research article portable bioactive paper-based...

Hindawi Publishing CorporationJournal of Analytical Methods in ChemistryVolume 2013 Article ID 932946 8 pageshttpdxdoiorg1011552013932946

Research ArticlePortable Bioactive Paper-Based Sensor forQuantification of Pesticides

Murat Kavruk12 Veli Cengiz Oumlzalp13 and Huumlseyin Avni Oumlktem12

1 NanoBiz Ltd Metu Technopolis Galyum Block Floor 2 No 18 06800 Ankara Turkey2Department of Biology Nanobiotechnology RampD Lab Middle East Technical University 06800 Ankara Turkey3 School of Medicine Istanbul Kemerburgaz University 34217 Istanbul Turkey

Correspondence should be addressed to Veli Cengiz Ozalp cengizozalpkemerburgazedutr

Received 14 May 2013 Revised 13 June 2013 Accepted 1 July 2013

Academic Editor Miren Lopez de Alda

Copyright copy 2013 Murat Kavruk et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A paper-based biosensor was developed for the detection of the degradation products of organophosphorus pesticides Thebiosensor quantifies acetylcholine esterase inhibitors in a fast disposable cheap and accurate formatWe specifically focused on theuse of sugar or protein stabilizer to achieve a biosensor with long shelf-life The new biosensor detected malathion with a detectionlimit of 25 ppm in 5min incubation time The operational stability was confirmed by testing 60 days storage at 4∘C when glucosewas used as stabilizer

1 Introduction

Detection of pesticide traces in food and water is an impor-tant safety issue due to intensive agricultural applicationsand their consequent toxicity Pesticides such as organophos-phates (OP) and carbamates (CM) have inhibitory effects oncholinesterases which are enzymes essential for the properfunctioning of the nervous system of vertebrates and insectsThe toxic action of organophosphate and carbamates arisefrom the inhibition of acetylcholinesterase activity leadingto accumulation of acetylcholine at the nerve endings andtherefore causing cholinergic overstimulation characterizedby severe consequences in humans including abdominalcramps muscular tremor hypotension breathing difficultydiarrhea slowing heartbeat (bradycardia) muscular fascic-ulation and paralysis [1] Therefore portable and accuratequantification of pesticides is essential for public health

The detection of pesticides or nerve agents has beentraditionally carried out in laboratory settings with large andexpensive instruments such as gas chromatography coupledwith mass spectroscopy (GC-MS) [2] spray mass spec-troscopy [3] or high performance liquid chromatography(HPLC) [4] Recent research efforts focused on developingbiosensors platforms that can be incorporated into mobile

detection devices In that respect paper attracts considerableattention as a matrix for developing low-cost analyticaldevices [5] Paper is affordable abundant disposable andhas high volume to surface ratio Paper-based biosensorsare usually fast-responding and low-cost diagnostic tools inhealth and environmental applications Bioactive papers areobtained by modification of paper matrix with biomoleculesin order to add sensor functionality One of the majoradvantages of bioactive paper sensors is that they are designedto operatewithout sophisticated equipment [6] Generally thesensitivity is tolerated at the expense of simple applicationscompared to other analytical methods In bioactive paperbiosensors enzyme-immobilized paper is thematrix for fluidsample transportation biological detection and the detectionin a single step process For paper-based biosensors a varietyof colorimetric formats have been developed including dip-stick techniques and lab-on-paper microfluidic systems

Acetylcholine is a neurotransmitter active in centralnervous systems and skeletal-muscle junction Acetylcholineesterase (AChE) is the hydrolase that degrades acetylcholinemolecules into choline and acetic acid thus terminatingimpulse transmission at cholinergic synapsis ThereforeAChE controls generation of nerve impulses in the postsy-naptic neurons Toxicity ofOP andCMdepends on inhibition

2 Journal of Analytical Methods in Chemistry

Plastic support

Filter paper

Color

10 cm

10

cm

(a)

(b) (c)

AChE + DTNB

(1) Dry

(2) Add ATCh + sample

Figure 1 Schematic representation of biosensor support construction (a) Munktell filter papers were cut and (b) fixed on a plastic support(c) The enzyme mixture (AChE and DTNB) was directly applied on the fixed paper and dried The samples with ATCh were directly appliedon dried paper strips for color formation

of AChE thus the enzyme is a common bioevaluator for thedetection of organophosphates and carbamates [7] Extentionof inhibition of this enzyme has been frequently used tomeasure quantitatively the presence of such pesticides AChEinhibition has been proven to be useful in monitoringorganophosphate and carbamate changes in samples withvarious platforms of sensors including surface plasmonresonance (SPR) [8] electrochemical [9 10] colorimetric[11] various nanomaterials-basedmethods [12ndash15] HoweverAChE is the most widely used biorecognition element inbiosensor development for pesticide detection [7] Papermatrix has been the improved material for some AChE-based detection devices such as sol-gel entrapment of goldnanoparticles for paper-dipstick sensor device [16] a lateralflow application [17] Moreover microfluidic paper devicescould be developed by patterning hydrophilic channels andhydrophobic barriers [18] Thus paper could be a low-cost ubiquitous material for developing alternative multiplexsensors for onsite pesticide determination for medical diag-nostics environmental monitoring or food quality analysis

