acetylcholinesterase based dipsticks with indoxylacetate as a substrate for assay of...

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This article was downloaded by: [University of Haifa Library] On: 19 August 2013, At: 07:58 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Analytical Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lanl20 Acetylcholinesterase Based Dipsticks with Indoxylacetate as a Substrate for Assay of Organophosphates and Carbamates Miroslav Pohanka a b a Faculty of Military Health Sciences, University of Defense, Hradec Kralove, Czech Republic b University Hospital Hradec Kralove, Hradec Kralove, Czech Republic Accepted author version posted online: 06 Jan 2012.Published online: 16 Mar 2012. To cite this article: Miroslav Pohanka (2012) Acetylcholinesterase Based Dipsticks with Indoxylacetate as a Substrate for Assay of Organophosphates and Carbamates, Analytical Letters, 45:4, 367-374, DOI: 10.1080/00032719.2011.644743 To link to this article: http://dx.doi.org/10.1080/00032719.2011.644743 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

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This article was downloaded by: [University of Haifa Library]On: 19 August 2013, At: 07:58Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Analytical LettersPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/lanl20

Acetylcholinesterase Based Dipstickswith Indoxylacetate as a Substratefor Assay of Organophosphates andCarbamatesMiroslav Pohanka a ba Faculty of Military Health Sciences, University of Defense, HradecKralove, Czech Republicb University Hospital Hradec Kralove, Hradec Kralove, CzechRepublicAccepted author version posted online: 06 Jan 2012.Publishedonline: 16 Mar 2012.

To cite this article: Miroslav Pohanka (2012) Acetylcholinesterase Based Dipsticks with Indoxylacetateas a Substrate for Assay of Organophosphates and Carbamates, Analytical Letters, 45:4, 367-374, DOI:10.1080/00032719.2011.644743

To link to this article: http://dx.doi.org/10.1080/00032719.2011.644743

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the“Content”) contained in the publications on our platform. However, Taylor & Francis,our agents, and our licensors make no representations or warranties whatsoever as tothe accuracy, completeness, or suitability for any purpose of the Content. Any opinionsand views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Contentshould not be relied upon and should be independently verified with primary sourcesof information. Taylor and Francis shall not be liable for any losses, actions, claims,proceedings, demands, costs, expenses, damages, and other liabilities whatsoever orhowsoever caused arising directly or indirectly in connection with, in relation to or arisingout of the use of the Content.

This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

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Bioanalytical

ACETYLCHOLINESTERASE BASED DIPSTICKS WITHINDOXYLACETATE AS A SUBSTRATE FOR ASSAY OFORGANOPHOSPHATES AND CARBAMATES

Miroslav Pohanka1Faculty of Military Health Sciences, University of Defense,Hradec Kralove, Czech Republic2University Hospital Hradec Kralove, Hradec Kralove, Czech Republic

Enzyme acetylcholinesterase (AChE) is an important part of cholinergic neurotransmission.

It is targeted by many toxins such as nerve agents, organophosphates, and carbamate pes-

ticides. Several drugs for treatment of Alzheimer’s disease and Myasthenia gravis are also

AChE inhibitors. The inhibitory effect can be used for assay purposes. The presented

experiment is devoted to the construction of a colorimetric dipstick with immobilized AChE

and using indoxylacetate as a chromogenic substrate. Standard qualitative cellulose filter

papers, high-performance TLC plates, cotton gauze, and parafilm were chosen as matrices

for the testing. The constructed dipsticks were created for assays of paraoxon as a model

organophosphate pesticide and neostigmine as a model carbamate. The assessed limit of

detection was 10�7mol/l for both inhibitors. It responds absolutely by detecting 4 pmol of

inhibitor when a sample volume of 40lL is considered. Long term stability and optimization

of immobilization were also done and practical importance is discussed. The prepared

dipsticks were also used for assays of paraoxon spiked tap and rain water. The suitability

of the dipsticks for practical performance was approved. Intensive color changes from white

to blue are suitable for scoring by a naked eye.

