan et al whoworkedwithAChEisolated from fish species and found out values above 01mM[26] and by Jiang et al who reported119870

119872to be 6385 120583Mwhen

measured with mosquito AChE [27] Recombinant enzymesexhibit slightly higher 119870

119872values compared to wild types as

showed in experiments with mice AChE carried out by Boydet al who found out119870

119872to be 46 120583M in wild type and 58120583M

for recombinant AChE [28]

34 Inhibitors Measurement AChE is sensitive to neurotoxiccompounds like drugs nerve agents or pesticides Some ofthese compounds are widely used in treatment of Alzheimerdisease [29] Galantamine and donepezil can be examples forthe currently available drugs [30] We performed calibrationcurve of galanthamine in concentration range from 0156to 25 120583M with limit of detection calculated to 149 nMand quantification limit of 05 120583M was achieved (Figure 6)Linearity of the assay is limited to 25 120583M when higherconcentrations appeared to be indistinguishable Comparingto the standard Ellmanrsquos assay it is only 10 times higherdetection limit when 183 nM of galanthamine was achievedin our experiment From previously published methodsfor galanthamine measurement based on AChE inhibitionwe can conclude that our method is competitive [21 22]Although detection limits are similar there is advantage infabrication time of pH strip with immobilized AChE whichis not time consuming

R = minus09975

1015 middot log c + 1292

minus10 minus05 05 1000log c galantamine (120583M)

10

15

20

ΔC

olor

inte

nsity

ΔColor intensity =

Figure 6 Calibration curve of galanthamine concentration is givenin logarithm Error bars represent standard error of the mean for 119899= 3

R = 09922ΔColor intensity = 3172 middot absorbance + 198

015625 120583M

03125 120583M

0625 120583M

125 120583M

25 120583M10

15

20

ΔC

olor

inte

nsity

(sm

artp

hone

assa

y)

03 04 05 0602Absorbance (Ellmanrsquos assay)

Figure 7 Validation of galanthamine assay compared to standardEllmanrsquos assay Error bars for smartphone assay represent standarderror of the mean and for Ellmanrsquos assay standard deviation for 119899 =3

Validation of method for galanthamine measurementwas done using standard Ellmanrsquos assay with correlationcoefficient of 09922 (Figure 7)

Method was verified using murine plasma Plasma sam-ples were pretreated by iso-OMPA selective inhibitor ofbutyrylcholinesterase [31] which naturally occurs in plasmaand has ability to split acetylcholine Then galanthamine wasspiked into plasma samples in appropriate concentrationsand smartphone assay has been performed (Figure 8) Formeasurement with plasma PBS buffer had to be replaced bywater because plasma strongly buffered itself then PBS bufferwas not required

In literature there are plenty of references about determi-nation of donepezil by chromatography techniques [32 33]Likewise there are electrochemical methods for donepezilmeasurementwith similar detection limits as ourmethod [23

International Journal of Analytical Chemistry 5

015625 120583M

03125 120583M

0625 120583M

125 120583M

25 120583M

R = 09673ΔColor intensity (plasma) = 136 middot ΔColor intensity (standard) + 030

15 2010ΔColor intensity (standard galanthamine solution)

15

20

25

30

35

ΔC

olor

inte

nsity

(spi

ked

plas

ma s

ampl

es)

Figure 8 Verifying of galanthamine assay in plasma samplescompared to standard galanthamine solution Error bars representstandard error of the mean for 119899 = 3

R = minus09844minus844 middot log c + 438

minus10 minus05minus15 00log c donepezil (120583M)

10

15

20

ΔC

olor

inte

nsity

ΔColor intensity =

Figure 9 Calibration curve of donepezil concentration is given inlogarithm Error bars represent standard error of the mean for 119899 =3

34] and some spectrophotometricmethods for quantificationof donepezil in pharmaceuticals or human plasma [35 36]However no evidence about determination of donepezil viainhibition of AChE was found Donepezil calibration curvewas done in concentration range from 0031 to 050 120583M andlimit of detection equal to 223 nMwas achievedwhile limit ofquantification was found to be 02120583M (Figure 9) Also abovecalibration range there is limited linearity For comparisondetection limit achieved by Ellmanrsquos assay in this experimentwas equal to 382 nM

Validation of method for donepezil measurement wasperformed using standard Ellmanrsquos assay with correlationcoefficient equal to 09895 (Figure 10)

Verifying of donepezil in plasma samples was done aswell Donepezil was spiked in appropriate concentrationsinto pretreated plasma samples and smartphone assay was

R = 098954372 middot absorbance + 682ΔColor intensity =

003125 120583M

00625 120583M

0125 120583M

025 120583M

05 120583M

01 02 0300Absorbance (Ellmanrsquos assay)

10

15

20

ΔC

olor

inte

nsity

(sm

artp

hone

assa

y)

Figure 10 Validation of donepezil assay compared to standardEllmanrsquos assay Error bars for smartphone assay represent standarderror of the mean and for Ellmanrsquos assay standard deviation for 119899 =3

003125 120583M

00625 120583M

0125 120583M

05 120583M

R = 09924ΔColor intensity (plasma) = 143 middot ΔColor intensity (standard) minus 044

025 120583M

10

15

20

25

30

ΔC

olor

inte

nsity

(spi

ked

plas

ma s

ampl

es)

15 2010ΔColor intensity (standard donepezil solution)

Figure 11 Verifying of donepezil assay in plasma samples comparedto standard donepezil solution Error bars represent standard errorof the mean for 119899 = 3

performed (Figure 11) PBS buffer was also replaced by waterfor reason mentioned in the principle based on pH changewhere a strong buffering could interfere It appears that bothmeasurements are in a good correlation

35 Organic Solvents Because AChE activity can be reducedby organic solvents in small concentration [37] we decidedto consider them as possible interferents in the AChEbased assay For this purpose ethanol isopropyl alcoholand dimethyl sulfoxide were tested all in 5 concentrationInfluence of DMSO to AChE activity has been investigatedbefore and results showed that DMSO slightly decreasesenzyme activity which was confirmed in our work [38]

6 International Journal of Analytical Chemistry

Table 1 Comparison of presented assay with Ellmanrsquos assay and literature

Detection limit Fabrication time Assay time Necessary equipmentPresented smartphoneassay

Galanthamine 1491 nMDonepezil 223 nM Aprox 1 hour 15min Smartphone

Standard Ellmanrsquos assay Galanthamine 183 nMDonepezil 382 nM NA 10min Spectrophotometer

Spectrophotometricassay [21] Galanthamine 005 nM Aprox 35 hours Aprox 20mn Spectrophotometer

Potentiometric assay[22] Galanthamine 54 nM Aprox 10 hours Aprox 10min Electrodes

potentiometerSquare-wavevoltammetry [23] Donepezil 151 nM NA Aprox 20min Electrodes

electrochemical analyser

G 25120583MD 05120583MNo inhibition EtOH IsoPr DMSO0

5

10

15

ΔC

olor

inte

nsity

Figure 12 Method interferences by organic solvents EtOH =ethanol IsoPr = isopropyl alcohol DMSO = dimethyl sulfoxideD 05 120583M = donepezil in 05120583M concentration and G 25 120583M= galanthamine in 25 120583M concentration Error bars representstandard error of the mean for 119899 = 3