In this study a paper-based sensor was developed as arapid and reliable monitoring method for organophosphatesand carbamates The model interaction between malathionand its specific inhibition of AChE was used to monitorquantitative changes in order to develop a mobile biosensorUnlike the previously reported acetylcholinesterase-basedpaper biosensors we evaluated sugar and protein stabilizersin order to develop a biosensor with improved shelf-life

2 Materials and Methods

21 Materials AChE (from electric eel) (551015840-dithiobis-(2-nitrobenzoic acid) (DTNB) acetylthiocholine iodide(ATCh) andmalathion were purchased from Sigma-AldrichThe chemicals used in the preparation of buffers werepurchased from Merck Munktell No 1 filter discs werepurchased fromMunktell (Falun Sweden)

22 Biosensor Construction A mixture of enzyme sub-strate chromophore and stabilizer solution was prepared forthe indicated final concentrations in each experiment Forpreparing biosensor strips Munktell filter paper discs wereprepared in 1 times 1 cm pieces and autoclaved at 120∘C for 25minutes prior to use (Figure 1(a)) Filter papers were thenfixed via two-sided plaster on plastic supports (Figure 1(b))A mixture of AChE and DTNB solution of 15 120583L was thenapplied on the filter paper by direct pipetting and drying in adesiccator atminus600mmHg for 20minutes Subsequently 15120583Lof ATCh solution was directly applied on the filter paper thatcontains enzyme-DNTBmixtureThe samples were includedin theATCh solutionswithout changing the final volumeThecolor intensity was measured at exactly 5 minutes after theaddition of ATCh containing solution (Figure 1(c))

23 Cholinesterase Inhibition Assay The ChE inhibitionassay is based on quantification of free sulfhydryl groupsof thiocholine which is the product of acetylthiocholinehydrolyzation by acetylcholine esterase Ellmanrsquos reagent 55-dithiobis-(2-nitrobenzoic acid) (DTNB) is used to generatea yellow chromophore detectable at the 405 nm [19] Thecolor development is at maximum level at the beginning andthe enzyme inhibitor addition reduces the final color devel-oped Color development change was converted to inhibitorconcentration by visual comparison or spectrophotometricmeasurements using malathion as standard inhibitor pHstudies were carried out between 10 and 130 with 10 gradeincrements Effect of temperature was investigated at 4∘C25∘C and 37∘C to determine optimum condition for thebiosensor to work efficiently In the shelf-life experiment4∘C and RT conditions were tested In all temperaturesbiosensors were incubated at that specific temperature for 5minutes after dropping of ATChI solution Malathion wasused as an inhibitor of enzyme and target molecule of thebiosensor Up to 200 ppm malathion was investigated todetermine the detection limit of the biosensor The stability

Journal of Analytical Methods in Chemistry 3

HS

S

O

ATCh

OO OH

SSS

Reaction 1

ACh esterase

Reaction 2

Yellow color

HS

O

OH

O

DTNBOO OH

S

O

OH

O

S

TChColorless TNB

OH

OAcetic acid

N+

N+

minusO

N+

N+

N+

N+

N+

minusO

Ominus

Ominus

Figure 2 Two-step sequential reactions of acetylthiocholine (ATCh) for production of yellow colored TNB In reaction 1 ATCh is broken toacetic acid and thiocholine (TCH)The free sulfhydryl group of TCh is quantified through Ellmanrsquos method in reaction 2The resulting TNBis used in a direct determination of the activity of ACh esterase

of the biosensor was compared in the presence of glucose (upto 15 wv) trehalose (up to 8 wv) or BSA (up to 5 wv)in order to improve the stability of the enzyme AChE Thestabilizers were added in the AChE-DNTB mixture and finalvolume was always 15 120583L The digital pictures of the sensormeasurements were obtained by putting the filter papersbetween two acetate papers and scanning them via CanonPixma MP610 multifunctional scanner with time intervalsThese digital pictures were converted into numerical values