Keywords: Acetylcholinesterase; Biosensor; Butyrylcholinesterase; Nerve agent; Pesticide

INTRODUCTION

Neurosynaptic junctions are the target of several toxins. Acetylcholinesterase(AChE; EC 3.1.1.7) is an unsubstitutable part of cholinergic neurotransmissionwhere it splits transmitter acetylcholine (Ballard et al. 2005). AChE is a target oftoxic compounds such as organophosphate pesticides, nerve agents, or drugs for

Received 3 February 2011; accepted 8 June 2011.

Ministry of Industry of the Czech Republic is gratefully acknowledged for project No.

FR-TI1=541.

Address correspondence to Dr. Miroslav Pohanka, Faculty of Military Health Sciences, University

of Defense, Trebesska 1575, 50001 Hradec Kralove, Czech Republic. E-mail: miroslav.pohanka@gmail.

com

Analytical Letters, 45: 367–374, 2012

Copyright # Taylor & Francis Group, LLC

ISSN: 0003-2719 print=1532-236X online

DOI: 10.1080/00032719.2011.644743

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treatment of Alzheimer’s disease (e.g., donepezil, rivastigmine, galantamine) andMyasthenia gravis (Schwarz et al. 1995). In addition to the physiological implicationof AChE inhibition, AChE is considered as a potent biorecognition element for theconstruction of analytical tools such as biosensors or disposable dipsticks for fastand reliable assays of AChE inhibitors (Pohanka, Musilek, and Kuca 2009).

The ability to construct a functional device is conditioned by the transductionof an enzyme reaction into a colorimetric, electrochemical, or other output. Themost common reaction uses 5,50-dithio-bis-2-nitrobenzoic acid (DTNB) as a chro-mogen and acetylthiocholine as a substrate. It was developed by Ellman andco-workers in the early 1960s (Ellman et al. 1961). The reaction is commonly knownas Ellman’s reaction. The Ellman’s reaction is well suited for assessment of AChEactivity in biological samples such as blood (Pohanka, Hrabinova, and Kuca2008). There are two main disadvantages of Ellman’s reaction when AChE is con-sidered as a biorecognition element. First, the color change is not well recognizablewhen assessed in artificial light by a naked eye. Second, compounds containing thiolsand=or oxime groups can cause color shifts that can be mistakenly considered as anenzyme activity and an AChE inhibitor assay can give a false negative (Petroianu2007). The Ellman’s reaction can be replaced by pH indication. The pH indicationis suitable for construction of colorimetric dipsticks (Pohanka et al. 2010); however,it is highly sensitive to interference by samples with high or low pH values.

Indoxylacetate is another option for the AChE assay. It is a fluorogenic as wellas chromogenic reagent acting also as a substrate of AChE and butyrylcholinester-ase. The hydrolyzed indoxylacetate undergo a reaction into indigo blue with anabsorption peak at 670 nm (Wu, Podust, and Guengerich 2005). In a previous experi-ment of our team, it was concluded that the indoxylacetate is suitable for the AChEactivity assay when used with standard spectrophotometry (Pohanka et al. 2011).The present experiment is aimed at construction of a disposable colorimetric dipstickbased on indoxylacetate for the assay of AChE inhibitors. It was decided to useindoxylacetate as a chromogen providing contrast color appropriate for simple vis-ual assessment of the presence of the inhibitors. Neostigmine and paraoxon werechosen as representative carbamate and organophosphate inhibitors for assay pur-poses. The investigated dipstick should be alternatives to the standard devices basedon DTNB and it is expected that the dipstick will be suitable for the fast field assaypurposes.