Ethanol has been reported to decrease AChE activity [37 39]however there is evidence that at low concentration it canenhance enzyme activity [38 40] No activity change againstuninhibited enzyme was observed in our work Isopropylalcohol as well as ethanol can drop AChE activity [41]which is in agreement with our results To compare usedinhibitors donepezil and galanthamine are included in graph(Figure 12)

4 Conclusion

Here the presented method proved its ability for deter-mination of neurotoxic compounds with promising limitsof detection for galanthamine and donepezil 149 nM and223 nM respectively Assay was successfully validated tothe standard Ellmanrsquos spectrophotometric test and showedfeasibility of measurement in plasma samples Additionallyno specialized equipment and trained personnel are requiredcombined with low cost portability easy preparation andminiaturization are considered as big advantages of the here

invented method Comparison of presented method withstandard Ellmanrsquos assay and literature is given in Table 1

Competing Interests

The authors declare that there is no conflict of interests withregard to the publication of this paper

Acknowledgments

A Long-Term Organization Development Plan 1011 (Facultyof Military Health Sciences University of Defense CzechRepublic) is gratefully acknowledged

References

[1] M Pohanka ldquoCholinesterases a target of pharmacology andtoxicologyrdquo Biomedical Papers vol 155 no 3 pp 219ndash230 2011

[2] M Pohanka ldquoBiosensors containing acetylcholinesterase andbutyrylcholinesterase as recognition tools for detection ofvarious compoundsrdquo Chemical Papers vol 69 no 1 pp 4ndash162015

[3] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961

[4] P M George and M H Abernethy ldquoImproved Ellman proce-dure for erythrocyte cholinesteraserdquo Clinical Chemistry vol 29no 2 pp 365ndash368 1983

[5] V Gorun I Proinov V Baltescu G Balaban and O BarzuldquoModified Ellman procedure for assay of cholinesterases incrude enzymatic preparationsrdquoAnalytical Biochemistry vol 86no 1 pp 324ndash326 1978

[6] M Pohanka ldquoVoltammetric assay of butyrylcholinesterase inplasma samples and its comparison to the standard spectropho-tometric testrdquo Talanta vol 119 pp 412ndash416 2014

[7] A Kostelnik A Cegan and M Pohanka ldquoElectrochemicaldetermination of activity of acetylcholinesterase immobilizedon magnetic particlesrdquo International Journal of ElectrochemicalScience vol 11 no 6 pp 4840ndash4849 2016

[8] F Morıs-Varas A Shah J Aikens N P Nadkarni J D Rozzelland D C Demirjian ldquoVisualization of enzyme-catalyzed reac-tions using pH indicators rapid screening of hydrolase librariesand estimation of the enantioselectivityrdquo Bioorganic andMedic-inal Chemistry vol 7 no 10 pp 2183ndash2188 1999

International Journal of Analytical Chemistry 7

[9] A Kostelnik A Cegan and M Pohanka ldquoColor change ofphenol red by integrated smart phone camera as a tool for thedetermination of neurotoxic compoundsrdquo Sensors vol 16 no9 article 1212 2016

[10] S Timur and A Telefoncu ldquoAcetylcholinesterase (AChE) elec-trodes based on gelatin and chitosan matrices for the pesticidedetectionrdquo Artificial Cells Blood Substitutes and Biotechnologyvol 32 no 3 pp 427ndash442 2004

[11] J Zhang J Zhang F Zhang et al ldquoGraphene oxide as a matrixfor enzyme immobilizationrdquo Langmuir vol 26 no 9 pp 6083ndash6085 2010

[12] M Mogharabi N Nassiri-Koopaei M Bozorgi-KoushalshahiN Nafissi-Varcheh G Bagherzadeh and M A FaramarzildquoImmobilization of laccase in alginate-gelatin mixed gel anddecolorization of synthetic dyesrdquo Bioinorganic Chemistry andApplications vol 2012 Article ID 823830 6 pages 2012

[13] R A Sheldon ldquoEnzyme immobilization the quest for optimumperformancerdquo Advanced Synthesis and Catalysis vol 349 no 8-9 pp 1289ndash1307 2007

[14] M Pohanka ldquoCholinesterases in biorecognition and biosensorsconstruction a reviewrdquo Analytical Letters vol 46 no 12 pp1849ndash1868 2013

[15] M Pohanka ldquoAcetylcholinesterase based dipsticks with indoxy-lacetate as a substrate for assay of organophosphates andcarbamatesrdquo Analytical Letters vol 45 no 4 pp 367ndash374 2012

[16] Y Zheng Z Liu Y Jing J Li and H Zhan ldquoAn acetyl-cholinesterase biosensor based on ionic liquid functionalizedgraphene-gelatin-modified electrode for sensitive detection ofpesticidesrdquo Sensors and Actuators B Chemical vol 210 pp389ndash397 2015

[17] K Grudpan S D Kolev S Lapanantnopakhun I D McKelvieand W Wongwilai ldquoApplications of everyday IT and commu-nications devices in modern analytical chemistry a reviewrdquoTalanta vol 136 pp 84ndash94 2015

[18] A Garcıa M M Erenas E D Marinetto et al ldquoMobile phoneplatform as portable chemical analyzerrdquo Sensors and ActuatorsB Chemical vol 156 no 1 pp 350ndash359 2011

[19] Y Lu W Shi J Qin and B Lin ldquoLow cost portable detectionof gold nanoparticle-labeled microfluidic immunoassay withcamera cell phonerdquo Electrophoresis vol 30 no 4 pp 579ndash5822009

[20] M Pohanka ldquoPhotography by cameras integrated in smart-phones as a tool for analytical chemistry represented by anbutyrylcholinesterase activity assayrdquo Sensors vol 15 no 6 pp13752ndash13762 2015

[21] B J White J A Legako and H J Harmon ldquoSpectrophoto-metric detection of cholinesterase inhibitors with an integratedacetyl-butyrylcholinesterase surfacerdquo Sensors and Actuators BChemical vol 89 no 1-2 pp 107ndash111 2003

[22] M Cuartero M S Garcıa F Garcıa-Canovas and J AOrtuno ldquoNew approach for the potentiometric-enzymatic assayof reversible- competitive enzyme inhibitors Application toacetylcholinesterase inhibitor galantamine and its determina-tion in pharmaceuticals and human urinerdquo Talanta vol 110 pp8ndash14 2013

[23] A Golcu and S A Ozkan ldquoElectroanalytical determination ofdonepezil HCl in tablets and human serum by differential pulseand osteryoung square wave voltammetry at a glassy carbonelectroderdquo Pharmazie vol 61 no 9 pp 760ndash765 2006

[24] Z Xu S Yao Y Wei et al ldquoMonitoring enzyme reaction andscreening of inhibitors of acetylcholinesterase by quantitative

matrix-assisted laser desorptionionization fourier transformmass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 19 no 12 pp 1849ndash1855 2008

[25] TWilleHThiermann and FWorek ldquoEffect of different bufferson kinetic properties of human acetylcholinesterase and theinteraction with organophosphates and oximesrdquo Archives ofToxicology vol 85 no 3 pp 193ndash198 2011

[26] L Shaonan X Xianchuan Z Guonian and T Yajun ldquoKineticcharacters and resistance to inhibition of crude and puri-fied brain acetylcholinesterase of three freshwater fishes byorganophosphatesrdquo Aquatic Toxicology vol 68 no 4 pp 293ndash299 2004