3 Results and Discussions

In this study a paper-based sensor device was developedas a rapid and reliable monitoring method for OP and CMpesticides The device is composed of dipstick paper sensorand a CCD camera for analysis in a portable format Themodel interaction between malathion and its specific inhibi-tion of AChE was used to monitor quantitative performanceof the sensor A biosensor for AChE inhibitory molecules hasbeen developed by immobilizing the enzyme its substrate(ATCh) and a chromophore (DTNB 2-nitro-5-thiobenzoicacid) in paper matrix through adsorption The chemicalreactions leading to inhibitor dependent colour developmentare summarized in Figure 2 Acetylcholine esterase inhibition

can be quantified by an assay method first described byEllman et al [19] DTNB is a water soluble chemical thatcan specifically react with free sulfhydryl groups AChEhydrolyses ATCh to produce thiocholine (TCh) and acetateThen the free sulfhydryl group of TCh reduces DTNB toTNB which is absorbed at 405 nm The difference betweeninitial AChE activity and that after incubation with inhibitorsis used to calculate the concentration of inhibitorymolecules

To determine the working amounts of sensing com-ponents a range of concentrations for each of them wassystematically optimized for the best combination of AChEas the enzyme DTNB as the chromophore and ATChIas the artificial substrate Optimum concentrations wereselected according to the quantified colour production andthe observation based on the naked eye since occasionallythe proposed biosensor would be used in the field withminimum instrumental help Figure 3 shows that increasingamount of biosensor components generally resulted in highercolour development as expected For the determinationof the optimal DTNB concentration up to 5120583gmL finalconcentrations were tested for color development with fixedamount of 12UmL enzyme and 4 120583gmL ATChI A higherdensity in yellow colour formation was observed with anincreasing ATChI concentration (Figure 3(a)) Experiments


(a)

Control

0 2 4 6 8 10 12 14 16

Col

or in

tens

ity (a

u)

0

10

20

30

40

50

(b)

0 1 2 3 4 5

Col

or in

tens

ity (a

u)

0

10

20

30

40

50

0 2 4 6

Col

or in

tens

ity (a

u)

0

10

20

30

40

50

60

(c)

[Acetylcholine esterase] (120583gmL)[Acetylthiocholine] (120583gmL)

[DTNB] (120583molmL)

Figure 3The response of biosensor (a) at different substrate (b) enzyme or (c) Ellmanrsquos reagent concentrations In each experiment ATChAChE and DTNB were mixed and adsorbed into paper matrixTheir fixed concentrations were AChE 12UmL DTNB 4 120583gmL and ATChI4 120583gmL The lower panel in (a) shows pictures of paper sensor upper row is performed with sensor mixture and lower row with controlmixture (lacking the enzyme component)

with 4 120583gmL gave a distinguishable yellow colour fromcontrol samples and had a response time faster than the otherconcentrations Thus 4120583gmL of DTNB as the final concen-tration for the chromophore in the final biosensor was chosenbased on this data Figure 3(b) is an enzyme titration curvewhen DTNB and ATChI were fixed at 4 120583gmL The colourintensity was directly proportional to the amount of enzymefor all concentrations used in this study Subsequently wetested ATChI for best signal by fixing DTNB concentrationat 4 120583gmL and 12UmL enzyme Figure 3(c) shows that4 120583gmL DTNB resulted in a biosensor with more rapidcolor formation The overall results from these experimentsresulted in the identification of optimum concentrations ofbiosensor mixture at (i) 4 120583gmLATChI (ii) 4120583gmLDNTBand (iii) 12UmL AChE All subsequent experiments wereconducted using there optimized concentrations of biosensorcomponents

The effect of pH on the biosensor platform was inves-tigated between 1 and 13 (Figure 4) since real samples

could have components changing acidity There was a slightresponse between the values of pH 2 to pH 8 Due to asmall activity loss or the degradation of the enzyme at pH 9slightly less yellow colour formationwas obtained At alkalinevalues more than 10 the yellow colour formation was instantafter pouring the ATChI on to the enzyme-chromophorestabilized part and the density of the yellow increasedimmenselyThis can be explained by cleavage of either DTNBor the ATChI Therefore the sensor would require well-buffered treatments especially for strongly alkaline samples

Thermal stability of AChE on paper was measured by aninvestigation at temperatures 4∘C 25∘C and 37∘C For alltemperatures the biosensors were incubated at that specifictemperature for 5 minutes after the addition of ATChIsolution to the mixture of other components Temperaturestudies showed that at 4∘C the activity of the enzyme ceasedand no colour formation was observed At room temperatureand at 37∘C the yellow colour formation occurred withsignificantly different colour units with respect to controlsensors


pH4 6 8 10 12 14

Col

or in

tens

ity (a

u)