MATERIAL AND METHODS

Chemicals

The AChE from electric eel (Electrophorus electricus) 16.7 mkat=mg protein,indoxylacetate, phosphate buffered saline (PBS), DTNB, acetylthiocholine chloride,neostigmine bromide, paraoxon ethyl, sepharose, c cyclodextrin, gelatin, bovineserum albumin, and propan-2-ol (isopropanol) were purchased from Sigma-Aldrich(Saint Louis, Missouri, USA). Ethanol was purchased from Penta (Prague, CzechRepublic). Deionized water was prepared by a MilliQ Ultrapure Water Purificationsystem (Millipore, Billerica, Massachusetts, USA). All other chemicals were obtainedfrom local sources in the analytical purity.

368 M. POHANKA

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Immobilization Procedure

The AChE was dissolved in PBS and the activity was measured and adjusted incompliance with the modified Ellman’s protocol as introduced earlier (Pohanka et al.2010). Immobilization solution was prepared by mixing AChE and one of the conse-quent stabilizing reagents: cyclodextrin, sepharose, gelatin, albumin (2% w=w), orPBS only. Final activity of AChE was 83.4 nkat (5U) in 20 mL of immobilization sol-ution. The following matrices were chosen for testing: standard qualitative cellulosefilter papers (Whatman, Kent, UK), high-Performance TLC plates (HPTP), cottongauze, and parafilm M (Fisher Scientific – Czech Republic branch, Prague, CzechRepublic). Matrices were cut into bands of 2.5� 1 cm. Twenty mL of 100mM indox-ylacetate and ethanol was injected into one edge of each band. The wet bands weredried at laboratory temperature for one hour. After that, 20 mL of immobilizationsolution was spread over the second edge of the bands. The control dipsticks wereprepared by spreading solution where AChE was replaced by albumin solution2mg=mL. Immobilization solution was dried in a cell culture incubator adjustedup to 37�C and atmosphere dried by silica gel. The prepared dipsticks were usedimmediately or stored in the dark and SATP conditions for testing of long termstability.

Dipsticks Performance

Paraoxon or neostigmine were prepared freshly before each assay. A cali-bration scale of inhibitors was prepared: 10�3, 10�4, 10�5, 10�6, 10�7, 10�8, and10�9mol=l in 5% (w=w) isopropanol or 5% isopropanol (control). The dipstick’sedge with immobilized AChE was covered with 40 mL of either saline solution orone of the calibration solutions. After that, the dipsticks were kept in a dry chamberfor 15 minutes. The reaction was started by folding the dipsticks in the middle andpressing the wet edges with immobilized AChE and indoxylacetate together for 30minutes. The coloration was scaled using the following arbitrary units: � no color-ing, þ low coloring, þþ middle coloring, þþþ intensive (maximal) coloring. Thearbitrary units scale can also be understood from Figure 1 introduced in the Resultsand Discussion part. All experiments were carried out in tetraplicate. All measure-ments were compared to the control dipsticks with AChE replaced by albumin.

RESULTS AND DISCUSSION

The principle of the reaction used for the dipstick construction is depicted inFigure 1 in compliance with the reference (Pohanka, Musilek, and Kuca 2009).The first part of experiment was aimed at immobilization procedures. The firstaim was to achieve a long term stable dipstick with immobilized AChE. It is expectedthat dipsticks will be disposable so there are no expected adverse effects such aswashing out or wiping of the enzyme that is disadvantageous in biosensors forrepeated use. Long term acting biosensors should include firmly bound sensitive ele-ments; on the other hand, disposable biosensors are not expected to persist for a longtime in action (Pohanka, Dobes, et al. 2009; Gough et al. 2010). When the immobi-lization protocols are compared, there are different efficacies in AChE entrapping.