[27] H Jiang S Liu P Zhao and C Pope ldquoRecombinant expres-sion and biochemical characterization of the catalytic domainof acetylcholinesterase-1 from the African malaria mosquitoAnopheles gambiaerdquo Insect Biochemistry andMolecular Biologyvol 39 no 9 pp 646ndash653 2009

[28] A E Boyd A B Marnett L Wong and P Taylor ldquoProbingthe active center gorge of acetylcholinesterase by fluorophoreslinked to substituted cysteinesrdquoThe Journal of Biological Chem-istry vol 275 no 29 pp 22401ndash22408 2000

[29] M Pohanka M Hrabinova J Fusek et al ldquoElectrochemicalbiosensor based on acetylcholinesterase and indoxylacetatefor assay of neurotoxic compounds represented by paraoxonrdquoInternational Journal of Electrochemical Science vol 7 no 1 pp50ndash57 2012

[30] J I da Silva M C de Moraes L C C Vieira A G CorreaQ B Cass and C L Cardoso ldquoAcetylcholinesterase capillaryenzyme reactor for screening and characterization of selectiveinhibitorsrdquo Journal of Pharmaceutical and Biomedical Analysisvol 73 pp 44ndash52 2013

[31] D R Liston J A Nielsen A Villalobos et al ldquoPharmacologyof selective acetylcholinesterase inhibitors implications for usein Alzheimerrsquos diseaserdquo European Journal of Pharmacology vol486 no 1 pp 9ndash17 2004

[32] N Yasui-Furukori R Furuya T Takahata and T TateishildquoDetermination of donepezil an acetylcholinesterase inhibitorin human plasma by high-performance liquid chromatographywith ultraviolet absorbance detectionrdquo Journal of Chromatog-raphy B Analytical Technologies in the Biomedical and LifeSciences vol 768 no 2 pp 261ndash265 2002

[33] C Apostolou Y Dotsikas C Kousoulos and Y L LoukasldquoQuantitative determination of donepezil in human plasma byliquid chromatographytandem mass spectrometry employingan automated liquid-liquid extraction based on 96-well formatplates Application to a bioequivalence studyrdquo Journal of Chro-matography B vol 848 no 2 pp 239ndash244 2007

[34] E M Ghoneim M A El-Attar and M M Ghoneim ldquoDeter-mination of donepezil hydrochloride in pharmaceutical formu-lation and human serum by square-wave adsorptive cathodicstripping voltammetryrdquo Chemia Analityczna vol 54 no 3 pp389ndash402 2009

[35] M Yasir and U Sara ldquoDevelopment of UV spectrophotometricmethod for the analysis of acetylcholinesterase inhibitorrdquo Inter-national Journal of Pharmacy and Pharmaceutical Sciences vol6 no 9 pp 128ndash131 2014

[36] C Patel N Patel and C Kothari ldquoQuantitative determina-tion of donepezil hydrochloride by a simple and accuratesynchronous spectrofluorimetric method in human plasmardquoJournal of Young Pharmacists vol 6 no 4 pp 47ndash50 2014

[37] M Pohanka J Fusek V Adam and R Kizek ldquoCarbofuranassay using gelatin based biosensor with acetylcholinesterase as

8 International Journal of Analytical Chemistry

a recognition elementrdquo International Journal of ElectrochemicalScience vol 8 no 1 pp 71ndash79 2013

[38] T Montesinos S Perez-Munguia F Valdez and J-L MartyldquoDisposable cholinesterase biosensor for the detection of pes-ticides in water-miscible organic solventsrdquo Analytica ChimicaActa vol 431 no 2 pp 231ndash237 2001

[39] S Andreescu T Noguer V Magearu and J-L Marty ldquoScreen-printed electrode based on ache for the detection of pesticidesin presence of organic solventsrdquo Talanta vol 57 no 1 pp 169ndash176 2002

[40] S SookW Portia and C Chang-Hwei ldquoBiochemical studies ofthe actions of ethanol on acetylcholinesterase activity ethnol-enzyme-solvent interactionrdquo International Journal of Biochem-istry vol 23 no 2 pp 169ndash174 1991

[41] A D C Obregon M R C Schetinger M M Correa etal ldquoEffects per se of organic solvents in the cerebral acetyl-cholinesterase of ratsrdquo Neurochemical Research vol 30 no 3pp 379ndash384 2005

International Journal of Analytical Chemistry 5

015625 120583M

03125 120583M

0625 120583M

125 120583M

25 120583M

R = 09673ΔColor intensity (plasma) = 136 middot ΔColor intensity (standard) + 030

15 2010ΔColor intensity (standard galanthamine solution)

15

20

25

30

35

ΔC

olor

inte

nsity

(spi

ked

plas

ma s

ampl

es)

Figure 8 Verifying of galanthamine assay in plasma samplescompared to standard galanthamine solution Error bars representstandard error of the mean for 119899 = 3

R = minus09844minus844 middot log c + 438

minus10 minus05minus15 00log c donepezil (120583M)

10

15

20

ΔC

olor

inte

nsity

ΔColor intensity =

Figure 9 Calibration curve of donepezil concentration is given inlogarithm Error bars represent standard error of the mean for 119899 =3

34] and some spectrophotometricmethods for quantificationof donepezil in pharmaceuticals or human plasma [35 36]However no evidence about determination of donepezil viainhibition of AChE was found Donepezil calibration curvewas done in concentration range from 0031 to 050 120583M andlimit of detection equal to 223 nMwas achievedwhile limit ofquantification was found to be 02120583M (Figure 9) Also abovecalibration range there is limited linearity For comparisondetection limit achieved by Ellmanrsquos assay in this experimentwas equal to 382 nM

Validation of method for donepezil measurement wasperformed using standard Ellmanrsquos assay with correlationcoefficient equal to 09895 (Figure 10)

Verifying of donepezil in plasma samples was done aswell Donepezil was spiked in appropriate concentrationsinto pretreated plasma samples and smartphone assay was

R = 098954372 middot absorbance + 682ΔColor intensity =

003125 120583M

00625 120583M

0125 120583M

025 120583M

05 120583M

01 02 0300Absorbance (Ellmanrsquos assay)

10

15

20

ΔC

olor

inte

nsity

(sm

artp

hone

assa

y)

Figure 10 Validation of donepezil assay compared to standardEllmanrsquos assay Error bars for smartphone assay represent standarderror of the mean and for Ellmanrsquos assay standard deviation for 119899 =3

003125 120583M

00625 120583M

0125 120583M

05 120583M

R = 09924ΔColor intensity (plasma) = 143 middot ΔColor intensity (standard) minus 044

025 120583M

10

15

20

25

30

ΔC

olor

inte

nsity

(spi

ked

plas

ma s

ampl

es)

15 2010ΔColor intensity (standard donepezil solution)

Figure 11 Verifying of donepezil assay in plasma samples comparedto standard donepezil solution Error bars represent standard errorof the mean for 119899 = 3

performed (Figure 11) PBS buffer was also replaced by waterfor reason mentioned in the principle based on pH changewhere a strong buffering could interfere It appears that bothmeasurements are in a good correlation