0

50

100

150

200

250

300

minus50

(a)

Temperature0 10 20 30 40 50

Col

or in

tens

ity (a

u)

0

20

40

60

80

100

120

140

160

minus10

(b)

Figure 4 (a) The response of biosensor at different pH values from 4 to 13 Red line (O) is the color intensity of DNTB and ATCh withAChE and black line (Δ) represents the color intensity without AChE (b) Effect of temperature on biosensor response Vertical bars indicatestandard error of mean

Enzyme-based biosensors have been known for theirselectivity specificity and catalytic signal amplification forthe development of biosensors [20 21] However the appli-cations of enzyme-based devices remain limited mostly dueto the liability for extreme physical conditions (eg pH hightemperature and shear force) Thus special procedures arerequired for increasing their stability during storage Immo-bilized enzymes can exhibit much better functional prop-erties when immobilization strategies are properly designedfor the enzyme [22] In our biosensor the stability of theAChE is the most labile part of the biosensor the otherimportant drawback is mentioned to be the instability ofthe artificial substrate ATCh The activity loss of the enzymeshould be controlled for a viable application of the biosensorTherefore several of the known enzyme stabilizers namelybovine serumalbumin (BSA) [23] glucose [24] and trehalose[25] were tested for their effect on the enzyme activity whenimmobilized in the paper In addition low concentration ofsodium azide was also used to maintain sterility as all thesestabilizers can be used as a carbon source bymicroorganismsAdsorption of some such as enzymes alkaline phosphatase(ALP) and horse radish peroxidase (HRP) has previouslybeen reported to increase thermal stability of enzymes by 2-3orders of magnitude For example ALP immobilized paperretained 60 of its activity after 48 hours of incubation at60∘C [26]

The stability of the biosensor was attempted to beimproved by adding BSA into sensor mixture at concen-trations up to 5 (wv) There was about a 40 increasein colour development for 5 BSA addition (Figure 5(a))However the negative controls with no enzyme exhibitedslight yellow colour formation (approximately 10) Thisnonspecific background reaction may be due to the sulfidemoieties in BSA and DTNB might be reacting and forming

colour The background colour development in control sen-sors can be confusing especially in visual determinationThusit was concluded that BSA could not be used as an enzymestabilizer in this biosensor development study Next effectof glucose on the stability of the biosensor was tested up to15 (wv) concentrations It was found that glucose increasedbiosensor colour development by about 35 for 15 glucoseaddition (Figure 5(c)) There was no colour development incontrol sensors with the addition of glucose

Trehalose is a dimer of two glucose molecules and hasrecently been shown to help retain the functional stabilityof enzymes [25] Among sugars trehalose has been knownfor its superior ability in protecting enzyme activity Thus wetested the effect of trehalose on the stability of our biosen-sor up to 8 (wv) (Figure 5(b)) The results showed thattrehalose stabilized the enzyme and improved the stabilityby 10 for 10 of trehalose but not as much as glucoseThus glucose was chosen as the stabilizer compound forthe rest of the studies The final format of the biosensorincluded one additional compound for protection againstmicroorganisms during storage Sodium azide is an antibac-terial agent commonly used in the biosensor developmentsIn this study 002 (wv) sodium azide was used for itsantibacterial effect and the possible effect on the activityof the enzyme was experimented 002 sodium azide didnot have a negative effect on the performance of biosensor(results are not shown) Therefore it was concluded thatsodium azide could be used as antibacterial agent in thebiosensor

The shelf-life of biosensors were tested after they wereincubated at 4 or 25∘C in nontransparent plastic bottlesin order to prevent direct light exposure because bothDTNB and ATCh are light sensitive molecules As seen inFigure 5(d) the biosensor response to ATChI decreased to


BSA ( wv)0 1 2 3 4 5 6

Col

or in

tens

ity (

)

0

10

20

30

40

50

(a)

Trehalose ( wv)0 2 4 6 8 10 12

Col

or in

tens

ity (

)

0

10

20

30

40

50

(b)

Glucose ( wv)0 5 10 15 20 25

Col

or in

tens

ity (

)

0

10

20

30

40

50

(c)

Shelf-life (days)0 10 20 30 40 50 60 70

Col

or in

tens

ity (

)

0

20

40

60

80

100

25∘C

4∘C

(d)

Figure 5 Effect of (a) BSA (b) trehalose (c) glucose and (d) temperature on ChE activity For (a) (b) and (c) black line is the color intensityfor reaction mixture without enzyme and red line is color intensity for reaction mixture with the enzyme

about 40 in one week at RT and to about 50 at 4∘C whenglucose was included in sensor mixture The 4∘C storagemaintained the biosensor efficiency at a constant around50 up to 60 days Similarly room temperature storagemaintained around 20 activity