COLORIMETRIC DIPSTICKS 369

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Compared to the dipsticks where the stabilizing reagent was not applied, applicationof sepharose, gelatin, cyclodextrin, or albumin is promising for AChE stabilization.Gelatin and albumin can be considered as the best. Regarding the tested matrix, theparafilm and filter paper should be favored to the HPTP and cotton gauze. TheHPTP was inconvenient because the silica gel layer peeled off of the aluminum sup-port sheet. Moreover, indoxylacetate spontaneously underwent a reaction intoindigo blue on the HPTP when stored for a few days. The cotton gauze was notapplicable for dipstick construction because there was no recognizable colorizingpresent. The lack of colorizing can be caused by instability of AChE; however, sub-strate diffusing out into fibers seems to be more plausible. The cotton gauze would beappropriate for dipstick construction after technological processing (nipping, fiberssplicing, etc.). Unfortunately, there was not equipment for such experiments.

In compliance with the experimental data, the filter paper with parafilm as thematrices and albumin with gelatin were scored as the most suitable for the nextexperiments. There was also an attempt to achieve better stability of the immobilizedAChE by adding glycerol that is well suited for macromolecules stabilization (DeLeeuw et al. 1993) including AChE (Millard et al. 2003). In the present experiments,glycerol addition in the range of 1–50% has no beneficial effect for AChE stabiliza-tion. To the contrary, it acted as an AChE inhibitor decreasing AChE activity.Application of glycerol was abandoned as a result of this investigation. The optimaltime interval for dipstick incubation was also researched. Intervals ranged from 10 to60 minutes. For gelatin and filter paper, the coloration was maximal after incubationlasting 30 minutes so this time was chosen for the next interval as an optimum fordipsticks performance.

In the next experiment, we evaluated long term (one month) stability of theprepared dipsticks (see Table 1). The freshly prepared dipsticks were kept at labo-ratory temperature in dry and dark (SATP) conditions. Stability of the prepared dip-sticks was proven for the whole time interval. Owing to the results, it can be statedthat AChE is well stabilized once transferred into the thin layer and successfullydried. It is in compliance with the known experiments (Pohanka, Dobes, et al.

Figure 1. Principle of AChE activity assay using indoxylacetate providing indigo blue color in presence of

air oxygen.

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2009; Vakurov et al. 2005; Ricci et al. 2003). The whole experiment was alsoperformed using dipsticks with AChE replaced by albumin. The control dipsticksprovided no significant coloration. The pertinent loss of enzyme activity may notbe recognized as the evaluation of coloring was carried out by arbitral units.On the other hand, stability of dipsticks can be further improved using vacuumpackaging and=or enclosing into plastic sheets.

The final performance of constructed dipsticks was aimed at estimation of ana-lytical parameters and limit of detection. As a consequence of the previous experi-ments, only dipsticks constructed from the filter paper as a matrix and gelatin as astabilizing reagent were performed. It is not possible to calculate a limit of detectionin a common way as the output values are arbitrary units. Shifting of one arbitraryunit against a control was considered as the proven limit. The whole assay was nega-tively and positively controlled. The negative control representing the enzymaticreaction was carried out by displacement of AChE by albumin (note: not confusenegative control of reaction and assay of analyte). Thus constructed dipsticks pro-vided no significant coloration (arbitrary unit �). The positive control, regardingAChE and providing coloration, was assessed by application of either saline solutionor 5% (w=w) isopropanol in saline solution. The positive controls were scored byþþþ arbitrary units. Assaying of paraoxon and neostigmine provided the same limitof detection: 10�7mol=l responding to absolute detection of 4 pmol of inhibitor whenconsidered the sample volume of 40 ml. Both the calibrations were repeated fourtimes (tetraplicate of measurement) with the same achieved results. The calibrationis depicted as Table 2 where photographs of dipsticks with arbitrary units scoringand molar concentrations of inhibitors are also included. The achieved limits ofdetection are quite low when considering that the coloring was assessed by the nakedeye. The limit of detection can be further improved by performance of reflectometer.For example, No (2007) achieved approximately a one hundred times lower limit ofdetection when indophenyl acetate was used as a substrate and reflectometer forreaction scoring. On the other hand, limits of detection were nearly the same as pro-ven here when the colorization would be scored by a naked eye. In another experi-ment, Luckham and Brennan (2010) achieved similar analytical parameters asreported here. They entrapped AChE via sol-gel nanotechnology and used systemacetylthiocholine as a substrate and AuIIIþ as a chromogen causing the colorchanges by gold containing nanoparticle growth. Comparing to the gold containingdipstick, our device is simpler and cheaper and would be more favorable for practicalperformance. Conversely, our dipstick was constructed in order to be scored visually.