35 Organic Solvents Because AChE activity can be reducedby organic solvents in small concentration [37] we decidedto consider them as possible interferents in the AChEbased assay For this purpose ethanol isopropyl alcoholand dimethyl sulfoxide were tested all in 5 concentrationInfluence of DMSO to AChE activity has been investigatedbefore and results showed that DMSO slightly decreasesenzyme activity which was confirmed in our work [38]

6 International Journal of Analytical Chemistry

Table 1 Comparison of presented assay with Ellmanrsquos assay and literature

Detection limit Fabrication time Assay time Necessary equipmentPresented smartphoneassay

Galanthamine 1491 nMDonepezil 223 nM Aprox 1 hour 15min Smartphone

Standard Ellmanrsquos assay Galanthamine 183 nMDonepezil 382 nM NA 10min Spectrophotometer

Spectrophotometricassay [21] Galanthamine 005 nM Aprox 35 hours Aprox 20mn Spectrophotometer

Potentiometric assay[22] Galanthamine 54 nM Aprox 10 hours Aprox 10min Electrodes

potentiometerSquare-wavevoltammetry [23] Donepezil 151 nM NA Aprox 20min Electrodes

electrochemical analyser

G 25120583MD 05120583MNo inhibition EtOH IsoPr DMSO0

5

10

15

ΔC

olor

inte

nsity

Figure 12 Method interferences by organic solvents EtOH =ethanol IsoPr = isopropyl alcohol DMSO = dimethyl sulfoxideD 05 120583M = donepezil in 05120583M concentration and G 25 120583M= galanthamine in 25 120583M concentration Error bars representstandard error of the mean for 119899 = 3

Ethanol has been reported to decrease AChE activity [37 39]however there is evidence that at low concentration it canenhance enzyme activity [38 40] No activity change againstuninhibited enzyme was observed in our work Isopropylalcohol as well as ethanol can drop AChE activity [41]which is in agreement with our results To compare usedinhibitors donepezil and galanthamine are included in graph(Figure 12)

4 Conclusion

Here the presented method proved its ability for deter-mination of neurotoxic compounds with promising limitsof detection for galanthamine and donepezil 149 nM and223 nM respectively Assay was successfully validated tothe standard Ellmanrsquos spectrophotometric test and showedfeasibility of measurement in plasma samples Additionallyno specialized equipment and trained personnel are requiredcombined with low cost portability easy preparation andminiaturization are considered as big advantages of the here

invented method Comparison of presented method withstandard Ellmanrsquos assay and literature is given in Table 1

Competing Interests

The authors declare that there is no conflict of interests withregard to the publication of this paper

Acknowledgments

A Long-Term Organization Development Plan 1011 (Facultyof Military Health Sciences University of Defense CzechRepublic) is gratefully acknowledged

References

[1] M Pohanka ldquoCholinesterases a target of pharmacology andtoxicologyrdquo Biomedical Papers vol 155 no 3 pp 219ndash230 2011

[2] M Pohanka ldquoBiosensors containing acetylcholinesterase andbutyrylcholinesterase as recognition tools for detection ofvarious compoundsrdquo Chemical Papers vol 69 no 1 pp 4ndash162015

[3] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961

[4] P M George and M H Abernethy ldquoImproved Ellman proce-dure for erythrocyte cholinesteraserdquo Clinical Chemistry vol 29no 2 pp 365ndash368 1983

[5] V Gorun I Proinov V Baltescu G Balaban and O BarzuldquoModified Ellman procedure for assay of cholinesterases incrude enzymatic preparationsrdquoAnalytical Biochemistry vol 86no 1 pp 324ndash326 1978

[6] M Pohanka ldquoVoltammetric assay of butyrylcholinesterase inplasma samples and its comparison to the standard spectropho-tometric testrdquo Talanta vol 119 pp 412ndash416 2014

[7] A Kostelnik A Cegan and M Pohanka ldquoElectrochemicaldetermination of activity of acetylcholinesterase immobilizedon magnetic particlesrdquo International Journal of ElectrochemicalScience vol 11 no 6 pp 4840ndash4849 2016

[8] F Morıs-Varas A Shah J Aikens N P Nadkarni J D Rozzelland D C Demirjian ldquoVisualization of enzyme-catalyzed reac-tions using pH indicators rapid screening of hydrolase librariesand estimation of the enantioselectivityrdquo Bioorganic andMedic-inal Chemistry vol 7 no 10 pp 2183ndash2188 1999

International Journal of Analytical Chemistry 7

[9] A Kostelnik A Cegan and M Pohanka ldquoColor change ofphenol red by integrated smart phone camera as a tool for thedetermination of neurotoxic compoundsrdquo Sensors vol 16 no9 article 1212 2016

[10] S Timur and A Telefoncu ldquoAcetylcholinesterase (AChE) elec-trodes based on gelatin and chitosan matrices for the pesticidedetectionrdquo Artificial Cells Blood Substitutes and Biotechnologyvol 32 no 3 pp 427ndash442 2004

[11] J Zhang J Zhang F Zhang et al ldquoGraphene oxide as a matrixfor enzyme immobilizationrdquo Langmuir vol 26 no 9 pp 6083ndash6085 2010

[12] M Mogharabi N Nassiri-Koopaei M Bozorgi-KoushalshahiN Nafissi-Varcheh G Bagherzadeh and M A FaramarzildquoImmobilization of laccase in alginate-gelatin mixed gel anddecolorization of synthetic dyesrdquo Bioinorganic Chemistry andApplications vol 2012 Article ID 823830 6 pages 2012

[13] R A Sheldon ldquoEnzyme immobilization the quest for optimumperformancerdquo Advanced Synthesis and Catalysis vol 349 no 8-9 pp 1289ndash1307 2007

[14] M Pohanka ldquoCholinesterases in biorecognition and biosensorsconstruction a reviewrdquo Analytical Letters vol 46 no 12 pp1849ndash1868 2013

[15] M Pohanka ldquoAcetylcholinesterase based dipsticks with indoxy-lacetate as a substrate for assay of organophosphates andcarbamatesrdquo Analytical Letters vol 45 no 4 pp 367ndash374 2012

[16] Y Zheng Z Liu Y Jing J Li and H Zhan ldquoAn acetyl-cholinesterase biosensor based on ionic liquid functionalizedgraphene-gelatin-modified electrode for sensitive detection ofpesticidesrdquo Sensors and Actuators B Chemical vol 210 pp389ndash397 2015

[17] K Grudpan S D Kolev S Lapanantnopakhun I D McKelvieand W Wongwilai ldquoApplications of everyday IT and commu-nications devices in modern analytical chemistry a reviewrdquoTalanta vol 136 pp 84ndash94 2015

[18] A Garcıa M M Erenas E D Marinetto et al ldquoMobile phoneplatform as portable chemical analyzerrdquo Sensors and ActuatorsB Chemical vol 156 no 1 pp 350ndash359 2011

[19] Y Lu W Shi J Qin and B Lin ldquoLow cost portable detectionof gold nanoparticle-labeled microfluidic immunoassay withcamera cell phonerdquo Electrophoresis vol 30 no 4 pp 579ndash5822009

[20] M Pohanka ldquoPhotography by cameras integrated in smart-phones as a tool for analytical chemistry represented by anbutyrylcholinesterase activity assayrdquo Sensors vol 15 no 6 pp13752ndash13762 2015