For evaluating the ability of developed sensor for quan-tifying AChE inhibitory effects a commonly used inhibitormalathion was used by preparing a standard paper sensorwith 4 120583gmL DNTB 12UmL AChE and 15 glucose(WV) The mixture was adsorbed in the paper dried andused immediately The biosensor response was tested by

application of various concentrations of malathion sampleswith 4 120583gmLATChI final concentrationMalathion presencedisplayed inhibition of AChE proportional to its concentra-tion (Figure 6) The inhibition curve approaches a constantvalue of 3 enzyme activity with malathion concentrationsover 25 ppm The limit of detection of the biosensor wasdetermined by testing the inhibitory effect of malathion ata range of concentrations between 1 and 200 ppm Above25 ppm malathion the biosensor response to ATChI wascompletely inhibited (Figure 6) Thus limit of detection wasdetermined to be 25 ppm for malathionThe enzyme activity


Malathion concentration (ppm)0 1 2 3 4 5 6

Enzy

me a

ctiv

ity (

)

0

20

40

60

80

100

Figure 6 Relationship between inhibition of AChE activity andmalathion

as quantifiedwith the biosensor did not reach zero valueThisshould be expected due to background colour development

4 Conclusions

Performance of a dipstick-type acetylcholine esteraseinhibitor sensor was investigated for determining optimalconcentrations of sensor components and relevant enzymestabilizers The results in this study suggest that sugarstabilizers can be a potentially useful component in paper-based acetylcholine esterase inhibitor sensors Among BSAglucose or trehalose glucose was the best stabilizer inimproving colour development and shelf-life at 4∘C forespecially visual tests BSA improved the stability at higherlevels compared to glucose but the background levels didalso increase to levels hindering visual quantification

References

[1] B W Wilson ldquoCholinesterase inhibitionrdquo in Encyclopedia ofToxicology P Wexler Ed pp 588ndash599 Elsevier New York NYUSA 2nd edition 2005

[2] H Schulze E Scherbaum M Anastassiades S Vorlova RD Schmid and T T Bachmann ldquoDevelopment validationand application of an acetylcholinesterase-biosensor test forthe direct detection of insecticide residues in infant foodrdquoBiosensors and Bioelectronics vol 17 no 11-12 pp 1095ndash11052002

[3] N Malaj Z Ouyang G Sindona and R G Cooks ldquoAnalysis ofpesticide residues by leaf spray mass spectrometryrdquo AnalyticalMethods vol 4 pp 1913ndash1919 2012

[4] G Zhao C Wang Q Wu and Z Wang ldquoDetermination ofcarbamate pesticides in water and fruit samples using carbonnanotube reinforced hollow fiber liquid-phase microextractionfollowed by high performance liquid chromatographyrdquo Analyt-ical Methods vol 3 no 6 pp 1410ndash1417 2011

[5] F Kong and Y F Hu ldquoBiomolecule immobilization techniquesfor bioactive paper fabricationrdquo Analytical and BioanalyticalChemistry vol 403 no 1 pp 7ndash13 2012

[6] R Pelton ldquoBioactive paper provides a low-cost platform fordiagnosticsrdquo TrAC-Trends in Analytical Chemistry vol 28 no8 pp 925ndash942 2009

[7] C S Pundir and N Chauhan ldquoAcetylcholinesterase inhibition-based biosensors for pesticide determination a reviewrdquoAnalyt-ical Biochemistry vol 429 pp 19ndash31 2012

[8] E Milkani C R Lambert and W G McGimpsey ldquoDirectdetection of acetylcholinesterase inhibitor binding with anenzyme-based surface plasmon resonance sensorrdquo AnalyticalBiochemistry vol 408 no 2 pp 212ndash219 2011

[9] M Cuartero J A Ortuno M S Garcıa and F Garcıa-CanovasldquoAssay of acetylcholinesterase activity by potentiometric mon-itoring of acetylcholinerdquo Analytical Biochemistry vol 421 pp208ndash212 2012

[10] D Lu J Wang L Wang et al ldquoA novel nanoparticle-baseddisposable electrochemical immunosensor for diagnosis ofexposure to toxic organophosphorus agentsrdquo Advanced Func-tional Materials vol 21 no 22 pp 4371ndash4378 2011

[11] H Li J Guo H Ping et al ldquoVisual detection of organophos-phorus pesticides represented by mathamidophos using Aunanoparticles as colorimetric proberdquo Talanta vol 87 no 1 pp93ndash99 2011