Table 1. Long term stability of prepared dipsticks

Days 0 2 4 7 14 21 28

filter paperþ gelatin þþþ þþþ þþþ þþþ þþþ þþþ þþþfilter paperþ albumin þþ þþ þþ þþ þþ þþ þþparafilmþ gelatin þþ þþ þþ þþ þþ þþ þþparafilmþ albumin þþ þþ þþ þþ þþ þþ þ

Arbitrary units: � no coloring, þ low coloring, þþ middle coloring, þþþ intensive (maximal) coloring

in 30 minutes reaction.

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Some problems could occur in attempts to digitalize signals due to dishomogeneityof papers.

Practical performance of the prepared dipstick was verified using spiked tapand rain water. Both the water samples were spiked with paraoxon to a final concen-tration of 10�5mol=l. The achieved colorings were compared with coloration of thedipstick used for assay of calibrant solution of 10�5mol=l and intact water. Therewere no significant changes between paraoxon assays in tap and rain water, andthe calibrant. This performance on spiked water supports suitability of the prepareddipsticks to assay neurotoxic pesticides in environmental samples.

The construction of simple dipsticks is desired for fast field tests. Disposabledipsticks for fast assay of organophosphorus neurotoxins were reported previously(Pohanka et al. 2010). The dipsticks were suitable for nerve agents such as sarinand were assayed with a limit of detection of 5 nmol=l. However, the pH sensitivedipsticks are sensitive to interference by acids and alkaline. Here, the proposed dip-sticks are not sensitive to such interference. Ellman’s reaction using dipsticks arecommercially available and some patents, for example, WO=1994=005808, are doneon that issue. Simple Ellman’s reaction using dipsticks are also expected to be usedfor assays of nerve agents and the other military relevant compounds (Hoskovcovaet al. 2009) and field diagnosis of organophosphates exposure (Ryhanen andHanninen 1987). Unlike the present dipsticks based on indoxylacetate, Ellman’sreaction-based tools work on the color change from white to yellow as the colorchange is poorly recorded by a naked eye.

CONCLUSIONS

Filter paper as the matrix and gelatin as a stabilizing reagent are suitable forconstruction of inexpensive dipsticks for the simple assay of AChE inhibitors. Thedipsticks functionality was verified using paraoxon as a model organophosphatepesticide and neostigmine as a representative carbamate derivative used in human

Table 2. Calibration for paraoxon and neostigmine using filter paper as matrix and gelatin as stabilizing

reagent

Inhibitor level (mol=l)

control

(no AChE) 10�3� 10�4 10�5 10�6�10�7 10�8� 10�9 0 (saline solution)

– – þ þþ þþþ þþþ

Arbitrary units: � no coloring, þ low coloring, þþ middle coloring, þþþ intensive (maximal) coloring

in 30 minutes reaction.

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medicine as drug for myasthenia gravis and for recovery after narcosis. The satisfac-tory limits of detection and long term stability confirm suitability of dipsticks forpractical performance. Intensive color change is another advantage, when the reac-tion is scored by a naked eye. The coloring is an advantage when comparing the dip-stick with the standard devices based on DTNB, and provides a good opportunityfor practical application. The practical impact of indoxylacetate-based dipsticksshould be expected and become the next use in field assays as the dipstick deviceis quite cheap and simple to operate.

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