[21] B J White J A Legako and H J Harmon ldquoSpectrophoto-metric detection of cholinesterase inhibitors with an integratedacetyl-butyrylcholinesterase surfacerdquo Sensors and Actuators BChemical vol 89 no 1-2 pp 107ndash111 2003

[22] M Cuartero M S Garcıa F Garcıa-Canovas and J AOrtuno ldquoNew approach for the potentiometric-enzymatic assayof reversible- competitive enzyme inhibitors Application toacetylcholinesterase inhibitor galantamine and its determina-tion in pharmaceuticals and human urinerdquo Talanta vol 110 pp8ndash14 2013

[23] A Golcu and S A Ozkan ldquoElectroanalytical determination ofdonepezil HCl in tablets and human serum by differential pulseand osteryoung square wave voltammetry at a glassy carbonelectroderdquo Pharmazie vol 61 no 9 pp 760ndash765 2006

[24] Z Xu S Yao Y Wei et al ldquoMonitoring enzyme reaction andscreening of inhibitors of acetylcholinesterase by quantitative

matrix-assisted laser desorptionionization fourier transformmass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 19 no 12 pp 1849ndash1855 2008

[25] TWilleHThiermann and FWorek ldquoEffect of different bufferson kinetic properties of human acetylcholinesterase and theinteraction with organophosphates and oximesrdquo Archives ofToxicology vol 85 no 3 pp 193ndash198 2011

[26] L Shaonan X Xianchuan Z Guonian and T Yajun ldquoKineticcharacters and resistance to inhibition of crude and puri-fied brain acetylcholinesterase of three freshwater fishes byorganophosphatesrdquo Aquatic Toxicology vol 68 no 4 pp 293ndash299 2004

[27] H Jiang S Liu P Zhao and C Pope ldquoRecombinant expres-sion and biochemical characterization of the catalytic domainof acetylcholinesterase-1 from the African malaria mosquitoAnopheles gambiaerdquo Insect Biochemistry andMolecular Biologyvol 39 no 9 pp 646ndash653 2009

[28] A E Boyd A B Marnett L Wong and P Taylor ldquoProbingthe active center gorge of acetylcholinesterase by fluorophoreslinked to substituted cysteinesrdquoThe Journal of Biological Chem-istry vol 275 no 29 pp 22401ndash22408 2000

[29] M Pohanka M Hrabinova J Fusek et al ldquoElectrochemicalbiosensor based on acetylcholinesterase and indoxylacetatefor assay of neurotoxic compounds represented by paraoxonrdquoInternational Journal of Electrochemical Science vol 7 no 1 pp50ndash57 2012

[30] J I da Silva M C de Moraes L C C Vieira A G CorreaQ B Cass and C L Cardoso ldquoAcetylcholinesterase capillaryenzyme reactor for screening and characterization of selectiveinhibitorsrdquo Journal of Pharmaceutical and Biomedical Analysisvol 73 pp 44ndash52 2013

[31] D R Liston J A Nielsen A Villalobos et al ldquoPharmacologyof selective acetylcholinesterase inhibitors implications for usein Alzheimerrsquos diseaserdquo European Journal of Pharmacology vol486 no 1 pp 9ndash17 2004

[32] N Yasui-Furukori R Furuya T Takahata and T TateishildquoDetermination of donepezil an acetylcholinesterase inhibitorin human plasma by high-performance liquid chromatographywith ultraviolet absorbance detectionrdquo Journal of Chromatog-raphy B Analytical Technologies in the Biomedical and LifeSciences vol 768 no 2 pp 261ndash265 2002

[33] C Apostolou Y Dotsikas C Kousoulos and Y L LoukasldquoQuantitative determination of donepezil in human plasma byliquid chromatographytandem mass spectrometry employingan automated liquid-liquid extraction based on 96-well formatplates Application to a bioequivalence studyrdquo Journal of Chro-matography B vol 848 no 2 pp 239ndash244 2007

[34] E M Ghoneim M A El-Attar and M M Ghoneim ldquoDeter-mination of donepezil hydrochloride in pharmaceutical formu-lation and human serum by square-wave adsorptive cathodicstripping voltammetryrdquo Chemia Analityczna vol 54 no 3 pp389ndash402 2009

[35] M Yasir and U Sara ldquoDevelopment of UV spectrophotometricmethod for the analysis of acetylcholinesterase inhibitorrdquo Inter-national Journal of Pharmacy and Pharmaceutical Sciences vol6 no 9 pp 128ndash131 2014

[36] C Patel N Patel and C Kothari ldquoQuantitative determina-tion of donepezil hydrochloride by a simple and accuratesynchronous spectrofluorimetric method in human plasmardquoJournal of Young Pharmacists vol 6 no 4 pp 47ndash50 2014

[37] M Pohanka J Fusek V Adam and R Kizek ldquoCarbofuranassay using gelatin based biosensor with acetylcholinesterase as

8 International Journal of Analytical Chemistry

a recognition elementrdquo International Journal of ElectrochemicalScience vol 8 no 1 pp 71ndash79 2013

[38] T Montesinos S Perez-Munguia F Valdez and J-L MartyldquoDisposable cholinesterase biosensor for the detection of pes-ticides in water-miscible organic solventsrdquo Analytica ChimicaActa vol 431 no 2 pp 231ndash237 2001

[39] S Andreescu T Noguer V Magearu and J-L Marty ldquoScreen-printed electrode based on ache for the detection of pesticidesin presence of organic solventsrdquo Talanta vol 57 no 1 pp 169ndash176 2002

[40] S SookW Portia and C Chang-Hwei ldquoBiochemical studies ofthe actions of ethanol on acetylcholinesterase activity ethnol-enzyme-solvent interactionrdquo International Journal of Biochem-istry vol 23 no 2 pp 169ndash174 1991

[41] A D C Obregon M R C Schetinger M M Correa etal ldquoEffects per se of organic solvents in the cerebral acetyl-cholinesterase of ratsrdquo Neurochemical Research vol 30 no 3pp 379ndash384 2005

6 International Journal of Analytical Chemistry

Table 1 Comparison of presented assay with Ellmanrsquos assay and literature

Detection limit Fabrication time Assay time Necessary equipmentPresented smartphoneassay

Galanthamine 1491 nMDonepezil 223 nM Aprox 1 hour 15min Smartphone

Standard Ellmanrsquos assay Galanthamine 183 nMDonepezil 382 nM NA 10min Spectrophotometer

Spectrophotometricassay [21] Galanthamine 005 nM Aprox 35 hours Aprox 20mn Spectrophotometer

Potentiometric assay[22] Galanthamine 54 nM Aprox 10 hours Aprox 10min Electrodes

potentiometerSquare-wavevoltammetry [23] Donepezil 151 nM NA Aprox 20min Electrodes

electrochemical analyser

G 25120583MD 05120583MNo inhibition EtOH IsoPr DMSO0

5

10

15

ΔC

olor

inte

nsity

Figure 12 Method interferences by organic solvents EtOH =ethanol IsoPr = isopropyl alcohol DMSO = dimethyl sulfoxideD 05 120583M = donepezil in 05120583M concentration and G 25 120583M= galanthamine in 25 120583M concentration Error bars representstandard error of the mean for 119899 = 3