[12] D Liu W Chen J Wei X Li Z Wang and X Jiang ldquoAhighly sensitive dual-readout assay based on gold nanoparticlesfor organophosphorus and carbamate pesticidesrdquo AnalyticalChemistry vol 84 no 9 pp 4185ndash4191 2012

[13] L Xiang C Zhao and J Wang ldquoNanomaterials-based electro-chemical sensors and biosensors for pesticide detectionrdquo SensorLetters vol 9 no 3 pp 1184ndash1189 2011

[14] R K Scheerle J Graszligmann and T Letzel ldquoEnzymatic conver-sion continuously monitored with a robotic nanoESI-MS toolexperimental statusrdquo Analytical Methods vol 3 no 4 pp 822ndash830 2011

[15] S Upadhyay M K Sharma G R Rao B K BhattacharyaV K Rao and R Vijayaraghavan ldquoApplication of bimetal-lic nanoparticles modified screen printed electrode for thedetection of organophosphate compounds using an enzymeinhibition approachrdquo Analytical Methods vol 3 no 10 pp2246ndash2253 2011

[16] R E Luckham and J D Brennan ldquoBioactive paper dip-stick sensors for acetylcholinesterase inhibitors based on sol-gelenzymegold nanoparticle compositesrdquoAnalyst vol 135 no8 pp 2028ndash2035 2010

[17] S M Z Hossain R E Luckham M J McFadden and JD Brennan ldquoReagentless bidirectional lateral flow bioactivepaper sensors for detection of pesticides in beverage and foodsamplesrdquo Analytical Chemistry vol 81 no 21 pp 9055ndash90642009

[18] X Li D R Ballerini and W Shen ldquoA perspective on paper-based microfluidics current status and future trendsrdquo Biomi-crofluidics vol 6 no 1 pp 11301ndash11313 2012

[19] 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

[20] F W Scheller U Wollenberger A Warsinke and F LisdatldquoResearch and development in biosensorsrdquo Current Opinion inBiotechnology vol 12 no 1 pp 35ndash40 2001

[21] K M Koeller and C-H Wong ldquoEnzymes for chemical synthe-sisrdquo Nature vol 409 no 6817 pp 232ndash240 2001

[22] C Mateo J M Palomo G Fernandez-Lorente J M Guisanand R Fernandez-Lafuente ldquoImprovement of enzyme activitystability and selectivity via immobilization techniquesrdquo Enzymeand Microbial Technology vol 40 no 6 pp 1451ndash1463 2007


[23] B S Chang and R R Mahoney ldquoEnzyme thermostabilizationby bovine serum albumin and other proteins evidence forhydrophobic interactionsrdquo Biotechnology and Applied Biochem-istry vol 22 part 2 pp 203ndash214 1995

[24] L S Taylor P York A C Williams et al ldquoSucrose reducesthe efficiency of protein denaturation by a chaotropic agentrdquoBiochimica et Biophysica Acta vol 1253 no 1 pp 39ndash46 1995

[25] J K Kaushik and R Bhat ldquoWhy is trehalose an exceptionalprotein stabilizer An analysis of the thermal stability ofproteins in the presence of the compatible osmolyte trehaloserdquoJournal of Biological Chemistry vol 278 no 29 pp 26458ndash26465 2003

[26] M S Khan X Li W Shen and G Garnier ldquoThermal stabilityof bioactive enzymatic papersrdquo Colloids and Surfaces B vol 75no 1 pp 239ndash246 2010

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Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


Plastic support

Filter paper

Color

10 cm

10

cm

(a)

(b) (c)

AChE + DTNB

(1) Dry

(2) Add ATCh + sample

Figure 1 Schematic representation of biosensor support construction (a) Munktell filter papers were cut and (b) fixed on a plastic support(c) The enzyme mixture (AChE and DTNB) was directly applied on the fixed paper and dried The samples with ATCh were directly appliedon dried paper strips for color formation

of AChE thus the enzyme is a common bioevaluator for thedetection of organophosphates and carbamates [7] Extentionof inhibition of this enzyme has been frequently used tomeasure quantitatively the presence of such pesticides AChEinhibition has been proven to be useful in monitoringorganophosphate and carbamate changes in samples withvarious platforms of sensors including surface plasmonresonance (SPR) [8] electrochemical [9 10] colorimetric[11] various nanomaterials-basedmethods [12ndash15] HoweverAChE is the most widely used biorecognition element inbiosensor development for pesticide detection [7] Papermatrix has been the improved material for some AChE-based detection devices such as sol-gel entrapment of goldnanoparticles for paper-dipstick sensor device [16] a lateralflow application [17] Moreover microfluidic paper devicescould be developed by patterning hydrophilic channels andhydrophobic barriers [18] Thus paper could be a low-cost ubiquitous material for developing alternative multiplexsensors for onsite pesticide determination for medical diag-nostics environmental monitoring or food quality analysis