Ethanol has been reported to decrease AChE activity [37 39]however there is evidence that at low concentration it canenhance enzyme activity [38 40] No activity change againstuninhibited enzyme was observed in our work Isopropylalcohol as well as ethanol can drop AChE activity [41]which is in agreement with our results To compare usedinhibitors donepezil and galanthamine are included in graph(Figure 12)

4 Conclusion

Here the presented method proved its ability for deter-mination of neurotoxic compounds with promising limitsof detection for galanthamine and donepezil 149 nM and223 nM respectively Assay was successfully validated tothe standard Ellmanrsquos spectrophotometric test and showedfeasibility of measurement in plasma samples Additionallyno specialized equipment and trained personnel are requiredcombined with low cost portability easy preparation andminiaturization are considered as big advantages of the here

invented method Comparison of presented method withstandard Ellmanrsquos assay and literature is given in Table 1

Competing Interests

The authors declare that there is no conflict of interests withregard to the publication of this paper

Acknowledgments

A Long-Term Organization Development Plan 1011 (Facultyof Military Health Sciences University of Defense CzechRepublic) is gratefully acknowledged

References

[1] M Pohanka ldquoCholinesterases a target of pharmacology andtoxicologyrdquo Biomedical Papers vol 155 no 3 pp 219ndash230 2011

[2] M Pohanka ldquoBiosensors containing acetylcholinesterase andbutyrylcholinesterase as recognition tools for detection ofvarious compoundsrdquo Chemical Papers vol 69 no 1 pp 4ndash162015

[3] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961

[4] P M George and M H Abernethy ldquoImproved Ellman proce-dure for erythrocyte cholinesteraserdquo Clinical Chemistry vol 29no 2 pp 365ndash368 1983

[5] V Gorun I Proinov V Baltescu G Balaban and O BarzuldquoModified Ellman procedure for assay of cholinesterases incrude enzymatic preparationsrdquoAnalytical Biochemistry vol 86no 1 pp 324ndash326 1978

[6] M Pohanka ldquoVoltammetric assay of butyrylcholinesterase inplasma samples and its comparison to the standard spectropho-tometric testrdquo Talanta vol 119 pp 412ndash416 2014

[7] A Kostelnik A Cegan and M Pohanka ldquoElectrochemicaldetermination of activity of acetylcholinesterase immobilizedon magnetic particlesrdquo International Journal of ElectrochemicalScience vol 11 no 6 pp 4840ndash4849 2016

[8] F Morıs-Varas A Shah J Aikens N P Nadkarni J D Rozzelland D C Demirjian ldquoVisualization of enzyme-catalyzed reac-tions using pH indicators rapid screening of hydrolase librariesand estimation of the enantioselectivityrdquo Bioorganic andMedic-inal Chemistry vol 7 no 10 pp 2183ndash2188 1999

International Journal of Analytical Chemistry 7

[9] A Kostelnik A Cegan and M Pohanka ldquoColor change ofphenol red by integrated smart phone camera as a tool for thedetermination of neurotoxic compoundsrdquo Sensors vol 16 no9 article 1212 2016

[10] S Timur and A Telefoncu ldquoAcetylcholinesterase (AChE) elec-trodes based on gelatin and chitosan matrices for the pesticidedetectionrdquo Artificial Cells Blood Substitutes and Biotechnologyvol 32 no 3 pp 427ndash442 2004

[11] J Zhang J Zhang F Zhang et al ldquoGraphene oxide as a matrixfor enzyme immobilizationrdquo Langmuir vol 26 no 9 pp 6083ndash6085 2010

[12] M Mogharabi N Nassiri-Koopaei M Bozorgi-KoushalshahiN Nafissi-Varcheh G Bagherzadeh and M A FaramarzildquoImmobilization of laccase in alginate-gelatin mixed gel anddecolorization of synthetic dyesrdquo Bioinorganic Chemistry andApplications vol 2012 Article ID 823830 6 pages 2012

[13] R A Sheldon ldquoEnzyme immobilization the quest for optimumperformancerdquo Advanced Synthesis and Catalysis vol 349 no 8-9 pp 1289ndash1307 2007

[14] M Pohanka ldquoCholinesterases in biorecognition and biosensorsconstruction a reviewrdquo Analytical Letters vol 46 no 12 pp1849ndash1868 2013

[15] M Pohanka ldquoAcetylcholinesterase based dipsticks with indoxy-lacetate as a substrate for assay of organophosphates andcarbamatesrdquo Analytical Letters vol 45 no 4 pp 367ndash374 2012

[16] Y Zheng Z Liu Y Jing J Li and H Zhan ldquoAn acetyl-cholinesterase biosensor based on ionic liquid functionalizedgraphene-gelatin-modified electrode for sensitive detection ofpesticidesrdquo Sensors and Actuators B Chemical vol 210 pp389ndash397 2015

[17] K Grudpan S D Kolev S Lapanantnopakhun I D McKelvieand W Wongwilai ldquoApplications of everyday IT and commu-nications devices in modern analytical chemistry a reviewrdquoTalanta vol 136 pp 84ndash94 2015

[18] A Garcıa M M Erenas E D Marinetto et al ldquoMobile phoneplatform as portable chemical analyzerrdquo Sensors and ActuatorsB Chemical vol 156 no 1 pp 350ndash359 2011

[19] Y Lu W Shi J Qin and B Lin ldquoLow cost portable detectionof gold nanoparticle-labeled microfluidic immunoassay withcamera cell phonerdquo Electrophoresis vol 30 no 4 pp 579ndash5822009

[20] M Pohanka ldquoPhotography by cameras integrated in smart-phones as a tool for analytical chemistry represented by anbutyrylcholinesterase activity assayrdquo Sensors vol 15 no 6 pp13752ndash13762 2015

[21] B J White J A Legako and H J Harmon ldquoSpectrophoto-metric detection of cholinesterase inhibitors with an integratedacetyl-butyrylcholinesterase surfacerdquo Sensors and Actuators BChemical vol 89 no 1-2 pp 107ndash111 2003

[22] M Cuartero M S Garcıa F Garcıa-Canovas and J AOrtuno ldquoNew approach for the potentiometric-enzymatic assayof reversible- competitive enzyme inhibitors Application toacetylcholinesterase inhibitor galantamine and its determina-tion in pharmaceuticals and human urinerdquo Talanta vol 110 pp8ndash14 2013

[23] A Golcu and S A Ozkan ldquoElectroanalytical determination ofdonepezil HCl in tablets and human serum by differential pulseand osteryoung square wave voltammetry at a glassy carbonelectroderdquo Pharmazie vol 61 no 9 pp 760ndash765 2006

[24] Z Xu S Yao Y Wei et al ldquoMonitoring enzyme reaction andscreening of inhibitors of acetylcholinesterase by quantitative

matrix-assisted laser desorptionionization fourier transformmass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 19 no 12 pp 1849ndash1855 2008

[25] TWilleHThiermann and FWorek ldquoEffect of different bufferson kinetic properties of human acetylcholinesterase and theinteraction with organophosphates and oximesrdquo Archives ofToxicology vol 85 no 3 pp 193ndash198 2011

[26] L Shaonan X Xianchuan Z Guonian and T Yajun ldquoKineticcharacters and resistance to inhibition of crude and puri-fied brain acetylcholinesterase of three freshwater fishes byorganophosphatesrdquo Aquatic Toxicology vol 68 no 4 pp 293ndash299 2004