In this study a paper-based sensor was developed as arapid and reliable monitoring method for organophosphatesand carbamates The model interaction between malathionand its specific inhibition of AChE was used to monitorquantitative changes in order to develop a mobile biosensorUnlike the previously reported acetylcholinesterase-basedpaper biosensors we evaluated sugar and protein stabilizersin order to develop a biosensor with improved shelf-life

2 Materials and Methods

21 Materials AChE (from electric eel) (551015840-dithiobis-(2-nitrobenzoic acid) (DTNB) acetylthiocholine iodide(ATCh) andmalathion were purchased from Sigma-AldrichThe chemicals used in the preparation of buffers werepurchased from Merck Munktell No 1 filter discs werepurchased fromMunktell (Falun Sweden)

22 Biosensor Construction A mixture of enzyme sub-strate chromophore and stabilizer solution was prepared forthe indicated final concentrations in each experiment Forpreparing biosensor strips Munktell filter paper discs wereprepared in 1 times 1 cm pieces and autoclaved at 120∘C for 25minutes prior to use (Figure 1(a)) Filter papers were thenfixed via two-sided plaster on plastic supports (Figure 1(b))A mixture of AChE and DTNB solution of 15 120583L was thenapplied on the filter paper by direct pipetting and drying in adesiccator atminus600mmHg for 20minutes Subsequently 15120583Lof ATCh solution was directly applied on the filter paper thatcontains enzyme-DNTBmixtureThe samples were includedin theATCh solutionswithout changing the final volumeThecolor intensity was measured at exactly 5 minutes after theaddition of ATCh containing solution (Figure 1(c))

23 Cholinesterase Inhibition Assay The ChE inhibitionassay is based on quantification of free sulfhydryl groupsof thiocholine which is the product of acetylthiocholinehydrolyzation by acetylcholine esterase Ellmanrsquos reagent 55-dithiobis-(2-nitrobenzoic acid) (DTNB) is used to generatea yellow chromophore detectable at the 405 nm [19] Thecolor development is at maximum level at the beginning andthe enzyme inhibitor addition reduces the final color devel-oped Color development change was converted to inhibitorconcentration by visual comparison or spectrophotometricmeasurements using malathion as standard inhibitor pHstudies were carried out between 10 and 130 with 10 gradeincrements Effect of temperature was investigated at 4∘C25∘C and 37∘C to determine optimum condition for thebiosensor to work efficiently In the shelf-life experiment4∘C and RT conditions were tested In all temperaturesbiosensors were incubated at that specific temperature for 5minutes after dropping of ATChI solution Malathion wasused as an inhibitor of enzyme and target molecule of thebiosensor Up to 200 ppm malathion was investigated todetermine the detection limit of the biosensor The stability


HS

S

O

ATCh

OO OH

SSS

Reaction 1

ACh esterase

Reaction 2

Yellow color

HS

O

OH

O

DTNBOO OH

S

O

OH

O

S

TChColorless TNB

OH

OAcetic acid

N+

N+

minusO

N+

N+

N+

N+

N+

minusO

Ominus

Ominus








(a)

Control

0 2 4 6 8 10 12 14 16

Col

or in

tens

ity (a

u)

0

10

20

30

40

50

(b)

0 1 2 3 4 5

Col

or in

tens

ity (a

u)

0

10

20

30

40

50

0 2 4 6

Col

or in

tens

ity (a

u)

0

10

20

30

40

50

60

(c)









pH4 6 8 10 12 14

Col

or in

tens

ity (a

u)

0

50

100

150

200

250

300

minus50

(a)


Col

or in

tens

ity (a

u)

0

20

40

60

80

100

120

140

160

minus10

(b)








BSA ( wv)0 1 2 3 4 5 6

Col

or in

tens

ity (

)

0

10

20

30

40

50

(a)

Trehalose ( wv)0 2 4 6 8 10 12

Col

or in

tens

ity (

)

0

10

20

30

40

50

(b)

Glucose ( wv)0 5 10 15 20 25

Col

or in

tens

ity (

)

0

10

20

30

40

50

(c)

Shelf-life (days)0 10 20 30 40 50 60 70

Col

or in

tens

ity (

)

0

20

40

60

80

100

25∘C

4∘C

(d)







Enzy

me a

ctiv

ity (

)

0

20

40

60

80

100



4 Conclusions


References





































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


HS

S

O

ATCh

OO OH

SSS

Reaction 1

ACh esterase

Reaction 2

Yellow color

HS

O

OH

O

DTNBOO OH

S

O

OH

O

S

TChColorless TNB

OH

OAcetic acid

N+

N+

minusO

N+

N+

N+

N+

N+

minusO

Ominus

Ominus








(a)

Control

0 2 4 6 8 10 12 14 16

Col

or in

tens

ity (a

u)

0

10

20

30

40

50

(b)

0 1 2 3 4 5

Col

or in

tens

ity (a

u)

0

10

20

30

40

50

0 2 4 6

Col

or in

tens

ity (a

u)

0

10

20

30

40

50

60

(c)









pH4 6 8 10 12 14

Col

or in

tens

ity (a

u)

0

50

100

150

200

250

300

minus50

(a)


Col

or in

tens

ity (a

u)

0

20

40

60

80

100

120

140

160

minus10

(b)








BSA ( wv)0 1 2 3 4 5 6

Col

or in

tens

ity (

)

0

10

20

30

40

50

(a)

Trehalose ( wv)0 2 4 6 8 10 12

Col

or in

tens

ity (

)

0

10

20

30

40

50

(b)

Glucose ( wv)0 5 10 15 20 25

Col

or in

tens

ity (

)

0

10

20

30

40

50

(c)

Shelf-life (days)0 10 20 30 40 50 60 70

Col

or in

tens

ity (

)

0

20

40

60

80

100

25∘C

4∘C

(d)







Enzy

me a

ctiv

ity (

)

0

20

40

60

80

100



4 Conclusions


References





































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


(a)

Control

0 2 4 6 8 10 12 14 16

Col

or in

tens

ity (a

u)

0

10

20

30

40

50

(b)

0 1 2 3 4 5

Col

or in

tens

ity (a

u)

0

10

20

30

40

50

0 2 4 6

Col

or in

tens

ity (a

u)

0

10

20

30

40

50

60

(c)









pH4 6 8 10 12 14

Col

or in

tens

ity (a

u)

0

50

100

150

200

250

300

minus50

(a)


Col

or in

tens

ity (a

u)

0

20

40

60

80

100

120

140

160

minus10

(b)








BSA ( wv)0 1 2 3 4 5 6

Col

or in

tens

ity (

)

0

10

20

30

40

50

(a)

Trehalose ( wv)0 2 4 6 8 10 12

Col

or in

tens

ity (

)

0

10

20

30

40

50

(b)

Glucose ( wv)0 5 10 15 20 25

Col

or in

tens

ity (

)

0

10

20

30

40

50

(c)

Shelf-life (days)0 10 20 30 40 50 60 70

Col

or in

tens

ity (

)

0

20

40

60

80

100

25∘C

4∘C

(d)







Enzy

me a

ctiv

ity (

)

0

20

40

60

80

100



4 Conclusions


References





































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


pH4 6 8 10 12 14

Col

or in

tens

ity (a

u)

0

50

100

150

200

250

300

minus50

(a)


Col

or in

tens

ity (a

u)

0

20

40

60

80

100

120

140

160

minus10

(b)








BSA ( wv)0 1 2 3 4 5 6

Col

or in

tens

ity (

)

0

10

20

30

40

50

(a)

Trehalose ( wv)0 2 4 6 8 10 12

Col

or in

tens

ity (

)

0

10

20

30

40

50

(b)

Glucose ( wv)0 5 10 15 20 25

Col

or in

tens

ity (

)

0

10

20

30

40

50

(c)

Shelf-life (days)0 10 20 30 40 50 60 70

Col

or in

tens

ity (

)

0

20

40

60

80

100

25∘C

4∘C

(d)







Enzy

me a

ctiv

ity (

)

0

20

40

60

80

100



4 Conclusions


References





































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


BSA ( wv)0 1 2 3 4 5 6

Col

or in

tens

ity (

)

0

10

20

30

40

50

(a)

Trehalose ( wv)0 2 4 6 8 10 12

Col

or in

tens

ity (

)

0

10

20

30

40

50

(b)

Glucose ( wv)0 5 10 15 20 25

Col

or in

tens

ity (

)

0

10

20

30

40

50

(c)

Shelf-life (days)0 10 20 30 40 50 60 70

Col

or in

tens

ity (

)

0

20

40

60

80

100

25∘C

4∘C

(d)







Enzy

me a

ctiv

ity (

)

0

20

40

60

80

100



4 Conclusions


References





































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



Enzy

me a

ctiv

ity (

)

0

20

40

60

80

100



4 Conclusions


References





































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

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