[27] H Jiang S Liu P Zhao and C Pope ldquoRecombinant expres-sion and biochemical characterization of the catalytic domainof acetylcholinesterase-1 from the African malaria mosquitoAnopheles gambiaerdquo Insect Biochemistry andMolecular Biologyvol 39 no 9 pp 646ndash653 2009

[28] A E Boyd A B Marnett L Wong and P Taylor ldquoProbingthe active center gorge of acetylcholinesterase by fluorophoreslinked to substituted cysteinesrdquoThe Journal of Biological Chem-istry vol 275 no 29 pp 22401ndash22408 2000

[29] M Pohanka M Hrabinova J Fusek et al ldquoElectrochemicalbiosensor based on acetylcholinesterase and indoxylacetatefor assay of neurotoxic compounds represented by paraoxonrdquoInternational Journal of Electrochemical Science vol 7 no 1 pp50ndash57 2012

[30] J I da Silva M C de Moraes L C C Vieira A G CorreaQ B Cass and C L Cardoso ldquoAcetylcholinesterase capillaryenzyme reactor for screening and characterization of selectiveinhibitorsrdquo Journal of Pharmaceutical and Biomedical Analysisvol 73 pp 44ndash52 2013

[31] D R Liston J A Nielsen A Villalobos et al ldquoPharmacologyof selective acetylcholinesterase inhibitors implications for usein Alzheimerrsquos diseaserdquo European Journal of Pharmacology vol486 no 1 pp 9ndash17 2004

[32] N Yasui-Furukori R Furuya T Takahata and T TateishildquoDetermination of donepezil an acetylcholinesterase inhibitorin human plasma by high-performance liquid chromatographywith ultraviolet absorbance detectionrdquo Journal of Chromatog-raphy B Analytical Technologies in the Biomedical and LifeSciences vol 768 no 2 pp 261ndash265 2002

[33] C Apostolou Y Dotsikas C Kousoulos and Y L LoukasldquoQuantitative determination of donepezil in human plasma byliquid chromatographytandem mass spectrometry employingan automated liquid-liquid extraction based on 96-well formatplates Application to a bioequivalence studyrdquo Journal of Chro-matography B vol 848 no 2 pp 239ndash244 2007

[34] E M Ghoneim M A El-Attar and M M Ghoneim ldquoDeter-mination of donepezil hydrochloride in pharmaceutical formu-lation and human serum by square-wave adsorptive cathodicstripping voltammetryrdquo Chemia Analityczna vol 54 no 3 pp389ndash402 2009

[35] M Yasir and U Sara ldquoDevelopment of UV spectrophotometricmethod for the analysis of acetylcholinesterase inhibitorrdquo Inter-national Journal of Pharmacy and Pharmaceutical Sciences vol6 no 9 pp 128ndash131 2014

[36] C Patel N Patel and C Kothari ldquoQuantitative determina-tion of donepezil hydrochloride by a simple and accuratesynchronous spectrofluorimetric method in human plasmardquoJournal of Young Pharmacists vol 6 no 4 pp 47ndash50 2014

[37] M Pohanka J Fusek V Adam and R Kizek ldquoCarbofuranassay using gelatin based biosensor with acetylcholinesterase as

8 International Journal of Analytical Chemistry

a recognition elementrdquo International Journal of ElectrochemicalScience vol 8 no 1 pp 71ndash79 2013

[38] T Montesinos S Perez-Munguia F Valdez and J-L MartyldquoDisposable cholinesterase biosensor for the detection of pes-ticides in water-miscible organic solventsrdquo Analytica ChimicaActa vol 431 no 2 pp 231ndash237 2001

[39] S Andreescu T Noguer V Magearu and J-L Marty ldquoScreen-printed electrode based on ache for the detection of pesticidesin presence of organic solventsrdquo Talanta vol 57 no 1 pp 169ndash176 2002

[40] S SookW Portia and C Chang-Hwei ldquoBiochemical studies ofthe actions of ethanol on acetylcholinesterase activity ethnol-enzyme-solvent interactionrdquo International Journal of Biochem-istry vol 23 no 2 pp 169ndash174 1991

[41] A D C Obregon M R C Schetinger M M Correa etal ldquoEffects per se of organic solvents in the cerebral acetyl-cholinesterase of ratsrdquo Neurochemical Research vol 30 no 3pp 379ndash384 2005

International Journal of Analytical Chemistry 7

[9] A Kostelnik A Cegan and M Pohanka ldquoColor change ofphenol red by integrated smart phone camera as a tool for thedetermination of neurotoxic compoundsrdquo Sensors vol 16 no9 article 1212 2016

[10] S Timur and A Telefoncu ldquoAcetylcholinesterase (AChE) elec-trodes based on gelatin and chitosan matrices for the pesticidedetectionrdquo Artificial Cells Blood Substitutes and Biotechnologyvol 32 no 3 pp 427ndash442 2004

[11] J Zhang J Zhang F Zhang et al ldquoGraphene oxide as a matrixfor enzyme immobilizationrdquo Langmuir vol 26 no 9 pp 6083ndash6085 2010

[12] M Mogharabi N Nassiri-Koopaei M Bozorgi-KoushalshahiN Nafissi-Varcheh G Bagherzadeh and M A FaramarzildquoImmobilization of laccase in alginate-gelatin mixed gel anddecolorization of synthetic dyesrdquo Bioinorganic Chemistry andApplications vol 2012 Article ID 823830 6 pages 2012

[13] R A Sheldon ldquoEnzyme immobilization the quest for optimumperformancerdquo Advanced Synthesis and Catalysis vol 349 no 8-9 pp 1289ndash1307 2007

[14] M Pohanka ldquoCholinesterases in biorecognition and biosensorsconstruction a reviewrdquo Analytical Letters vol 46 no 12 pp1849ndash1868 2013

[15] M Pohanka ldquoAcetylcholinesterase based dipsticks with indoxy-lacetate as a substrate for assay of organophosphates andcarbamatesrdquo Analytical Letters vol 45 no 4 pp 367ndash374 2012

[16] Y Zheng Z Liu Y Jing J Li and H Zhan ldquoAn acetyl-cholinesterase biosensor based on ionic liquid functionalizedgraphene-gelatin-modified electrode for sensitive detection ofpesticidesrdquo Sensors and Actuators B Chemical vol 210 pp389ndash397 2015

[17] K Grudpan S D Kolev S Lapanantnopakhun I D McKelvieand W Wongwilai ldquoApplications of everyday IT and commu-nications devices in modern analytical chemistry a reviewrdquoTalanta vol 136 pp 84ndash94 2015

[18] A Garcıa M M Erenas E D Marinetto et al ldquoMobile phoneplatform as portable chemical analyzerrdquo Sensors and ActuatorsB Chemical vol 156 no 1 pp 350ndash359 2011

[19] Y Lu W Shi J Qin and B Lin ldquoLow cost portable detectionof gold nanoparticle-labeled microfluidic immunoassay withcamera cell phonerdquo Electrophoresis vol 30 no 4 pp 579ndash5822009

[20] M Pohanka ldquoPhotography by cameras integrated in smart-phones as a tool for analytical chemistry represented by anbutyrylcholinesterase activity assayrdquo Sensors vol 15 no 6 pp13752ndash13762 2015

[21] B J White J A Legako and H J Harmon ldquoSpectrophoto-metric detection of cholinesterase inhibitors with an integratedacetyl-butyrylcholinesterase surfacerdquo Sensors and Actuators BChemical vol 89 no 1-2 pp 107ndash111 2003

[22] M Cuartero M S Garcıa F Garcıa-Canovas and J AOrtuno ldquoNew approach for the potentiometric-enzymatic assayof reversible- competitive enzyme inhibitors Application toacetylcholinesterase inhibitor galantamine and its determina-tion in pharmaceuticals and human urinerdquo Talanta vol 110 pp8ndash14 2013

[23] A Golcu and S A Ozkan ldquoElectroanalytical determination ofdonepezil HCl in tablets and human serum by differential pulseand osteryoung square wave voltammetry at a glassy carbonelectroderdquo Pharmazie vol 61 no 9 pp 760ndash765 2006

[24] Z Xu S Yao Y Wei et al ldquoMonitoring enzyme reaction andscreening of inhibitors of acetylcholinesterase by quantitative

matrix-assisted laser desorptionionization fourier transformmass spectrometryrdquo Journal of the American Society for MassSpectrometry vol 19 no 12 pp 1849ndash1855 2008

[25] TWilleHThiermann and FWorek ldquoEffect of different bufferson kinetic properties of human acetylcholinesterase and theinteraction with organophosphates and oximesrdquo Archives ofToxicology vol 85 no 3 pp 193ndash198 2011

[26] L Shaonan X Xianchuan Z Guonian and T Yajun ldquoKineticcharacters and resistance to inhibition of crude and puri-fied brain acetylcholinesterase of three freshwater fishes byorganophosphatesrdquo Aquatic Toxicology vol 68 no 4 pp 293ndash299 2004

[27] H Jiang S Liu P Zhao and C Pope ldquoRecombinant expres-sion and biochemical characterization of the catalytic domainof acetylcholinesterase-1 from the African malaria mosquitoAnopheles gambiaerdquo Insect Biochemistry andMolecular Biologyvol 39 no 9 pp 646ndash653 2009

[28] A E Boyd A B Marnett L Wong and P Taylor ldquoProbingthe active center gorge of acetylcholinesterase by fluorophoreslinked to substituted cysteinesrdquoThe Journal of Biological Chem-istry vol 275 no 29 pp 22401ndash22408 2000

[29] M Pohanka M Hrabinova J Fusek et al ldquoElectrochemicalbiosensor based on acetylcholinesterase and indoxylacetatefor assay of neurotoxic compounds represented by paraoxonrdquoInternational Journal of Electrochemical Science vol 7 no 1 pp50ndash57 2012

[30] J I da Silva M C de Moraes L C C Vieira A G CorreaQ B Cass and C L Cardoso ldquoAcetylcholinesterase capillaryenzyme reactor for screening and characterization of selectiveinhibitorsrdquo Journal of Pharmaceutical and Biomedical Analysisvol 73 pp 44ndash52 2013

[31] D R Liston J A Nielsen A Villalobos et al ldquoPharmacologyof selective acetylcholinesterase inhibitors implications for usein Alzheimerrsquos diseaserdquo European Journal of Pharmacology vol486 no 1 pp 9ndash17 2004

[32] N Yasui-Furukori R Furuya T Takahata and T TateishildquoDetermination of donepezil an acetylcholinesterase inhibitorin human plasma by high-performance liquid chromatographywith ultraviolet absorbance detectionrdquo Journal of Chromatog-raphy B Analytical Technologies in the Biomedical and LifeSciences vol 768 no 2 pp 261ndash265 2002

[33] C Apostolou Y Dotsikas C Kousoulos and Y L LoukasldquoQuantitative determination of donepezil in human plasma byliquid chromatographytandem mass spectrometry employingan automated liquid-liquid extraction based on 96-well formatplates Application to a bioequivalence studyrdquo Journal of Chro-matography B vol 848 no 2 pp 239ndash244 2007

[34] E M Ghoneim M A El-Attar and M M Ghoneim ldquoDeter-mination of donepezil hydrochloride in pharmaceutical formu-lation and human serum by square-wave adsorptive cathodicstripping voltammetryrdquo Chemia Analityczna vol 54 no 3 pp389ndash402 2009

[35] M Yasir and U Sara ldquoDevelopment of UV spectrophotometricmethod for the analysis of acetylcholinesterase inhibitorrdquo Inter-national Journal of Pharmacy and Pharmaceutical Sciences vol6 no 9 pp 128ndash131 2014

[36] C Patel N Patel and C Kothari ldquoQuantitative determina-tion of donepezil hydrochloride by a simple and accuratesynchronous spectrofluorimetric method in human plasmardquoJournal of Young Pharmacists vol 6 no 4 pp 47ndash50 2014

[37] M Pohanka J Fusek V Adam and R Kizek ldquoCarbofuranassay using gelatin based biosensor with acetylcholinesterase as

8 International Journal of Analytical Chemistry

a recognition elementrdquo International Journal of ElectrochemicalScience vol 8 no 1 pp 71ndash79 2013

[38] T Montesinos S Perez-Munguia F Valdez and J-L MartyldquoDisposable cholinesterase biosensor for the detection of pes-ticides in water-miscible organic solventsrdquo Analytica ChimicaActa vol 431 no 2 pp 231ndash237 2001

[39] S Andreescu T Noguer V Magearu and J-L Marty ldquoScreen-printed electrode based on ache for the detection of pesticidesin presence of organic solventsrdquo Talanta vol 57 no 1 pp 169ndash176 2002

[40] S SookW Portia and C Chang-Hwei ldquoBiochemical studies ofthe actions of ethanol on acetylcholinesterase activity ethnol-enzyme-solvent interactionrdquo International Journal of Biochem-istry vol 23 no 2 pp 169ndash174 1991

[41] A D C Obregon M R C Schetinger M M Correa etal ldquoEffects per se of organic solvents in the cerebral acetyl-cholinesterase of ratsrdquo Neurochemical Research vol 30 no 3pp 379ndash384 2005

8 International Journal of Analytical Chemistry

a recognition elementrdquo International Journal of ElectrochemicalScience vol 8 no 1 pp 71ndash79 2013

[38] T Montesinos S Perez-Munguia F Valdez and J-L MartyldquoDisposable cholinesterase biosensor for the detection of pes-ticides in water-miscible organic solventsrdquo Analytica ChimicaActa vol 431 no 2 pp 231ndash237 2001

[39] S Andreescu T Noguer V Magearu and J-L Marty ldquoScreen-printed electrode based on ache for the detection of pesticidesin presence of organic solventsrdquo Talanta vol 57 no 1 pp 169ndash176 2002

[40] S SookW Portia and C Chang-Hwei ldquoBiochemical studies ofthe actions of ethanol on acetylcholinesterase activity ethnol-enzyme-solvent interactionrdquo International Journal of Biochem-istry vol 23 no 2 pp 169ndash174 1991

[41] A D C Obregon M R C Schetinger M M Correa etal ldquoEffects per se of organic solvents in the cerebral acetyl-cholinesterase of ratsrdquo Neurochemical Research vol 30 no 3pp 379ndash384 2005